WO2016100376A1 - Nanothérapeutique pour le traitement d'infections provoquées par des agents pathogènes intracellulaires et extracellulaires - Google Patents

Nanothérapeutique pour le traitement d'infections provoquées par des agents pathogènes intracellulaires et extracellulaires Download PDF

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WO2016100376A1
WO2016100376A1 PCT/US2015/065873 US2015065873W WO2016100376A1 WO 2016100376 A1 WO2016100376 A1 WO 2016100376A1 US 2015065873 W US2015065873 W US 2015065873W WO 2016100376 A1 WO2016100376 A1 WO 2016100376A1
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mxf
mesoporous silica
silica particles
msn
composition
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Inventor
Marcus A. Horwitz
Jeffrey I. Zink
Zilu LI
Bai-Yu Lee Clemens
Daniel L. Clemens
Angela An-Chi HWANG
Min XUE
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B37/00—Compounds having molecular sieve properties but not having base-exchange properties
    • C01B37/02—Crystalline silica-polymorphs, e.g. silicalites dealuminated aluminosilicate zeolites

Definitions

  • the field of the currently claimed embodiments of this invention relate to compositions and methods for treating infectious diseases caused by intracellular pathogens within host cells.
  • MSNs Mesoporous silica nanoparticles
  • Embodiments of the invention include a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein, said plurality of mesoporous silica particles comprising capping structures that prevent release of said antibiotic prior to being exposed to an activation stimulus present in said host cells; and an antibiotic loaded into said pores of said plurality of mesoporous silica particles and contained therein by said capping structures to be available to be released therefrom when said plurality of mesoporous silica particles are exposed to said activation stimulus, wherein said antibiotic comprises at least one antibiotic selected from the fluoroquinolone group of antibiotics, and wherein said composition has a ratio of weight of said plurality of mesoporous silica particles to weight of said antibiotic loaded into said pores and contained therein and available to be released of at least 5%.
  • Embodiments of the invention include a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein, said plurality of mesoporous silica particles comprising capping structures that prevent release of said antibiotic prior to being exposed to an activation stimulus present in said host cells; and an antibiotic loaded into said pores of said plurality of mesoporous silica particles and contained therein by said capping structures to be available to be released therefrom when said plurality of mesoporous silica particles are exposed to said activation stimulus, wherein said antibiotic comprises at least one antibiotic selected from the fluoroquinolone group of antibiotics, and wherein said composition has a ratio of weight of said plurality of mesoporous silica particles to weight of said antibiotic loaded into said pores and contained there
  • Embodiments of the invention include a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores that are suitable to contain a first antibiotic loaded therein, said first plurality of mesoporous silica particles comprising capping structures that prevent release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells; a first antibiotic loaded into said pores of said first plurality of mesoporous silica particles and contained therein by said capping structures to be available to be released therefrom when said first plurality of mesoporous silica particles are exposed to said activation stimulus; a second plurality of mesoporous silica particles defining pores that are suitable to contain a second antibiotic loaded therein, said second plurality of mesoporous silica particles comprising capping structures that prevent release of said second antibiotic prior to being exposed to an activation stimulus present in said host cells; and a second antibiotic loaded into said pores
  • Embodiments of the invention include a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said chemical linker prevents release of said antibiotic prior to being exposed to an activation stimulus present in said host cells.
  • Embodiments of the invention include a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said chemical linker prevents release of said antibiotic prior to being exposed to an activation stimulus present in said host cells.
  • Embodiments of the invention include a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores; a first chemical linker attached to said first plurality of mesoporous silica particles; a first antibiotic attached to said first plurality of mesoporous silica particles by said first chemical linker; a second plurality of mesoporous silica particles defining pores; a second chemical linker attached to said second plurality of mesoporous silica particles; and a second antibiotic attached to said second plurality of mesoporous silica particles by said second chemical linker; wherein said first chemical linker prevents release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells, and wherein said second chemical linker prevents release of said second antibiotic prior to being exposed to said activation stimulus present in said host cells.
  • Embodiments of the invention include a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores; a first chemical linker attached to said first plurality of mesoporous silica particles; a first antibiotic attached to said first plurality of mesoporous silica particles by said first chemical linker; a second plurality of mesoporous silica particles defining pores; a second chemical linker attached to said second plurality of mesoporous silica particles; and a second antibiotic attached to said second plurality of mesoporous silica particles by said second chemical linker; wherein said first chemical linker prevents release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells, and wherein said second chemical linker prevents release of said second antibiotic prior to being
  • Embodiments of the invention include a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores; a first chemical linker attached to said first plurality of mesoporous silica particles; a first antibiotic attached to said first plurality of mesoporous silica particles by said first chemical linker; a second plurality of mesoporous silica particles defining pores; a second chemical linker attached to said second plurality of mesoporous silica particles; and a second antibiotic attached to said second plurality of mesoporous silica particles by said second chemical linker; wherein said first chemical linker prevents release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells, and wherein said second chemical linker prevents release of said second antibiotic prior to being exposed to said activation stimulus present in said host cells.
  • Embodiments of the invention include a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores that are suitable to contain a first antibiotic loaded therein, said first plurality of mesoporous silica particles comprising capping structures that prevent release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells; a first antibiotic loaded into said pores of said first plurality of mesoporous silica particles and contained therein by said capping structures to be available to be released therefrom when said first plurality of mesoporous silica particles are exposed to said activation stimulus; a second plurality of mesoporous silica particles defining pores; a chemical linker attached to said second plurality of mesoporous silica particles; and a second antibiotic attached to said second plurality of mesoporous silica particles by said chemical linker; wherein said chemical linker prevents release of said second
  • Embodiments of the invention include a composition for targeting a nanoparticle to a particular organ in a subject, comprising: a plurality of mesoporous silica particles defining pores; and a compound attached to said plurality of mesoporous silica particles for directing said plurality of mesoporous silica particles to a particular organ.
  • Embodiments of the invention include a method of targeting a nanoparticle to a particular organ in a subject comprising administering a composition for targeting a nanoparticle to a particular organ in a subject, comprising: a plurality of mesoporous silica particles defining pores; and a compound attached to said plurality of mesoporous silica particles for directing said plurality of mesoporous silica particles to a particular organ.
  • FIG. 1 shows a depiction of the drug trapping and intracellular release mechanism of
  • FIG 2A is a schematic showing a silane stalk (3-mercaptopropyl) trimethoxysilane is attached to the surface of the MSN.
  • FIG. 2B shows that the disulfide bond on the thread is cleaved by the reducing agent, 2-mercaptoethanol in the laboratory or glutathione inside cells, removing the bulky ⁇ -CD cap and releasing MXF from the pores of the nanoparticle.
  • FIG. 3 is a graph showing MSN-SS-MXF is released by MXF in DI water when 2- mercaptoethanol is added and cleaves the disulfide bond.
  • FIG. 4 is a graph showing Hoechst dye release from MSN-SS snap-top by physiological concentrations of GSH. Snap-Top nanoparticles (1 mg/mL) loaded with the membrane permeant DNA stain Hoechst 33342 were incubated with various concentrations of GSH ranging from 0 - 16 mM, as indicated, overnight at room temperature.
  • FIG. 5 is a fluorescent image showing that MSN-SS-Hoechst but not their PBS eluates stain the nuclei of THP-1 cells.
  • FIG. 6 A shows PMA-differentiated THP-1 macrophages infected with F. tularensis
  • FIG. 6B shows results with eluates prepared from MSN-SS-MXF incubated in aqueous PBS with and without reducing agent 2-mercaptoethanol (PME)sand with eluates prepared in DMSO with ⁇ .
  • FIG. 6C shows results with free MXF.
  • FIG. 6D shows a scale showing the impact of MSN-SS-MXF and MXF treatment on bacterial viability compared using median-effect analysis.
  • Median-effect curves generated by CompuSyn for free MXF and an equivalent amount of MXF on the nanoparticle (MSN) were plotted in the same graph.
  • Log(D) is dose of free MXF or MXF equivalent of MSN-SS-MXF in logarithm;
  • Log(Fa/Fu) is the division of the fraction of bacteria killed (Fa) by the fraction of bacteria surviving (Fu) in logarithm.
  • FIG. 7A shows percentage change in weight of mice.
  • FIG. 7B shows percentage change in weight of mice.
  • FIG. 8A is a graph showing bacterial burden in the lung monitored over the course of infection.
  • FIG. 8B is a graph showing bacterial burden in the lung monitored over the course of infection.
  • FIG. 8C is a graph showing the effect of each treatment on F. tularensis burden in lung, liver, and spleen as determined by assaying the bacterial CFU one day after the final treatment.
  • FIG. 8A is a graph showing bacterial burden in the lung monitored over the course of infection.
  • FIG. 8B is a graph showing bacterial burden in the lung monitored over the course of infection.
  • FIG. 8C is a graph showing the effect of each treatment on F. tularensis burden in lung, liver, and spleen as determined by assaying the bacterial CFU one day after the final treatment.
  • 8D is a graph showing the effect of each treatment on F. tularensis burden in lung, liver, and spleen as determined by assaying the bacterial CFU one day after the final treatment.
  • FIG. 9A is a graph showing the distribution of i.v. administered MSN-SS-MXF in lung, liver, spleen, heart and kidney after a single injection.
  • FIG. 9B is a graph showing the distribution of i.v. administered MSN-SS-MXF in lung, liver, spleen, heart and kidney after a three injections over 6 days.
  • FIG. 9C is a graph showing control results.
  • FIG. 10 is a spectroscopy graph showing adamantyl group attachment.
  • FIG. 11 A is a graph showing dose dependent inhibition of F. novicida growth by
  • FIG. 1 IB is a graph showing MXF concentrations plotted against the difference in OD540 readings between an F. novicida culture not treated with MXF and a culture treated with MXF in the amounts indicated.
  • FIG. 11C is a linear standard curve converted from the log value of MXF concentrations plotted against the difference in OD540 reading between an F. novicida culture not treated with MXF and an F. novicida culture treated with MXF.
  • FIG. 12 shows graphs showing median-effect plots to compare efficacy of MXF administered as free drug vs. MSN-SS-MXF.
  • FIG. 13 is a graphic showing gated nanoparticles carry large quantities of moxifloxicin into macrophages, release the cargo and kill intracellular F. tularensis both in cultures and in mice.
  • FIG. 14 shows chemical structures of the stalks (top) and caps (bottom) of two nanovalves.
  • FIG. 15A shows attachment of two different pH-sensitive nanovalves on MCM-41 surface.
  • FIG. 15B shows MSN-MBI-MXF drug release profile. There is no leakage at pH 7, as indicated by the flat baseline; drug release starts when the pH is lowered to 5 by addition of acid.
  • FIG. 15C is a TEM image of MCM-41 showing its hexagonal pore structure.
  • FIG. 16A is a graph showing uptake capacity of MSN-MBI with different inner mesopore charges and stalk synthetic pathways.
  • FIG. 16B is a schematic showing MSN mesopores modified (left to right) with amine (+), unmodified silanol (-), or phosphonate (-).
  • FIG. 17A-17E show confocal microscopy images demonstrating avid uptake of
  • FIG. 18A is a graph showing human THP-1 macrophages infected with F. tularensis
  • FIG. 18B is a graph showing human THP-1 macrophages infected with F. tularensis LVS and treated with MSN-ANA-MXF.
  • FIG. 18C is a graph showing human THP-1 macrophages infected with F. tularensis LVS and treated with MSN-MBI-MXF. Viable bacteria were determined by enumerating colony forming units (CFU) of F. tularensis in the macrophage monolayer.
  • FIG. 18D is a graph showing impact of the drug released from MSN-ANA-MXF.
  • FIG. 18E is a graph showing impact of the drug released from MSN-MBI-MXF.
  • FIG. 19 is a graph showing uptake and release capacity of negatively charged MSN-
  • MBI loaded at pH 4 or 7 and positively charged MSN-MBI loaded at pH 7, 10, or 12 MXF aqueous solution.
  • FIG. 20 shows uptake capacity, uptake efficiency and release capacity of phosphonated MSN-MBI loaded in 20 mM MXF aqueous solution (pH 7), 20 mM MXF PBS solution (pH 7.4) and 40 mM MXF PBS solution (pH 7.4).
  • FIG. 21 A is a graph showing that release profiles show that the more times the MSN are washed, the lower the amount of residual and release capacity.
  • FIG. 21B is a graph showing the amount of MXF washed away each time decreases as the number of washes increases; the decrease for each step is -30 %.
  • FIG. 22A shows results from experiment 1 where treatment with MSN-MBI-MXF prevents weight loss caused by pneumonic tularemia.
  • FIG. 22B shows results from experiment 2 where treatment with MSN-MBI-MXF prevents weight loss caused by pneumonic tularemia.
  • FIG. 23 A shows results of mice infected with F. tularensis LVS by the intranasal route.
  • FIG. 23B shows results of mice infected with F. tularensis LVS by the intranasal route.
  • FIG. 23 C shows bacterial numbers in the lung, liver, and spleen.
  • FIG. 23D shows bacterial numbers in the lung, liver, and spleen.
  • FIG. 24 is a graph showing MSN-MBI-MXF release profile.
  • FIG. 25 is a graph showing dynamic light scattering (DLS) measurement of MSN with pH sensitive nanovalve.
  • FIG. 26 is a graph showing the uptake efficiency of MSN-MBI-MXF loading with 5 mM and 10 mM MXF aqueous solution for 24, 48 and 72 hours.
  • FIG. 27 shows median-effect plots to compare efficacy of MSN-MBI-MXF with
  • FIG. 28 is a schematic showing disulfide snap-top system synthesis.
  • FIG. 29 is a schematic showing a MSN with disulfide snap-top release mechanism.
  • FIG. 30 is a TEM image of MSN with disulfide snap-top that shows structure integrity preserved after surface modification.
  • FIG. 31 shows dynamic light scattering (DLS) measurement of MSN with disulfide snap-top in PBS. It shows that mean hydrodynamic diameter of the modified nanoparticle is around 740 nm due to disulfide formation among MSN.
  • DLS dynamic light scattering
  • FIG. 32 is a graph shwoing UV-Vis spectrum of moxifloxacin in PBS.
  • FIG. 33 is a graph showing moxifloxacin loaded MCM-41 with disulfide snap-top release profile.
  • FIG. 34 shows graphs showing standard curves (left panels) for MXF established by spectrophotometry used to calculate the amount of MXF present in the aqueous eluates prepared from MSN-SS-MXF in PBS with and without ⁇ -mercaptoethanol reducing reagent (right panels).
  • FIG. 35 is a median-effect plot of MXF standards generated by CompuSyn.
  • Logarithmic plot of log(Fa/Fu) vs. log(D) serves as a standard curve for calculating MXF loading on nanoparticles in the F. tularensis LVS bioassay.
  • FIG. 36A is a graph showing dose dependent inhibition of F. novicida growth by
  • FIG. 36B is a graph showing MXF concentrations plotted against the difference in OD540 readings between an F. novicida culture without MXF and a culture treated with standard amounts of MXF.
  • FIG. 36C is a linear standard curve converted from the log value of MXF concentrations plotted against the difference in OD540 reading between an F. novicida culture not treated with MXF and an F. novicida culture treated with a standard amount of MXF.
  • FIG. 37A is a graph showing that free MXF kill F. tularensis LVS in human macrophages in a dose-dependent manner.
  • FIG. 37B is a graph showing that disulfide snap-top MSN-SS-MXF kill F. tularensis LVS in human macrophages in a dose-dependent manner.
  • FIG. 38 shows that MSN-SS-Hoechst but not their PBS eluates stain the nuclei of
  • FIG. 39A is a graph showing killing of intracellular F. tularensis LVS by MXF. GI.
  • 39B is a graph showing killing of intracellular F. tularensis LVS by eluates prepared from MSN- SS-MXF.
  • FIG. 40A is a graph showing killing of F. tularensis LVS by MSN-SS-MXF in human macrophages.
  • FIG. 40B shows median-effect curves generated by CompuSyn for free MXF (MXF) and an equivalent amount of MXF on the nanoparticle (MSN) plotted in the same graph.
  • FIG. 41 is a graph showing weight changes in infected mice.
  • FIG. 42A is a graph showing bacterial burdens in the lung.
  • FIG. 42B is a graph showing bacterial burden in the liver.
  • FIG. 42C is a graph showing bacterial burden in the spleen.
  • FIG. 43 is a graph showing weight changes in infected mice.
  • FIG. 44A is a graph showing bacterial burden in the lung.
  • FIG. 44B is a graph showing bacterial burden in the liver.
  • FIG. 44C is a graph showing bacterial burden in the spleen.
  • FIG. 45 is a schematic of the pH sensitive nanovalve mechanism.
  • FIG. 46 is a schematic of pH sensitive nanovalve (MBI) system synthesis.
  • FIG. 47 is a TEM image of MSN with pH sensitive nanovalve that shows structural integrity preserved after all surface modifications and surfactant temple extraction
  • FIG. 48 shows dynamic light scattering (DLS) measurement of MSN with pH sensitive nanovalve. It shows that mean hydrodynamic diameter of the modified nanoparticle is around 100 nm
  • FIG. 49A is a 13 C-CPMS NMR of MBI MSN.
  • FIG. 49B is a 29 Si-CPMS NMR spectra of MBI MSN. It shows bulk silica band and attached thread containing a Si-C bond-band, proving the attachment of the MBI compound.
  • FIG. 50 is a UV-Vis spectrum of moxifloxacin under pH 1 and 7.4
  • FIG. 51 is a graph showing Moxifloxacin loaded MCM-41 with MBI pH sensitive nanovalve release profile.
  • FIG. 52 is an example of a MXF standard curve used to calculate drug loading on nanoparticles eluted under neutral pH or acidic pH conditions.
  • FIG. 53 is an example of a MXF standard curve used for calculating drug loading on nanoparticles after sequential elution under neutral and acidic pH conditions.
  • FIG. 54A is a graph showing that free MXF kill F. tularensis LVS in human macrophages in a dose dependent manner.
  • FIG. 54B is a graph showing that pH-gated MSN1-MXF kill F. tularensis LVS in human macrophages in a dose dependent manner.
  • FIG. 54C is a graph showing that MSN2-MXF kill F. tularensis LVS in human macrophages in a dose dependent manner.
  • FIG. 55A is a graph showing that acid eluates of pH-gated MSN1-MXF reduce the number of F. tularensis LVS in macrophages.
  • FIG. 55B is a graph showing that MSN2-MXF do not reduce the number of F. tularensis LVS in macrophages.
  • FIG. 56A shows THP-1 macrophages infected with M tuberculosis and treated with various doses of MXF.
  • FIG. 56B shows THP-1 macrophages infected with M tuberculosis and treated with various doses of MSNl-MXF.
  • FIG. 56C shows THP-1 macrophages infected with M tuberculosis and treated with various doses of eluates prepared from MSNl-MXF in acidified DMSO.
  • FIG. 57 is a chart showing the percentage change in weight of the F. tularensis- infected mice that were sham-treated, treated with the broad spectrum antibiotic MXF administered as a free drug, or treat with pH-gated MSNl-MXF were monitored over the course of treatment.
  • FIG. 58A is a graph showing bacterial burdens in the liver.
  • FIG. 58B is a graph showing bacterial burdens in the lung.
  • FIG. 58C is a graph showing bacterial burdens in the spleen.
  • FIG. 59 is a graph showing percentage change in weight of the F. tularensis-miected mice that were sham-treated, treated with the broad spectrum antibiotic MXF administered as a free drug, or treated with pH-gated MSNl-MXF was monitored over the treatment period.
  • FIG. 60A is a graph showing that pH-gated MSNl-MXF treatment reduces bacterial burden in the lung of F. tularensis-miected mice.
  • FIG. 60B is a graph showing that pH-gated MSNl-MXF treatment reduces bacterial burden in the liver of F. tularensis-m ' iected mice.
  • FIG. 60C is a graph showing that pH-gated MSNl-MXF treatment reduces bacterial burden in the spleen of F. tularensis-m ' iected mice.
  • FIG. 61 is a graph showing percentage change in weight of the F. tularensis-miected mice that were sham-treated, treated with MXF administered as a free drug, or treated with pH- gated MSNl-MXF was monitored over the treatment period.
  • FIG. 62A is a graph showing bacterial burden in lung.
  • FIG. 62B is a graph showing bacterial burden in the liver.
  • FIG. 62C is a graph showing bacterial burden in the spleen.
  • FIG. 63 is a diagram of isoniazid (INH) attaching to the surface of the aldehyde- modified nanoparticles to form the 'pro-drug' MSN.
  • FIG. 64A is a TEM image of INH-CHO-PEI-PEG- SMSNs.
  • FIG. 64B is a TEM image of INH-CHO-PEI-PEG- MSNs.
  • FIG. 65 is a graph showing Isoniazid standard curve measured at 262 nm to measure loading and release.
  • FIG. 66A is a UV-vis spectra of supernatant after washing INH-loaded nanoparticles
  • FIG. 66B is a UV-vis spectra of supernatant after washing INH-loaded nanoparticles (black trace) and the release of INH (red trace) for SMSN.
  • FIG. 67 A is a graph showing that after 24 hours INH-CHO-MSNs are primarily in the liver.
  • FIG. 67B is a graph showing that after 24 hours INH-CHO-SMSNs are well distributed throughout the body.
  • FIG. 67C is a graph showing that after two weeks of accumulation, INH- MSNs are still primarily in the liver.
  • FIG. 67D is a graph showing that after two weeks of accumulation, INH-SMSNs have higher quantities of silica in the lung, liver, and spleen.
  • FIG. 68 is an example of an INH standard curve used to calculate drug loading on nanoparticles eluted under neutral pH or acidic pH conditions.
  • FIG. 69 is an example of an INH standard curve used for calculating drug loading on nanoparticles after sequential elution under neutral and acidic pH conditions.
  • FIG. 70 A is a graph showing that INH kills M tuberculosis in human macrophages in a dose dependent manner.
  • FIG. 70B is a graph showing that MSN-CHO-INH kill M tuberculosis in human macrophages in a dose dependent manner.
  • FIG. 71 is a graph showing that acid eluates of MSN-CHO-INH kill M. tuberculosis in macrophages to a similar extent as the nanoparticle.
  • FIG. 72 is a graph showing that MSN-CHO-INH is stable at 4°C for at least one month.
  • FIG. 73 A is a graph showing killing of M. tuberculosis by INH.
  • FIG. 73B is a graph showing killing of M. tuberculosis by MSN-CHO-INH.
  • FIG. 73C is a graph showing killing of M. tuberculosis by SMSN-CHO-INH.
  • FIG. 73D is a graph showing killing of M tuberculosis by MSN- CHO-INH under neutral or acidic pH conditions.
  • FIG. 73E is a graph showing killing of M.
  • SMSN-CHO-INH under neutral or acidic pH conditions.
  • FIG. 74A is a graph showing bacterial burdens in the lung throughout the course of infection.
  • FIG. 74B is a graph showing the effect of the treatments on M tuberculosis burden in the lung.
  • FIG. 74C is a graph showing the effect of the treatments on M tuberculosis burden in the liver.
  • FIG. 74D is a graph showing the effect of the treatments on M tuberculosis burden in the spleen.
  • FIG. 75 A is a chart showing weights of infected mice that were sham-treated, treated with the anti-TB drug INH administered as a free drug or delivered by MSN-CHO-INH.
  • FIG. 75B is a chart showing weights of infected mice that were sham-treated, treated with the anti-TB drug rifampin (RTF) as free drug or delivered by MSN-PEI-RTF.
  • RTF anti-TB drug rifampin
  • FIG. 76A is a graph showing in sham-treated mice, bacterial burden in the lung was assayed on the first day after infection (Day 1) and bacterial burden in all organs was assayed two weeks later at the start of the treatment period (Day 14) and three weeks and 3 days later (Day 38), 3 days after the conclusion of the three week treatment period.
  • FIG. 76B is a graph showing the effect of various treatments on M tuberculosis burden in the lung.
  • FIG. 76C is a graph showing the effect of various treatments on M tuberculosis burden in the liver.
  • FIG. 76D is a graph showing the effect of various treatments on M tuberculosis burden in the spleen.
  • FIG. 77 is a graph showing lung tubercle lesion counts.
  • FIG. 78A shows bacterial burdens in the lung throughout the course of infection.
  • FIG. 78B is a graph showing the effect of the various treatments on M. tuberculosis burden in lung.
  • FIG. 78C is a graph showing the effect of the various treatments on M. tuberculosis burden in liver.
  • FIG. 78D is a graph showing the effect of the various treatments on M tuberculosis burden in spleen.
  • FIG. 79A is a chart showing weights of infected mice that were sham-treated or treated with the anti-TB drug INH as free drug or delivered by SMSN-CHO-INH.
  • FIG. 79B is a chart showing weights of infected mice that were sham-treated or treated with the anti-TB drug RTF as free drug or delivered by MSN-Z-RTF.
  • FIG. 80A is a graph showing bacterial burden in the lung throughout the course of infection.
  • FIG. 80B is a graph showing the effect of the treatments on M. tuberculosis burden in lung.
  • FIG. 80C is a graph showing the effect of the treatments on M tuberculosis burden in liver.
  • FIG. 80D is a graph showing the effect of the treatments on M. tuberculosis burden in spleen.
  • FIG. 81 A is a graph showing that after 24 hours, INH-CHO-SMSNs lacking a targeting molecule are located primarily in the spleen, followed by the lung.
  • FIG. 8 IB is a graph showing that targeted nanoparticles APP2-INH-CHO-SMSNs show much greater localization to the lung.
  • FIG. 81C show that after 2 weeks of dosing, non-targeted INH-CHO-SMSNs are primarily localized in the liver with negligible amounts in the lung.
  • FIG. 8 ID shows that targeted APP2- INH-CHO-SMSNs show greatly increased localization in the lung compared with the non-targeted nanoparticles.
  • FIG. 82A shows animal organs (liver, spleen, heart, lungs, and kidneys, as indicated) photographed under normal light.
  • FIG. 82B shows animal organs (liver, spleen, heart, lungs, and kidneys, as indicated) imaged for near infra-red emission using the IVIS Imaging System.
  • FIG. 83 is a graph showing silica nanoparticle distribution over a period of 24 hours
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein, said plurality of mesoporous silica particles comprising capping structures that prevent release of said antibiotic prior to being exposed to an activation stimulus present in said host cells; and an antibiotic loaded into said pores of said plurality of mesoporous silica particles and contained therein by said capping structures to be available to be released therefrom when said plurality of mesoporous silica particles are exposed to said activation stimulus, wherein said antibiotic comprises at least one antibiotic selected from the fluoroquinolone group of antibiotics, and wherein said composition has a ratio of weight of said plurality of mesoporous silica particles to weight of said antibiotic loaded into said pores and contained therein and available to be released of at least 5%.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said capping structure is a redox -responsive disulfide snap-top structure that releases said antibiotic loaded into said pores in response to a reducing environment in said host cells.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein further comprising a capping structure is a pH-responsive valve structure that releases said antibiotic loaded into said pores in response to a pH environment in said host cells.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said antibiotic is selected from the group consisting of ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, and norfloxacin.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said antibiotic is moxifloxacin.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said ratio is at least 10%.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said ratio is at least 20%.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said ratio is at least 30%.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said ratio is at least 40%.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said ratio is at least 45%.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said ratio is about 50%.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said ratio is substantially a maximum amount that can be loaded into said pores.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 20 nm and less than 2 ⁇ .
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 25 nm and less than 400 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 30 nm and less than 300 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 50 nm and less than 200 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 50 nm and less than 100 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said plurality of mesoporous silica particles further comprise a compound attached to said plurality of mesoporous silica particles for directing said plurality of mesoporous silica particles to a particular organ.
  • the invention relates to a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein.
  • the invention relates to a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said administering is at least one of administering orally, administering intravenously, administering subcutaneously, administering intramuscularly, administering with a patch, administering with a cream, or administering by inhalation.
  • the invention relates to a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores that are suitable to contain an antibiotic loaded therein wherein said administering further comprises administering at least one additional therapeutic agent to said subject.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores that are suitable to contain a first antibiotic loaded therein, said first plurality of mesoporous silica particles comprising capping structures that prevent release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells; a first antibiotic loaded into said pores of said first plurality of mesoporous silica particles and contained therein by said capping structures to be available to be released therefrom when said first plurality of mesoporous silica particles are exposed to said activation stimulus; a second plurality of mesoporous silica particles defining pores that are suitable to contain a second antibiotic loaded therein, said second plurality of mesoporous silica particles comprising capping structures that prevent release of said second antibiotic prior to being exposed to an activation stimulus present in said host cells; and a second antibiotic loaded into
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said chemical linker prevents release of said antibiotic prior to being exposed to an activation stimulus present in said host cells.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said chemical linker is a pH-responsive chemical element that releases said antibiotic in response to a pH environment in said host cells.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said chemical linker comprises an aldehyde group.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said antibiotic is isoniazid.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 20 nm and less than 2 ⁇ .
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 25 nm and less than 400 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 30 nm and less than 300 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 50 nm and less than 200 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said plurality of mesoporous silica particles have a mean hydrodynamic diameter of at least 50 nm and less than 100 nm.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said plurality of mesoporous silica particles further comprise a compound attached to said plurality of mesoporous silica particles for directing said plurality of mesoporous silica particles to a particular organ.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said plurality of mesoporous silica particles further comprise a copolymer attached to said plurality of mesoporous silica particles.
  • this polymer is
  • the invention relates to a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker.
  • the invention relates to a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said administering is at least one of administering orally, administering intravenously, administering subcutaneously, administering intramuscularly, administering with a patch, administering with a cream, or administering by inhalation.
  • the invention relates to a method of treating an infectious disease caused by an intracellular pathogen within host cells in a subject comprising administering an effective amount of a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a plurality of mesoporous silica particles defining pores; a chemical linker attached to said plurality of mesoporous silica particles; and an antibiotic attached to said plurality of mesoporous silica particles by said chemical linker, wherein said administering further comprises administering at least one additional therapeutic agent to said subject.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores; a first chemical linker attached to said first plurality of mesoporous silica particles; a first antibiotic attached to said first plurality of mesoporous silica particles by said first chemical linker; a second plurality of mesoporous silica particles defining pores; a second chemical linker attached to said second plurality of mesoporous silica particles; and a second antibiotic attached to said second plurality of mesoporous silica particles by said second chemical linker; wherein said first chemical linker prevents release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells, and wherein said second chemical linker prevents release of said second antibiotic prior to being exposed to said activation stimulus present in said host cells.
  • the invention relates to a composition for treating infectious diseases caused by intracellular pathogens within host cells, comprising: a first plurality of mesoporous silica particles defining pores that are suitable to contain a first antibiotic loaded therein, said first plurality of mesoporous silica particles comprising capping structures that prevent release of said first antibiotic prior to being exposed to an activation stimulus present in said host cells; a first antibiotic loaded into said pores of said first plurality of mesoporous silica particles and contained therein by said capping structures to be available to be released therefrom when said first plurality of mesoporous silica particles are exposed to said activation stimulus; a second plurality of mesoporous silica particles defining pores; a chemical linker attached to said second plurality of mesoporous silica particles; and a second antibiotic attached to said second plurality of mesoporous silica particles by said chemical linker; wherein said chemical linker prevents release of
  • the invention relates to a composition for targeting a nanoparticle to a particular organ in a subject, comprising: a plurality of mesoporous silica particles defining pores; and a compound attached to said plurality of mesoporous silica particles for directing said plurality of mesoporous silica particles to a particular organ.
  • the invention relates to a method of targeting a nanoparticle to a particular organ in a subject comprising administering a composition for targeting a
  • nanoparticle to a particular organ in a subject comprising: a plurality of mesoporous silica particles defining pores; and a compound attached to said plurality of mesoporous silica particles for directing said plurality of mesoporous silica particles to a particular organ.
  • the release capacity is dependent upon the mass and internal pore volume of the nanoparticle and the mass and dimensions of the cargo molecule.
  • the pore volume is calculated from its dimensions measured by transmission electron microscopy, BET.
  • the mass and size of the cargo molecule is known from its chemical structure and x-ray crystallographic analysis.
  • the mass of the nanoparticles is based on the density of amorphous silica. Based on these factors, the theoretical upper limit of release capacity (after the nanoparticle is loaded with drug capped and washed) is 30-50 weight percent for moxifloxacin.
  • tularemia Effective and rapid treatment of tularemia, especially the inhalational form, is needed to reduce morbidity and mortality of this serious and potentially fatal infectious disease.
  • the etiologic agent of tularemia Francisella tularensis, is a facultative intracellular bacterial pathogen which infects and multiplies to high numbers in macrophages.
  • Nanotherapeutics are particularly promising for treatment of infectious diseases caused by intracellular pathogens whose primary host cells are macrophages because nanoparticles preferentially target and are avidly internalized by macrophages.
  • MSN mesoporous silica nanoparticle
  • disulfide snap-tops that has high drug loading and selectively releases drug intracellularly in response to the intracellular redox potential.
  • These nanoparticles when loaded with Hoechst fluorescent dye, release their cargo exclusively intracellularly and stain the nuclei of macrophages.
  • We demonstrate the utility of the nanoparticles by comparing the efficacy of the antibiotic moxifloxacin delivered by MSNs vs. administered as free drug in macrophages infected with F. tularensis and in a mouse model of pneumonic tularemia. The MSNs loaded with moxifloxacin killed F.
  • Francisella tularensis is a highly infectious bacterium that causes a life threatening disease, tularemia. Inhalation of as few as 25 bacteria is sufficient to cause severe illness. 1 Because of its extremely high infectivity, ease of dissemination by the air borne route, and capacity to cause severe disease, F. tularensis was developed as a biological weapon by Japan during World War 2 2 and by both the U.S. and the former Soviet Union during the cold war, 3 and it is classified as a Tier 1 Select Agent. Although effective antibiotics for treatment of tularemia are available, intensive care is frequently required and the infection can be fatal even with appropriate treatment. It has been estimated that deliberate dispersal of F.
  • a delivery strategy that targets macrophages and delivers high concentrations of antibiotic to the macrophages has the potential to provide more effective treatment.
  • nanoparticles are avidly taken up by macrophages of the mononuclear phagocyte system in the lung, liver, and spleen. 6"8 Because these are the cells infected by F. tularensis, a nanoparticle delivery system has the potential to deliver high
  • Nanoparticles also have several other advantages over free drug, including shielding the drug from metabolism and excretion and providing more favorable pharmacokinetics. While several different nanoparticle delivery platforms have been studied for antibiotic delivery, including liposomes, solid lipid particles, poly-L-lactide (PLGA), and biological materials such as gelatin, chitosan, and alginates, 9 10 mesoporous silica nanoparticles (MSNs) offer several important advantages, including structural and chemical stability, uniformity, inherent lack of toxicity, capacity to encapsulate exceptionally high concentrations of different types of cargo, and versatility in incorporating rationale design features, including stimulus responsive drug release systems. In this work, we have developed a stimulus-responsive MSN platform for treatment of tularemia that delivers the antibiotic moxifloxacin (MXF) intracellularly in response to the intracellular redox potential. .
  • MXF antibiotic moxifloxacin
  • redox couples that are kept primarily in the reduced state by metabolic processes such as glycolysis, mitochondrial electron transport, and the pentose phosphate pathway.
  • These redox couples include NADH/NAD; NADPH/NADP; thioredoxin/oxidized-thioredoxin, cysteine/cystine, and glutathione (GSH)/GSSG, with the latter redox couple being quantitatively the most abundant inside cells, with cytosolic GSH concentrations in the 1 -10 mM range.
  • GSH glutathione
  • Disulfide snap-top MSNs release cargo selectively intracellularly because the redox potential is much lower in the intracellular than in the extracellular environment. 12 13 On the basis of the intracellular glutathione/glutathione disulfide ratio, the redox potential is estimated to range from - 250 mV in rapidly dividing cells to -200 mV in differentiating cells to -160 mV in cells undergoing apoptosis. 14 Different compartments within the cell also maintain different ambient potentials; for example, based on the thioredoxin redox poise, the cytoplasm, nucleus, and mitochondria exhibit redox potentials of -280, -300, and -340 mV, respectively.
  • the GSH/GSSG redox couple in plasma has a redox potential of -140 mV 15 and the much more abundant cysteine- cystine is even more oxidized, with a redox potential of -80 mV.
  • 16 A similar situation is replicated in cell culture model systems, as human cell lines regulate the redox state of the cysteine-cystine couple in their culture medium to approximately -80 mV. 17
  • cysteine-free RPMI-1640 Prior to addition to cultured cells, cysteine-free RPMI-1640 has a relatively high redox potential of -37 mV and RPMI supplemented with 0.45 mM cysteine has a redox potential of -182 mV.
  • Ciprofloxacin has been used successfully both in animal models of tularemia 19 and in the treatment of clinical tularemia infections.
  • 20 In a mouse model of pneumonic tularemia comparing ciprofloxacin, gatifloxacin, and MXF, while all three fluorquinolones showed efficacy during the treatment phase, both MXF and gatifloxacin were superior to ciprofloxacin in preventing relapse, indicating greater efficacy in eradicating the F.
  • tularensis 21 Because of its potent antimicrobial activity against F.
  • MSN-SS-MXF redox-responsive disulfide snap-top MSNs
  • FIG. 1 shows a depiction of the drug trapping and intracellular release mechanism of MSN-SS-MXF.
  • MSN-SS-MXF is a mesoporous silica nanoparticle functionalized with disulfide snap-tops that carries a large quantity of the broad spectrum antibiotic moxifloxacin within its pores.
  • the snap-top has a bulky ⁇ -cyclodextrin cap that blocks the pores but is detached by reducing agents, releasing the cargo.
  • MSN-SS-MXF naturally targets macrophages, releases the antibiotic in response to the intracellular redox potential, and kills intracellular bacterial pathogens, such as Francisella tularensis, in vitro and in vivo.
  • FIG. 2A is a schematic showing a silane stalk (3-mercaptopropyl) trimethoxysilane is attached to the surface of the MSN. Subsequently, 1-adamantanethiol is reacted with the silane linker in the presence of the oxidant thiocyanogen to form a disulfide bond.
  • Disulfide modified MSNs are then loaded with MXF, followed by the addition of ⁇ -cyclodextrin ( ⁇ -CD) as the capping molecule.
  • FIG 2B shows that the disulfide bond on the thread is cleaved by the reducing agent, 2- mercaptoethanol in the laboratory or glutathione inside cells, removing the bulky ⁇ -CD cap and releasing MXF from the pores of the nanoparticle.
  • FIG. 10 is a spectroscopy graph showing adamantyl group attachment.
  • FIG. 11 shows MXF loading on MSN-SS-MXF can be calculated from a MXF standard curve generated in the F. novicida bioassay.
  • FIG. 11 A is a graph showing dose dependent inhibition of F. novicida growth by MXF at the concentrations indicated.
  • FIG. 1 IB is a graph showing MXF concentrations plotted against the difference in OD540 readings between an F.
  • FIG. 11C is a linear standard curve converted from the log value of MXF concentrations plotted against the difference in OD540 reading between an F. novicida culture not treated with MXF and an F. novicida culture treated with MXF.
  • Disulfide modified MSN was then mixed with MXF PBS solution for 24 hours, followed by adding ⁇ -cyclodextrin ( ⁇ -CD) as the capping molecule which formed a stable complex with the adamantyl group.
  • ⁇ -CD ⁇ -cyclodextrin
  • the disulfide bond is cleaved and cargo is released.
  • the strong binding affinity between the adamantyl group and ⁇ -CD ensure that cargo is trapped inside the pores and prevents premature leakage before reaching target cells.
  • MXF is a fourth generation fluoroquinolone active against both Gram-positive and Gram-negative bacteria. It has a UV-Vis maximum absorption peak at 288 nm in PBS allowing measurement of its concentration.
  • FOG. 3 We measured the absorption of MXF in solution before and after loading the nanoparticles (FIG. 3) and used the difference in concentration to calculate the amount of MXF taken up by the particles (including inside pore channels and on external surfaces).
  • FIG. 3 is a graph showing MSN-SS-MXF is released by MXF in DI water when 2-mercaptoethanol is added and cleaves the disulfide bond.
  • the mass of MXF taken up by particles divided by the mass of MSNs is defined as "uptake capacity” (expressed in wt %).
  • uptake capacity expressed in wt %.
  • the nanoparticles were dispersed in deionized water or PBS and then an excess amount of 2-mercaptoethanol was added to cleave the disulfide bond and release the drug.
  • release capacity expressed in wt %
  • Release capacity of a nanoparticle delivery system is an important factor that impacts in vivo efficacy, as a higher release capacity allows a greater amount of drug to be delivered to target cells with the same number of MSNs.
  • MXF has two ionizable groups with pKa' s of 6.3 and 9.3, and the extent to which the drug is positively charged, neutral, or negatively charged is pH-dependent. Hence, the pH of the loading solution markedly impacts uptake capacity. In PBS buffer with pH 7.4, 87.8% of MXF molecules are zwitterionic species, 7.3% molecules are positively charged, and 4.8% are negatively charged.
  • We modified the inner pores of MSNs with either amine groups or phosphonate groups to make the inner environment positively or negatively charged, respectively. Positively charged cargo interacts electrostatically with negatively charged inner pores, thereby increasing the uptake capacity;
  • Inner pore modification was achieved by co-condensation of two silanes, in which diethylphosphatoethyltriethoxysilane (DEPETS) was mixed with tetraethyl orthosilicate (TEOS) and then added to heated base solution in a dropwise fashion.
  • DEPETS diethylphosphatoethyltriethoxysilane
  • TEOS tetraethyl orthosilicate
  • MSN-SS with a more concentrated MXF PBS solution (40 mM MXF in a volume of lmL PBS vs. 10 mM MXF in a volume of lmL PBS).
  • Table IE the highest release capacity yet obtained
  • MSN-SS-MXF released 9 wt% MXF in pure PBS, and after adding 2-mercaptoethanol, released a total of 21 wt% MXF.
  • ⁇ -CD dissociates from the adamantyl group because of hydrophobic-hydrophobic interaction with DMSO.
  • Table 2 by UV-Vis measurement, MSN-SS-MXF released 73 wt% MXF in pure DMSO, and the release capacity increased further to 133 wt% upon the addition of a reducing agent to cleave the disulfide bond.
  • GSH is the major reducing agent in cells, with intracellular concentrations of approximately 10 mM in healthy cells. 25 ' 26
  • disulfide snap-top MSNs with Hoechst 33342, a membrane permeant probe for double-stranded DNA, and incubated them with 0 - 16 mM GSH in PBS for 18 hours at room temperature.
  • the nanoparticles were pelleted by centrifugation and the supernates were diluted 20-fold with RPMI culture medium and added to monolayers of human macrophage-like THP-1 cells.
  • FIG. 4 is a graph showing Hoechst dye release from MSN-SS snap-top by physiological concentrations of GSH.
  • Snap-Top nanoparticles (1 mg/mL) loaded with the membrane permeant DNA stain Hoechst 33342 were incubated with various concentrations of GSH ranging from 0 - 16 mM, as indicated, overnight at room temperature.
  • the nanoparticles were pelleted by centrifugation and the supernate was diluted 20-fold with RPMI culture medium and added to THP-1 cells.
  • Cells were incubated for 3 hours at 37 °C, stained with WGA-AlexaFluor 633, fixed, and examined by fluorescence microscopy with fixed exposure and gain settings. Data are relative fluorescence intensity of the Hoechst staining per cell as quantitated using CellProfiler.
  • FIG. 5 is a fluorescent image showing that MSN-SS-Hoechst but not their PBS eluates stain the nuclei of THP-1 cells.
  • THP-1 macrophages were incubated with snap-top MSNs loaded with the membrane permeant DNA stain Heochst 33342 (MSN-SS-Hoechst) or the PBS anteate from MSN-SS-Hoechst for 18 h, fixed with 4% paraformaldehyde, and incubated with Alexa Fluor 633 -conjugated wheat germ agglutanin (WGA) to stain the plasma membrane of the cells.
  • WGA agglutanin
  • macrophages with F. tularensis Live Vaccine Strain (LVS) and treated the infected macrophages with serial two-fold increasing concentrations of MSN-SS-MXF or free MXF.
  • the infected macrophages that were not treated were lysed at 3 hours and 1 day post infection to monitor bacterial growth. All infected macrophages that were treated were lysed at 1 day post infection to determine the impact of each treatment on the bacterial viability in macrophages by enumerating colony forming units (CFU).
  • CFU colony forming units
  • MSN-SS-MXF (6.25 - 400 ng/mL) or MXF (1 - 64 ng/mL) reduced bacterial CFU in macrophages in a dose-dependent manner (FIG. 6A and C).
  • the amount of releasable drug loaded on the disulfide snap-top MSN was determined by the level of bacterial killing using the supernatants prepared from the MSN under a) aqueous PBS non-reducing condition; b) aqueous PBS with reducing agent 2-mercaptoethanol; and c) organic DMSO with reducing agent 2-mercaptoethanol.
  • FIG. 6A shows PMA-differentiated THP-1 macrophages infected with F.
  • FIG. 6B shows results with eluates prepared from MSN-SS-MXF incubated in aqueous PBS with and without reducing agent 2- mercaptoethanol (PME)sand with eluates prepared in DMSO with ⁇ .
  • FIG. 6C shows results with free MXF. Bacterial colony forming units (CFU) in the macrophages with or without treatment were determined at 30 min and 24 hours post infection.
  • FIG. 6D shows a scale showing the impact of MSN-SS-MXF and MXF treatment on bacterial viability compared using median-effect analysis.
  • MSN-SS-MXF is Much More Efficacious Than an Equivalent Amount of Free
  • mice were infected by the intranasal route (i.n.) with 4000 CFU of F. tularensis LVS, a dose equivalent to about 6 times the LD50.
  • the bacterial number in the lung increased by 1.5 logs. Without treatment, the bacteria continued to grow in the lung and disseminate to other organs.
  • the bacterial number reached approximately 10 7 in the lung and 10 5 - 10 6 in the liver and spleen (FIG. 8 A and 8C).
  • mice were treated with 50, 100 or 200 ⁇ g of free MXF or 260 ⁇ g of the MSN-SS-MXF (loaded with 91 ⁇ g free MXF) per dose by tail vein injection every other day for a total of 3 treatment doses.
  • sham (PBS)- treated control mice suffered significant weight loss, whereas mice treated with free MXF or MSN-SS-MXF maintained their body weights (FIG. 7 A and 7B).
  • Treatment with MSN-SS-MXF prevents weight loss in mice infected with F. tularensis. Mice with pneumonic tularemia were weighed daily during the course of treatment.
  • mice 7A and 7B show percentage change in weight of mice in two independent experiments.
  • the mice were sham-treated, treated with three different doses of the broad spectrum antibiotic MXF administered as a free drug, or treated with one or two doses of MSN-SS-MXF, as indicated.
  • MSN-SS-MXF is more efficacious than an equivalent amount of free MXF in the lung, spleen, and liver with an efficacy ratio (MSN-SS-MXF : free MXF) of -3-4 : 1 in the lung and spleen, and ⁇ 1 : 1 in the liver (FIG. 12, left panel).
  • FIG. 8A-D show In vivo efficacy of MSN-SS-MXF in two independent experiments, Experiment 1 (8A and 8C) and Experiment 2 (8B and 8D). Mice were infected with F. tularensis LVS by the intranasal route.
  • FIG. 8A is a graph showing bacterial burden in the lung monitored over the course of infection.
  • FIG. 8B is a graph showing bacterial burden in the lung monitored over the course of infection.
  • mice were sham-treated, treated with one of the three doses of free MXF as indicated, or treated with MXF delivered by the disulfide snap-top MSN (MSN-SS-MXF) by tail vein injection.
  • 8C and 8D are graphs showing the effect of each treatment on F. tularensis burden in lung, liver, and spleen as determined by assaying the bacterial CFU one day after the final treatment.
  • the equivalent amount of free MXF for the MSN-SS-MXF is shown in parenthesis.
  • Statistics were analyzed using one-way ANOVA with Bonferroni post-test correction. **p ⁇ 0.01, ***p ⁇ 0.001. Error bars represent standard errors with 3 mice per group. ⁇ Bacterial CFU below limit of detection.
  • FIG. 12 shows graphs showing median-effect plots to compare efficacy of MXF administered as free drug vs. MSN-SS-MXF.
  • the efficacy of MSN-SS-MXF in the lung, spleen, and liver was compared to that of free MXF in a median-effect plot for mouse Experiments 1 and 2.
  • an upward shift, as indicated by the red arrows paralleling the y-axis denotes a greater / , tularensis killing efficacy.
  • Fa Fraction of bacteria killed
  • Fu Fraction of bacteria surviving
  • D Dose of MXF in micrograms.
  • mice treated with 50, 150, and 300 ⁇ g of MXF had 5.2-, 4.2-, and 3.6-logs CFU, respectively. Although it did not reach statistical significance, CFU in the lung of mice treated with 230 ⁇ g of the MSN-SS-MXF (containing 117 ⁇ g releasable MXF) was 0.75 logs lower than that of mice treated with 300 ⁇ g free MXF, the highest dose of free MXF tested in the experiment ( Figure 7B).
  • MXF delivered by the disulfide snap-top MSN is more efficacious than 3-fold the equivalent amount of free MXF in the lung.
  • MSN-SS-MXF is much more efficacious than an equivalent amount of free MXF in the lung, spleen, and liver with an efficacy ratio (MSN-SS-MXF : free MXF) of ⁇ 5 : 1 in the lung, ⁇ 3 : 1 in the spleen, and ⁇ 3 : 1 in the liver (Figure 12, right panel).No space here
  • FIG. 9A is a graph showing the distribution of i.v. administered MSN-SS-MXF in lung, liver, spleen, heart and kidney after a single injection.
  • FIG. 9B is a graph showing the distribution of i.v. administered MSN-SS-MXF in lung, liver, spleen, heart and kidney after a three injections over 6 days.
  • FIG. 9C is a graph showing control results. Data represent means ⁇ standard errors of results from 3 mice per experimental condition with 3 technical repeats per mouse.
  • a nanoparticle delivery platform that releases drug exclusively intracellularly has the potential to release high concentrations of drug into infected cells, thus providing for a greater killing efficacy relative to free drug and at the same time limiting systemic exposure to the drug and off-target toxicities.
  • the nanoparticle delivery platform also has the potential to improve the pharmacokinetic profile of the drug by shielding it from excretion and metabolism before it reaches its target cells.
  • Key to the success of such a nanoparticle delivery system is a nanovalve mechanism that releases the drug cargo only after uptake of the nanoparticle into the host cell.
  • ⁇ - interferon (often elevated in infections) has been shown to lower GSH levels in macrophages. 39
  • lysosomes have a powerful ⁇ -interferon-inducible lysosomal thiol reductase (GILT) 40 capable of cleaving disulfide linkages, including those present in ⁇ -CD-based polyrotaxanes therapeutics for lysosomal storage disease.
  • GILT ⁇ -interferon-inducible lysosomal thiol reductase
  • Modification of the mesopores with phosphonate groups has allowed us to increase the loading and release capacity of our MSNs and functionalization of the MSN with a disulfide-cleavable capping system provides for very tight closure of the mesopores that prevents premature release of drug cargo yet opens readily in response to the intracellular environment. While redox -responsive disulfide gate mechanisms have been described, 12 18 they have not previously been tested in vitro or in vivo for safety or efficacy in the delivery of an antibiotic for treatment of an intracellular pathogen. Ma et al.
  • the MSN-delivered MXF can achieve higher levels in the infected tissues and host cells than free MXF.
  • MSN-encapsulated drug is shielded from metabolism and excretion, it is likely to have a more favorable Area Under the Curve/Minimal Inhibitory Concentration (AUC/MIC) ratio compared with free drug.
  • AUC/MIC Area Under the Curve/Minimal Inhibitory Concentration
  • the MSNs passively target infected macrophages, but it is likely that even greater enhancement of therapeutic efficacy can be achieved by surface modifications (e.g. targeting to specific cellular receptors) that further enhance targeting to infected tissues and uptake by macrophages or by use of an aerosol delivery device that delivers the MSNs directly to the lung, as has recently been demonstrated for liposomally encapsulated ciprofloxacin in treatment of tularemia. 54
  • Cetyltrimethylammonium bromide 250 mg, 0.7 mmol was dissolved in H 2 0 (120 mL) and NaOH (875 uL, 2 M). The mixture was heated to 80 °C and kept stable for 30 minutes, followed by adding a mixture of tetraethyl orthosilicate (TEOS, 1.2 mL) and diethylphosphatoethyltriethoxysilane (DEPETS) (0.2 mL) drop-wise into the solution while stirring vigorously. The solution was kept at 80 °C for 2 hours and as-synthesized
  • nanoparticles were centrifuged and washed thoroughly with methanol.
  • MCM-41 100 mg was dispersed into dry toluene (10 mL), mixed with (3-mercaptopropyl) trimethoxysilane (24 ⁇ , O. lmmol), and refluxed for 12 hours under nitrogen atmosphere.
  • Thiol group modified MCM-41 100 mg was washed and dispersed again in anhydrous toluene (10 mL) in a second step.
  • lead thiocyanate 800 mg was dispersed in 10 mL chloroform and titrated by bromine (200 ⁇ ) in chloroform (10 mL).
  • the titration product mixture was filtered and the filtrate containing thiocyanogen in chloroform was light yellowish.
  • 1-adamantanethiol (17 mg, 0.1 mmole) and as-synthesized thiocyanogen were added into the MSN toluene suspension.
  • the disulfide oxidation reaction took four days under 4 °C and nitrogen gas atmosphere.
  • As-synthesized material was yellowish and washed thoroughly with toluene, methanol and water.
  • MCM-41 (10 mg) with disulfide snap-tops was suspended in 1 mL of 40 mM MXF in PBS solution and rotated overnight, ⁇ -cyclodextrin (40 mg) was added into the solution as capping agent to prevent the drug from leaking out. After mixing the solution for another 12 hours, MXF loaded MCM-41 with disulfide snap-tops (MSN-SS-MXF) was dried under vacuum overnight.
  • MXF from MSN-SS-MXF in solution was measured by fluorescence spectroscopy using a 5 mW 377 nm laser beam to excite MXF in solution within a glass vial and a charge coupled device (CCD) connected to a computer to detect and collect emitted fluorescence.
  • CCD charge coupled device
  • the dried MSN-SS-MXF powder was put at a corner of the bottom of the glass vial containing 10 mL DI water. Baseline fluorescence spectra were collected for 1 hour to establish that there was no MXF leakage, and then 2-mercaptoethanol (200 ⁇ ) was added to the suspension. This resulted in a dramatic increase in fluorescence emission in the supernatant fluid, indicating release of MXF.
  • a release profile was constructed by integration of MXF emission peak area from 480 nm to 520 nm. After collecting data for 17 hours, by which time the MXF was released completely, the MXF concentration in the solution was calculated based on the UV-Vis spectrum and standard curve by Beer's law.
  • LVS Bacteria. Francisella tularensis subsp. holarctica Live Vaccine Strain (LVS) was obtained from the Centers for Disease Control and Prevention (Atlanta, GA). For in vitro experiments, LVS was grown from frozen stock on GCII chocolate agar plates for 3 days prior to being used to infect macrophages. For in vivo experiments, pre-titered LVS frozen stock was used directly to infect mice and was serially diluted and plated on agar plates after infection to confirm bacterial CFU in the stock. For use in the bioassay, F. tularensis subsp. novicida strain Utah 112 (F. novicida) was grown at 37 °C with aeration in trypticase soy broth supplemented with 0.2% cysteine (TSBC).
  • TSBC cysteine
  • F. novicida Bioassay MXF was eluted from 1 mg/ml of MSN-SS-MXF under a) aqueous conditions by PBS; b) aqueous reducing conditions by PBS and 2-mercaptoethanol; and c) organic reducing conditions by DMSO and 2-mercaptoethanol; mixed by end-to-end rotation for 1 hour at room temperature; and centrifuged at 10,000 g for 10 min. The supernates (1.5 ⁇ ) were added to F. novicida in 3 ml trypticase soy broth supplemented with 0.2% L-cysteine (TSBC) at a starting optical density (O.D.) at 540 nm of 0.05.
  • TSBC L-cysteine
  • novicida broth cultures were grown at 37 °C with shaking at 200 rpm for 6 h. At the end of the incubation, the O.D. of the bacterial broth cultures was measured. The amount of releasable MXF from the nanoparticles was determined by comparing the O.D. of the bacterial cultures treated with the supernates to the O.D. of the cultures treated with standard concentrations of MXF.
  • Macrophages Human monocytic THP-1 cells (ATCC TIB 202) were maintained in RPMI-1640 (Lonza) with 10% fetal bovine serum (Cellgro), 2 mM GlutaMAX (Life Technology), penicillin (100 IU) and streptomycin (100 ⁇ g/mL). Prior to use, the TFIP-1 cells were suspended in culture medium without antibiotics and treated with 100 nM phorbol 12-myristate 13- acetate (PMA; Sigma) for 3 days to mature the cells into a macrophage-like cell type.
  • PMA phorbol 12-myristate 13- acetate
  • TFIP-1 macrophages were infected with F. tularensis LVS at a multiplicity of infection ratio of 10 bacteria to 1 THP-1 cell for 90 min at 37 °C, 5% C0 2 - 95% air atmosphere. Infected monolayers were washed to remove extracellular bacteria. Fresh medium with or without MXF or MSN-SS- MXF was added to the infected macrophage monolayer. The cultures were incubated in the continued presence of the treatment for one day. F. tularensis LVS was harvested from untreated cultures at 30 min and 1 day post infection to determine bacterial growth without treatment and from infected cultures at 1 day to assess the effect of treatment.
  • the bacteria were harvested by lysing the infected macrophages with 1% saponin in PBS and the lysate was serially diluted and plated on GCII chocolate agar plates. Bacterial CFU on agar plates were counted after incubation at 37 °C, 5% C0 2 - 95% air atmosphere for 3 days.
  • mice Eight-week old, female, pathogen-free Balb/c mice purchased from Taconic were acclimated for one week. Mice were infected by the intranasal route with 4000 - 8,000 CFU of F. tularensis LVS, a dose equivalent to about 6-12 times the LD50, respectively. Two mice were euthanized 5 hours after infection (day 0) to establish the number of bacteria in the lung at the start of the experiment. An additional 3 mice were euthanized one day later (day 1) to determine bacterial growth over that time period.
  • mice per group were then sham-treated or treated with either free MXF or MSN- SS-MXF by tail vein injection every other day for a week (days 1, 3, and 5 for a total of 3 treatments). Mice were euthanized one day after the last treatment (day 6). Lungs, livers, and spleens from infected mice that were sham treated or treated with free MXF or MSN-SS-MXF were homogenized in PBS, pH 7.4.
  • the organ homogenates were serially diluted and plated on GCII chocolate agar plates containing sulfamethoxazole (40 ⁇ g/mL), trimethoprim (8 ⁇ g/mL), and erythromycin (50 ⁇ g/mL) to prevent growth of contaminants.
  • the agar plates were incubated at 37 °C for 4 days at which time the number of bacterial colonies on each plate was counted.
  • mice that were either sham-treated or treated with MSN-SS-MXF were homogenized in PBS, digested with 0.1% HNO3, and analyzed by ICP-OES (ICPE-9000,
  • a median-effect plot 27 for MXF or MSN-SS-MXF was generated using MXF or MXF equivalent (MSN) dose in base- 10 logarithm as the X-axis and the fraction of surviving bacteria divided by the fraction of killed bacteria in base-10 logarithm as the Y-axis.
  • Bonferroni' s post-test correction A P value of 0.05 or less was considered statistically significant.
  • MSNs mesoporous silica nanoparticles
  • MXF moxifloxacin
  • MSNs mesoporous silica nanoparticles
  • MXF moxifloxacin
  • ANA anilinoalkane
  • MSNs Mesoporous silica nanoparticles
  • 1"4 MSNs readily accommodate stimulus-responsive functionalizations to enable on-command release of drug cargo in response to a variety of stimuli, including pH, 5"8 light, 9 and remote magnetic actuation, 10 and have shown superiority over free drug both in cell culture, 11"13 and in animal models.
  • release capacity An important parameter that influences the amount of MSNs that must be administered to animals or humans for therapeutic efficacy is the "release capacity", defined as the ratio between the masses of releasable drug and of silica.
  • the uptake and release capacity of a MSN platform depends on the properties of both the nanoparticles and the cargo molecules, including the cargo molecule size, charge in various solutions, and
  • MSNs hydrophilic/hydrophobic properties.
  • MXF moxifloxacin
  • Francisella tularensis is a facultative intracellular bacterial pathogen that causes tularemia, a serious and potentially fatal disease. 15 Because / , tularensis has extraordinarily high infectivity, causes serious morbidity and mortality, is readily cultured on a large scale, is relatively easily dispersed, and was developed as a biological weapon during World War II by Japan and in the Cold War by both the U.S. and the former Soviet Union , 16"18 it is classified as a Tier 1 Select Agent.
  • Nanoparticles are attractive as drug delivery platforms for tularemia treatment because the nanoparticles are avidly taken up by cells of the mononuclear phagocyte system - such cells are the primary host cells in which F. tularensis resides and multiplies. By releasing high concentrations of antibiotic in the host cells that are infected by F.
  • nanoparticles have the potential to have a greater efficacy than free drug while simultaneously limiting off-target toxicities.
  • Nanoparticle delivery platforms also have the advantage of shielding the drug from metabolism and clearance, thereby providing more favorable pharmacokinetics than free drug.
  • MSN-MBI-MXF fluoroquinolone antibiotic
  • Intravenously injected nanoparticles, or nanoparticles delivered by other routes of administration are preferentially taken up by macrophages of the mononuclear phagocyte (reticuloendothelial) system and accumulate in liver, spleen and lung, 22"24 a distribution that mirrors the tissues infected by F. tularensis and many other important intracellular pathogens that cause serious human diseases, including those that cause tuberculosis, Legionnaires' disease, Q-fever, Salmonellosis, Listeriosis, Leishmaniasis, and chlamydial, mycoplasmal, and rickettsial infections.
  • macrophages of the mononuclear phagocyte (reticuloendothelial) system and accumulate in liver, spleen and lung, 22"24 a distribution that mirrors the tissues infected by F. tularensis and many other important intracellular pathogens that cause serious human diseases, including those that cause tuberculosis, Legionnaires'
  • FIG. 13 is a graphic showing gated nanoparticles carry large quantities of moxifloxicin into macrophages, release the cargo and kill intracellular F. tularensis both in cultures and in mice.
  • the second nanovalve system has a 1 -methyl- 1-H-benzimidazole (MB I) stalk with pK a about 6, and ⁇ -CD as the capping molecule because of its suitable cavity size and stable association with the benzimidazole moiety at physiological pH 7.4 (FIG. 14).
  • MB I 1 -methyl- 1-H-benzimidazole
  • ⁇ -CD the capping molecule because of its suitable cavity size and stable association with the benzimidazole moiety at physiological pH 7.4 (FIG. 14).
  • benzimidazole is protonated at pH 6 or lower, the binding affinity between benzimidazole and ⁇ -CD decreases, leading to dissociation of the cyclodextrin.
  • Both nanovalves are closed tightly at physiological pH 7.4 and only open and release cargo at pH 6 and lower when the hydrophobic interaction between cyclodextrin and the organic stalk moiety is weakened and interrupted.
  • FIG. 14 shows chemical structures of the stalks (top) and caps (bottom) of two nanovalves. Left: the ANA (stalk) and a-CD (cap); Right: the MBI (stalk) and ⁇ -CD (cap)
  • MSN-ANA and MSN-MBI nanoparticles were loaded in MXF aqueous/PBS solution overnight and then the a-CD or ⁇ -CD capping molecule, respectively, was added to the mixture with stirring overnight.
  • the MXF solution concentrations before and after loading were measured and calculated based on UV-Vis
  • the amount of MXF released was calculated based on the supernatant MXF concentration measured by UV-Vis.
  • the mass of released MXF divided by the mass of particle is defined as "release capacity" (expressed in wt %).
  • release capacity expressed in wt %).
  • the porous structure is preserved after these modifications (Figure 15C) and the hydrodynamic diameter is around 100 nm ( Figure 25).
  • FIG. 15A shows attachment of two different pH-sensitive nanovalves on MCM-41 surface.
  • the cap molecule a-CD or ⁇ -CD dissociates from it due to the decrease of the binding constant between them.
  • FIG. 15B shows MSN-MBI-MXF drug release profile. There is no leakage at pH 7, as indicated by the flat baseline; drug release starts when the pH is lowered to 5 by addition of acid.
  • FIG. 15C is a TEM image of MCM-41 showing its hexagonal pore structure.
  • FIG. 24 is a graph showing M SN-MB I-MXF release profile. There is no leakage at pH 7 evidenced by the flat baseline. Drug release starts at when the pH is lower than 6. The release rate can be further increased by lowering pH to 4.5.
  • FIG. 25 is a graph showing dynamic light scattering (DLS) measurement of MSN with pH sensitive nanovalve.
  • the mean hydrodynamic diameter of the modified nanoparticle is around 100 nm.
  • MXF is a fourth generation fluoroquinolone used to treat various bacterial infections including F. tularensis. It has two ionizable groups with pK a of 6.3 and 9.3. Based on the
  • MXF has a positive net charge at neutral pH.
  • a negatively modified inner pore readily attracts positive cargo molecules, but the release may be slow and incomplete after the cap dissociates due to the electrostatic interaction between cargo molecules and inner pores at the pH of acidifying endosomal compartments. 26
  • a positively charged inner pore surface will lead to lower uptake capacity than when negatively charged but may promote expulsion of the positive cargo molecules upon protonation.
  • MSN-MBI (10 mg) was dispersed in 2 ml of a 5 mM MXF aqueous solution and uptake capacity was measured as described above.
  • Amine modified MSN-MBI (indicated as "+") had a very low uptake capacity compared with that of phosphonate modified MSN-MBI (indicated as "-") ( Figure 16 A). This result indicates that MXF with positive net charge diffuse poorly into positively charged inner mesopores, resulting in very low uptake and release capacities.
  • Phosphonated particles show much greater uptake of MXF, potentially providing a much greater release capacity.
  • FIG. 16A is a graph showing uptake capacity of MSN-MBI with different inner mesopore charges and stalk synthetic pathways. From left to right, samples are: slightly negatively charged underivatized MSN with stalk MBI synthesized by pathway I; negatively charged MSN- MBI by pathway I; positively charged MSN-MBI by pathway I; negatively charged MSN-MBI by pathway II; and positively charged MSN-MBI by pathway II. Pathyway I: synthesize the whole stalk first and then attach it on MCM-41 ; pathway II: attach first part of stalk on MCM-41 first and then synthesize the whole stalk.
  • FIG. 16B is a schematic showing MSN mesopores modified (left to right) with amine (+), unmodified silanol (-), or phosphonate (-).
  • chloromethyltrimethoxysilane to produce the MBI stalk, and then covalently attached this to the MCM-41 surface.
  • This method has the disadvantage that, in the presence of small amounts of water or moisture, the MBI stalk readily hydrolyses and undergoes self-condensation prior to coupling to the nanoparticle.
  • chloromethyltrimethoxysilane to the silica surface first and then coupled it with benzimidazole to form the MBI stalk.
  • MSN-MBI-MXF Because negatively charged inner pores provided greater uptake of MXF, we used phosphonated MCM-41 and compared the uptake and release of MXF of MSN-ANA-MXF, which has a-CD as cap, and MSN-MBI-MXF which has ⁇ -CD as cap. The same amount of phosphonated MCM-41 with one or the other nanovalve was loaded in 1 ml 10 mM MXF PBS solutions and stirred for one day. MSN-MBI-MXF had a much higher uptake capacity (7.4 wt%) and release capacity (1.02 wt%) than MSN-ANA-MXF (Table 4).
  • the superior uptake and release capacity of the MSN-MBI-MXF is likely attributable to better trapping of the MXF within the pores.
  • the ⁇ -CD has a 15.6 A outer diameter compared with 14.6 A for a-CD while MCM-41 has an average pore diameter of 22 A. 6
  • the larger ⁇ -CD has more steric hindrance and blocks the MSN pores more effectively than the smaller a-CD.
  • MSN-MBI has a shorter stalk length that positions the ⁇ -CD cap closer to the MSN surface, again providing more effective steric hindrance to prevent MXF leakage.
  • FIG. 17A-E show confocal microscopy images demonstrating avid uptake of RITC- labeled MSN-MBI by F. tularensis- infected THP-1 macrophages.
  • Human macrophage-like THP-1 cells were infected with GFP-expressing F. tularensis for 90 min, washed, and incubated with 12.5 ⁇ g/mL of RITC -labeled 100 nm MSN-MBI. After 3 hours, the cells were washed; the plasma membrane was stained with WGA-AlexaFluor 633; the cells were fixed; and nuclei were stained with DAPI.
  • MSN-MBI-MXF at 1 ⁇ g/mL reduced bacterial colony forming units (CFU) by 3.4 logs compared with the level in the untreated group at one day, whereas the same concentration of MSN-ANA-MXF reduced bacterial CFU by only 0.2 logs compared with the untreated control group.
  • the minimal inhibitory concentration in our macrophage assay is 4 ⁇ g/mL for MSN-ANA-MXF and it falls to between 0.25 and 0.5 ⁇ g/mL for MSN-MBI-MXF (Table 6).
  • FIG. 18 shows In vitro efficacy of MXF-loaded MSNs functionalized with two different types of pH-sensitive nanovalves.
  • FIG. 18A is a graph showing human THP-1
  • FIG. 18B is a graph showing human THP-1 macrophages infected with F. tularensis LVS and treated with MSN-ANA-MXF.
  • FIG. 18C is a graph showing human THP-1 macrophages infected with F. tularensis LVS and treated with MSN-MBI-MXF. Viable bacteria were determined by enumerating colony forming units (CFU) of F. tularensis in the macrophage monolayer.
  • FIG. 18D is a graph showing impact of the drug released from MSN-ANA-MXF.
  • FIG. 18E is a graph showing impact of the drug released from MSN-MBI-MXF.
  • MSN-MBI-MXF or MSN-ANA-MXF at neutral pH had no effect in the infected macrophage bioassay.
  • This study demonstrates that 1) the pH operative valves on MSN-MBI-MXF are tightly closed at neutral pH and open at acidic pH, 2) MXF eluted under acidic pH retains biological activity, 3) MSN-ANA- MXF and MSN-MBI-MXF kill F. tularensis LVS in macrophages in a dose-dependent fashion, and 4) MSN-MBI-MXF has greater efficacy than MSN-ANA-MXF, most likely because of its higher
  • Acid-released solution obtained from 1 ⁇ g/mL of MSN-MBI-MXF exerted the same inhibitory effect on F. tularensis as 0.016 ⁇ g/mL MXF in our macrophage bioassay, indicating a 1.6% (wt/wt) aqueous acid release capacity. Based on this estimation, 0.5 ⁇ g/mL of MSN-MBI- MXF could release 0.008 ⁇ g of MXF in the acidified endolysosomes. In our F.
  • MSN-MBI-MXF at 0.5 ⁇ g/mL had a biological effect equivalent to that exerted by free MXF at a concentration of 0.016 ⁇ g/mL, indicating an efficacy ratio of 2 (MSN-MBI-MXF : free MXF), as nanoparticle-delivered drug appeared to have an efficacy twice that of the same amount of free drug in killing F. tularensis in macrophages in vitro.
  • this efficacy ratio is likely an over-estimation since some of the yellowish color of MXF still remained on MSN-MBI- MXF after maleate treatment.
  • FIG. 19 is a graph showing uptake and release capacity of negatively charged MSN- MBI loaded at pH 4 or 7 and positively charged MSN-MBI loaded at pH 7, 10, or 12 MXF aqueous solution.
  • MXF has positive net charge in solution at pH ⁇ 7 and MCM-41 is negatively charged. Decreasing the loading pH from 7 to 4 increases uptake capacity, but, not release capacity because the nanovalve is open at pH 6 and particles must be transferred to neutral solution before capping. Most of MXF diffuses out of the pores because of these extra steps.
  • positively charged MCM-41 repels MXF and leads to very low uptake and release capacities.
  • MXF has negative charge when solution pH > 7, and increasing pH dramatically improves uptake capacities. However, loading at pH 12 does not lead to highest release capacity because particles degrade in base solution within 24 hours.
  • FIG. 20 shows uptake capacity, uptake efficiency and release capacity of phosphonated MSN-MBI loaded in 20 mM MXF aqueous solution (pH 7), 20 mM MXF PBS solution (pH 7.4) and 40 mM MXF PBS solution (pH 7.4).
  • PBS loading increases the uptake more than 10 times than neutral water and release capacity got increased to 6.2 wt%, which is more than 3 times of 1.7 wt% from neutral loading.
  • MSN-MBI loaded with 40 mM MXF in PBS had an uptake capacity twice that of MSN-MBI loaded with 20 mM MXF in PBS, and the release capacity reached 8.1 wt% compared with 6.2 wt% for MSN-MBI loaded with 20 mM MXF.
  • uptake efficiency which is defined as the percentage of MXF taken up by MSN from the original solution (expressed in percent)
  • almost 70% of MXF in high concentration solution was taken up by nanoparticles.
  • a loading time of 24 hours was appropriate to allow MXF to diffuse into pore channels and reach equilibrium.
  • FIG. 26 is a graph showing the uptake efficiency of MSN-MBI-MXF loading with 5 mM and 10 mM MXF aqueous solution for 24, 48 and 72 hours. 24 hours loading yielded the highest uptake efficiency for both low and high MXF concentrations.
  • FIG. 21 A is a graph showing that release profiles show that the more times the MSN are washed, the lower the amount of residual and release capacity. When particles were washed 15 times, there was negligible residual drug detected from the particle surface (no fluorescence detected). A small amount of residual was observed when drug loaded particles were washed 8 times.
  • FIG. 21B is a graph showing the amount of MXF washed away each time decreases as the number of washes increases; the decrease for each step is -30 %. The first eight washes contribute -95 % to the total amount of MXF ultimately removed by washing.
  • FIG. 22A shows results from experiment 1 where treatment with MSN-MBI-MXF prevents weight loss caused by pneumonic tularemia.
  • FIG. 22B shows results from experiment 2 where treatment with MSN-MBI-MXF prevents weight loss caused by pneumonic tularemia.
  • Percentage change in weight of F. tularensis-mfected mice was monitored over the course of the experiments. The mice were sham treated, treated with one of three doses of MXF as a free drug, as indicated, or treated with MSN-MBI-MXF (loaded with 138 ⁇ g MXF in Experiment 1 and 50 ⁇ g MXF in Experiment 2).
  • FIG. 23 shows In vivo efficacy of MSN-MBI-MXF assessed by assay of F.
  • FIG. 23 A shows results of mice infected with F. tularensis LVS by the intranasal route.
  • FIG. 23B shows results of mice infected with F. tularensis LVS by the intranasal route. Bacterial burden in the lung was monitored over the course of infection.
  • mice were sham treated, treated with one of three doses of free MXF, as indicated, or treated with MSN-MBI-MXF (loaded with 138 ⁇ g in Experiment 1 shown in A and 50 ⁇ g in Experiment 2 shown in B) by tail vein injection on days 1, 3, and 5.
  • FIG. 23 C shows bacterial numbers in the lung, liver, and spleen.
  • FIG. 23D shows bacterial numbers in the lung, liver, and spleen. ⁇ Bacterial CFU below limit of detection. *P ⁇ 0.05 by one-tailed t-test.
  • FIG. 27 shows median-effect plots to compare efficacy of MSN-MBI-MXF with
  • MXF administered as free drug The efficacy of MSN-MBI-MXF in the lung, spleen, and liver was compared with that of free MXF in a median-effect plot of the results of mouse Experiments 1 and 2. For a given dose of MXF, an upward shift as indicated by the red arrows on the y-axis indicates greater / , tularensis killing efficacy of the MSN-MBI-MXF. Fa: Fraction of bacteria killed; Fu: Fraction of bacteria surviving; D: Dose of MXF in micrograms.
  • mice were infected with -4000 CFU of F. tularensis LVS (-6 x LD50) by the intranasal route. One day later, mice were sham-treated or treated with 640 ⁇ g of MSN-MBI-MXF (with - 50 ⁇ g of releasable MXF) or with one of the three doses of MXF (50, 100, and 200 ⁇ g) equal to lx, 2x, and 4x the amount of the releasable MXF from 640 ⁇ g of MSN-MBI-MXF by acidic DMSO.
  • mice suffered substantial weight loss but mice treated with free MXF or MSN-MBI-MXF did not (Figure 9B).
  • MSN-MBI-MXF treatment reduced the bacterial burden by 2.8 logs in the lung, 3.2 logs in the liver, and 3.3 logs in the spleen to a level close to that achieved by 100 ⁇ g free MXF ( Figure 23D).
  • MSN-MBI-MXF had an efficacy twice the equivalent amount of free MXF in the lung, spleen, and liver (Table S2 and Figure 27, right panel). Again, we observed no toxicity in the mice from MSN-MBI-MXF treatment.
  • Intracellular pathogens that reside in mononuclear phagocytes present an ideal target for nanotherapeutics because nanoparticles are readily taken up by cells of the Mononuclear Phagocyte System and have the potential to deliver high concentrations of antibiotics selectively to the intracellular compartment, thereby providing increased efficacy with reduced systemic exposure and off-target side effects.
  • MSN-MBI-MXF tularensis-miected macrophages and that it was 2.7 fold more effective than the amount of free drug released from the particles by aqueous acid.
  • MSN-MBI-MXF was well tolerated and was more effective than a 2- to 4-fold greater dose of free MXF in reducing bacterial load in the lung.
  • Our MSN-MBI-MXF delivery system has the potential to provide more effective treatment than free drug, shortening the duration of treatment of intracellular infectious diseases such as tularemia, tuberculosis, Q-fever, and Legionnaires' disease and reducing systemic toxicity of the MXF.
  • the nanoparticle delivered drug By providing high concentrations of antibiotic directly to the site of infection, the nanoparticle delivered drug also has the potential to decrease the emergence of drug resistance. Further optimization of our platform may be possible by incorporation of additional functionalizations to increase targeting to infected tissues and macrophages, employment of different delivery modalities, such as aerosol delivery, or utilization of other internal and external stimulus-response systems.
  • MCM-41 Synthesis of MCM-41.
  • CTCAB Cetyltrimethyl ammonium bromide
  • H20 120 mL
  • NaOH 875 ⁇ iL, 2M
  • TEOS tetraethyl orthosilicate
  • phosphonated MCM-41 3-(trihydroxysilyl)propyl methylphosphonate (315 pL) was added into the solution 15 minutes after adding TEOS.
  • N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane (90 %) was mixed with TEOS before adding to CTAB solution.
  • the solution was kept at 80 °C for 2 hours.
  • the synthesized nanoparticles were centrifuged and washed thoroughly with methanol. The successful synthesis of nanoparticles is very sensitive to the temperature and stirring speed.
  • IPTMS IPTMS, 20 uL, O. lmmol
  • the IPTMS modified nanoparticles were washed with toluene to remove unreacted agents and re-dispersed in anhydrous toluene, and mixed with p-anisidine (123.2 mg, 1 mmol) and triethylamine (TEA, 420 pL, 3 mmol).
  • p-anisidine (123.2 mg, 1 mmol
  • TEA triethylamine
  • the solution was refluxed under N 2 for another 24 hours.
  • the final product was centrifuged and washed with toluene, methanol and water to be ready for drug/dye loading process.
  • MCM-41 (100 mg) was washed and dispersed in anhydrous toluene, mixed with chloromethyltrimethoxysilane (15 pL) and refluxed for 12 hours.
  • the modified MCM-41 was washed by toluene and dimethyoformamide (DMF) and dispersed in 8 ml DMF.
  • Tetrabutyammonium iodide (2 mg), benzimidazole (12 mg) and triethylamine (150 pL) were added into the solution and the mixture was heated up to 70 °C under N 2 for 24 hours.
  • Nanovalve-modified MCM-41 (100 mg) was dispersed in methanol (60 mL), mixed with concentrated HC1 (12 M, 2.3 mL) and refluxed for 8 hours under N 2 , and then washed extensively with methanol and water.
  • Release capacity (wt %) ( Wreleased MXF / Wparticle ) ⁇ 100 %.
  • the release profile was the plot of the integrated emission peak area between 480 nm to 520 nm as a function of time.
  • TEM Transmission electron microscopy
  • JEOL JEM1200-EX
  • Particle size and zeta potential were measured by ZetaSizer Nano (Malvern Instruments Ltd, Worcestershire, UK) with 50 ⁇ g/mL MSN dispersed in DI water.
  • LVS glycerol stocks were prepared as described and stored at -80°C. 30 ' 31
  • a vial of the LVS frozen glycerol stock was thawed in a 37 °C water bath and cultivated on GCII chocolate agar plates for 3 days before use.
  • LVS-GFP superfolder green fluorescent protein
  • Macrophages Human peripheral blood monocytes were prepared from the blood of healthy donors and cultivated in Teflon wells for 5 days to differentiate them into monocyte derived macrophages. 31 Human THP-1 monocytic cells (American Type Culture Collection, TH3-202) were maintained in RPMI-1640 (Lonza) supplemented with 10% fetal bovine serum (Mediatech), 2 mM GlutaMAX (Life Technology), penicillin (100 IU) and streptomycin (100 ⁇ g/mL) at 37 °C, 5% C0 2 - 95% air atmosphere. Prior to usage, THP-1 cells were differentiated into macrophages with 100 nM phorbol 12-myristate 13-acetate (PMA; Sigma) in antibiotic-free RPMI with 10% fetal bovine serum.
  • PMA phorbol 12-myristate 13-acetate
  • the infected macrophage cultures were incubated in the continued presence of the treatment for one day. Thereafter, F. tularensis LVS was harvested from the infected macrophages to assess the effect of treatment.
  • the bacteria were harvested by lysing the macrophage monolayers with 1% saponin in PBS for 5 min at room temperature, serially diluted, and plated on GCII chocolate agar. Bacterial colony forming units (CFU) on agar plates were enumerated after incubation at 37 °C for 3 days.
  • CFU Bacterial colony forming units
  • mice Female Balb/c mice (Taconic) of approximately 18 g were provided with standard diet ad libitum and acclimated for one week. Mice were infected by the intranasal route with -8000 (Experiment 1) or -4000 (Experiment 2) CFU of F. tularensis LVS. Two mice were euthanized 5 hours after intranasal infection (day 0) to determine the number of bacteria delivered to the lung at the start of the experiment.
  • mice An additional 3 mice were euthanized one day later (day 1) to determine bacterial growth during that period of time. Mice were then sham-treated or treated with MXF or MSN-MBI-MXF by tail vein injection every other day (day 1, day 3, and day 5) for a total of 3 treatments. Mice were euthanized one day after the last treatment (day 6).
  • Lungs, livers, and spleens from infected mice that were sham-treated or treated with MXF or MSN-MBI-MXF were homogenized and serially diluted for plating on GCII chocolate agar containing sulfamethoxazole (40 ⁇ g/mL), trimethoprim (8 ⁇ g/mL), and erythromycin (50 ⁇ g/mL).
  • Bacterial CFU on the agar plates were enumerated after incubation at 37 °C for 4 days.
  • a median-effect plot for MSN-MBI-MXF or MXF was generated using MXF or MXF equivalent (MSNs) dose in base-10 logarithm as the X-axis and the fraction of surviving bacteria divided by the fraction of killed bacteria in base-10 logarithm as the Y-axis.
  • a Francisella tularensis live vaccine strain (LVS) mutant with a deletion in capB, encoding a putative capsular biosynthesis protein, is significantly more attenuated than LVS yet induces potent protective immunity in mice against F. tularensis challenge.
  • One embodiment of this invention is a composition for treating infections caused by pathogens sensitive to the antibiotic moxifloxacin (MXF), including Tularemia, Tuberculosis (TB) and other mycobacterial diseases (e.g. Mycobacterium kansasii infection, Mycobacterium intracellular infection, disseminated BCG, etc.) comprising MXF loaded into a mesoporous silica nanoparticle (MSN) equipped with a disulfide snap-top valve. MXF releases inside host cells in response to a reducing and hydrophobic environment inside the host cell.
  • MXF mesoporous silica nanoparticle
  • Another embodiment is a method for treating tularemia, TB, other mycobacterial diseases, and infections caused by intracellular pathogens in general utilizing this technology.
  • MSN Mesoporous silica nanoparticles
  • MSNs are synthesized by first dissolving a surfactant, cetyltrimethylammonium bromide (250 mg,
  • CTAB into a basic solution (120 mL, pH 12) and heating up to 80 °C.
  • the silica precursor mixture of tetraethyl orthosilicate (1.2 mL, TEOS) and diethylphosphatoethyltriethoxysilane (0.2 ml, DEPETS)
  • TEOS tetraethyl orthosilicate
  • DEPETS diethylphosphatoethyltriethoxysilane
  • MCM-41 (100 mg) was dispersed into dry toluene (10 ml), mixed with (3-mercaptopropyl) trimethoxysilane (24 ⁇ , O. lmmol) and refluxed for 12 hours under nitrogen atmosphere as shown in Figure 1.
  • Thiol group modified MCM-41(100 mg) was washed and dispersed again in anhydrous toluene (10 ml) in second step.
  • lead thiocyanate 800 mg was dispersed in 10 ml chloroform and titrated by bromine (200 ⁇ ) in chloroform (10ml). The titration product mixture was filtered and the supernatant containing thiocyanogen in chloroform was light yellowish.
  • 1-adamantanethiol (17 mg, O. lmmole) and as-synthesized thiocyanogen were added into MSN toluene dispersion.
  • the disulfide oxidation reaction took four days under 4°C and nitrogen gas atmosphere.
  • As-synthesized material was yellowish and washed thoroughly with toluene, methanol and water.
  • FIG. 28 is a schematic showing disulfide snap-top system synthesis.
  • the disulfide snap-top consists of an adamantyl group which binds with ⁇ cyclodextrin and when nanoparticles are endocytosed into cells the disulfide bond can be reduced by glutathione. Then adamantly group will be removed together with cyclodextrin and drug will be released as shown in FIG. 29.
  • FIG. 29 is a schematic showing a MSN with disulfide snap-top release mechanism.
  • TEM images of MSNs were obtained using a JEM1200-EX (JEOL) instrument (JEOL USA, Inc., Peabody, MA). Particle size was measured by
  • UV-Vis spectra of moxiflxoacin were collected by a Cary 500 UV- vis-NTR spectrophotometer. The release profile was obtained by time-resolved fluorescence spectroscopy.
  • FIG. 30 is a TEM image of MSN with disulfide snap-top that shows structure integrity preserved after surface modification.
  • FIG. 31 shows dynamic light scattering (DLS) measurement of MSN with disulfide snap-top in PBS. It shows that mean hydrodynamic diameter of the modified nanoparticle is around 740 nm due to disulfide formation among MSN.
  • DLS dynamic light scattering
  • FIG. 32 is a graph shwoing UV-Vis spectrum of moxifloxacin in PBS.
  • FIG. 33 is a graph showing moxifloxacin loaded MCM-41 with disulfide snap- top release profile. After adding reducing agent 2-mercaptothanol, moxifloxacin was immediately released and finally reached a plateau. The beginning fluorescence increase is due to external surface drug desorption.
  • Moxifloxacin was released from 100 nm disulfide snap-top MSN-SS-
  • MXF MXF by the reducing agent ⁇ -mercaptoethanol and measured by spectrophotometry at 288 nm or by Francisella bioassays.
  • MSN-SS-MXF in 1 ml of phosphate buffered saline (PBS), pH 7.4 with and without ⁇ - mercaptoethanol (20 ⁇ ), mixed by end-to-end rotation overnight at room temperature, and centrifuged at 10,000 g for 10 min.
  • the supernate was diluted 1 : 150 to 1 : 1200 in PBS to a final volume of 1 ml, and the absorbance read at 288 nm (Table 7).
  • standards with known amounts of MXF were also prepared in PBS, pH 7.4, and used to calculate MXF
  • FIG. 34 shows graphs showing standard curves (left panels) for MXF established by spectrophotometry used to calculate the amount of MXF present in the aqueous eluates prepared from MSN-SS-MXF in PBS with and without ⁇ -mercaptoethanol reducing reagent (right panels).
  • the amount of MXF present in the eluates prepared from MSN-SS-MXF under reducing and non-reducing conditions can also be determined by comparing the amount of killing of Francisella tularensis subsp. holarctica Live Vaccine Strain (LVS) in macrophages by drug eluted from the nanoparticles to the amount of killing by standard concentrations of MXF.
  • LVS holarctica Live Vaccine Strain
  • tularensis LVS and determining bacterial colony forming units (CFU) in macrophage monolayers are described in detail below in the section on the efficacy assay in infected macrophages.
  • CFU bacterial colony forming units
  • FIG. 35 is a median-effect plot of MXF standards generated by CompuSyn.
  • Logarithmic plot of log(Fa/Fu) vs. log(D) serves as a standard curve for calculating MXF loading on nanoparticles in the F. tularensis LVS bioassay.
  • D is dose of MXF; Fa is the fraction of bacteria killed; Fu is the fraction of bacteria surviving.
  • F. tularensis subsp. novicida grows faster than F. tularensis LVS and serves as an alternative bioassay for MXF.
  • MXF was eluted from 1 mg/ml of MSN-SS-MXF under an 1) aqueous condition by PBS, 2) aqueous reducing condition by PBS with ⁇ - mercaptoethanol, and 3) organic reducing condition by DMSO with ⁇ -mercaptoethanol; mixed by end-to-end rotation for 1 hour at room temperature; and centrifuged at 10,000 g for 10 min. Eluates (1.5 ⁇ ) were added to F.
  • novicida in 3 ml trypticase soy broth containing 0.2% cysteine (TSBC) at a starting optical density at 540 nm of 0.05.
  • F. novicida broth cultures were grown at 37°C, with shaking at 200 rpm for 6 hours.
  • the amount of releasable MXF from the nanoparticles was determined by comparing the optical density of the cultures treated with MXF eluted from the nanoparticles to the optical density of the cultures treated with standard concentrations of MXF.
  • FIG. 36 A-C show MXF loading on MSN-SS-MXF can be calculated from a
  • FIG. 36A is a graph showing dose dependent inhibition of F. novicida growth by MXF.
  • FIG. 36B is a graph showing MXF concentrations plotted against the difference in OD540 readings between an F. novicida culture without MXF and a culture treated with standard amounts of MXF.
  • FIG. 36C is a linear standard curve converted from the log value of MXF concentrations plotted against the difference in OD540 reading between an F. novicida culture not treated with MXF and an F. novicida culture treated with a standard amount of MXF.
  • tularensis subsp. holarctica was grown from frozen stocks on GCII chocolate agar at 37°C for 3 days prior to being used to infect macrophages.
  • the human monocytic THP-1 cell line was differentiated with phorbol 12-myristate 13-acetate (PMA) for 3 days to mature the cells into a macrophage-like cell type and infected with F. tularensis LVS at a multiplicity of infection ratio of about 10 bacteria to 1 THP-1 cell for 90 min at 37°C, 5% C0 2 - 95% air atmosphere.
  • Infected monolayers were washed to remove extracellular bacteria. Fresh medium with or without MXF or MXF loaded nanoparticles was added to the infected monolayer.
  • F. tularensis LVS was harvested from infected but not treated cultures at 2 hours and 1 day post infection to determine bacterial growth and from infected cultures that were treated at 1 day to assess the effect of treatment.
  • To harvest the bacteria we lysed the infected macrophages with 1% saponin in PBS and serially diluted the lysate for plating on GCII chocolate agar plates.
  • Bacterial colony forming units (CFU) on agar plates were counted after incubation at 37°C, 5% C0 2 - 95% air atmosphere for 3 days.
  • MSN-SS-MXF at serial two-fold increases in concentration.
  • the MSN-SS-MXF tested had 2.69% (wt/wt) releasable drug in PBS with ⁇ -mercaptoethanol as determined by the spectrophotometry assay.
  • the amount of releasable MXF at each concentration of MSN-SS-MXF tested in this study was calculated according to the 2.69% drug loading and shown in Table 7.
  • MSN-SS-MXF Treatment with MXF or MSN-SS-MXF reduced bacterial CFU in macrophages in a dose-dependent manner (Figure 37).
  • MSN-SS-MXF killed more F. tularensis LVS than an equivalent amount of free MXF.
  • MSN-SS-MXF was at least two fold more efficacious than an equivalent amount of free MXF in killing F.
  • FIG. 37A is a graph showing that free MXF kill F. tularensis LVS in human macrophages in a dose-dependent manner.
  • FIG. 37B is a graph showing that disulfide snap- top MSN-SS-MXF kill F. tularensis LVS in human macrophages in a dose-dependent manner.
  • THP-1 macrophages were infected with F. tularensis LVS and treated for one day before lysing and plating for bacterial CFU. Infected but untreated macrophages were lysed at 2 hours and at one day to determine the extent of bacterial growth during this time period.
  • FIG. 38 shows that MSN-SS-Hoechst but not their PBS eluates stain the nuclei of THP-1 cells.
  • THP-1 macrophages were incubated with MSN-SS-MXF or the PBS anteate from MSN-SS-MXF for 18 h, fixed with 4% paraformaldehyde, and incubated with Alexa Fluor 633- conjugated wheat germ agglutanin (WGA) to stain the plasma membrane of the cells. Images were acquired with a Nikon Optishot microscope equipped with SPOT RKT camera using SPOT software and fixed exposure and gain settings.
  • the infected macrophages that were not treated were lysed at 2 h and 1 day post infection, and all infected macrophages that were treated were lysed at 1 day post infection to determine bacterial CFU in the macrophages.
  • the infected macrophages were lysed as described and plated on GCII chocolate agar. Bacterial colonies on the plates were counted after incubating for 3 days at 37°C, 5% C0 2 - 95% air atmosphere.
  • macrophages treated with MSN-SS-MXF exhibited no evidence of toxicity by morphology.
  • MXF may absorb to mesoporous silica nanoparticles through hydrophobic interactions
  • releasable drug loaded on MSN-SS-MXF was measured using eluates prepared from MSN-SS-MXF under an 1) aqueous PBS non-reducing condition, 2) aqueous PBS with reducing agent ⁇ -mercaptoethanol, and 3) organic DMSO with reducing agent ⁇ - mercaptoethanol.
  • eluates from MSN-SS-MXF in organic DMSO with ⁇ - mercaptoethanol were much more effective in killing LVS than eluates from MSN-SS-MXF in aqueous PBS with ⁇ -mercaptoethanol.
  • a hydrophobic environment such as DMSO or an intracellular environment, is also required for complete release of MXF from the nanoparticle carrier.
  • FIG. 39A is a graph showing killing of intracellular F. tularensis LVS by MXF.
  • GI. 39B is a graph showing killing of intracellular F. tularensis LVS by eluates prepared from MSN-SS-MXF.
  • TFIP-1 macrophages were infected with F. tularensis LVS and treated with various doses of MXF (A) or with eluates (B) prepared from MSN-SS-MXF incubated in aqueous PBS with and without reducing agent ⁇ -mercaptoethanol ( ⁇ ) or in DMSO with ⁇ - ⁇ .
  • FIG. 40A is a graph showing killing of F. tularensis LVS by MSN-SS-MXF in human macrophages.
  • THP-1 macrophages were infected with F. tularensis LVS and treated with various doses of MSN-SS-MXF (A).
  • FIG. 40B shows median-effect curves generated by
  • D is dose of free MXF or MXF equivalent of MSN-SS-MXF; Fa is the fraction of bacteria killed; Fu is the fraction of bacteria surviving.
  • mice were infected by the intranasal route with about 4000 CFU of F. tularensis LVS, a dose equivalent to about 6 times the LD50. Five hours later (day 0), two mice were euthanized to establish the number of bacteria in the lung at the start of the experiment. One day later (day 1), an additional three mice were euthanized to determine bacterial growth over that time period.
  • mice per group were then either sham treated or treated with 50, 100 or 200 ⁇ g of MXF or 260 ⁇ g of disulfide snap-top MSN-SS-MXF by tail vein injection every other day (Friday, Sunday, and Tuesday) for a week.
  • the 260 ⁇ g of MSN-SS-MXF had about 91 ⁇ g of MXF (Table 9).
  • Mice were euthanized one day after the last treatment (day 6).
  • Table 9 LVS burden (Log CFU) in the organs of infected mice 1 day after the last treatment dose
  • mice showed no toxicity from MSN-SS-MXF nanoparticles. During the course of infection, sham control (PBS treated) mice lost more than 20% of their body weight, whereas mice treated with free MXF or MSN-SS-MXF did not (Figure 41).
  • FIG. 41 is a graph showing weight changes in infected mice. The percentage change in weight of infected mice that were sham-treated, treated with the broad spectrum antibiotic MXF administered as a free drug, or treated with MSN-SS-MXF over the course of treatment.
  • Lungs, livers, and spleens from infected mice that were untreated or treated with free MF or MSN-SS-MXF were homogenized.
  • the organ homogenates were serially diluted and plated on GCII chocolate agar containing sulfamethoxazole (40 ⁇ g/ml), trimethoprim (8 ⁇ g/ml), and erythromycin (50 ⁇ g/ml) to prevent growth of contaminants.
  • the agar plates were incubated at 37°C for 3 days at which time the number of bacterial colonies on each plate was counted.
  • MSN-SS-MXF reduced bacterial burden in the liver to a level below that of free MXF at a dose of 100 ⁇ g.
  • FIG. 42A-C show that MSN-SS-MXF kills more F. tularensis LVS than equivalent amount of free MXF in infected mice. Mice were infected with F. tularensis LVS and either sham treated or treated with MXF or MSN-SS-MXF.
  • FIG. 42A is a graph showing bacterial burdens in the lung.
  • FIG. 42B is a graph showing bacterial burden in the liver.
  • FIG. 42C is a graph showing bacterial burden in the spleen. All tissues were monitored throughout the course of infection.
  • mice were euthanized to determine the initial bacterial burden in the lung.
  • day 1 three additional mice were euthanized to determine bacterial growth over this time period.
  • mice per group were then either sham treated or treated with various concentrations of MSN-SS-MXF or free MXF by tail vein injection every other day, 3 days a week (Friday, Sunday and Tuesday) for one week. Mice were euthanized one day (day 6) after the last treatment.
  • This batch of MSN-SS-MXF had 51% (wt/wt) drug release capacity under organic reducing conditions.
  • the amount of MXF for each dose of MSN-SS-MXF used for treatment was calculated according to the drug release capacity and shown in Table 10.
  • mice Over the course of the F. tularensis infection, sham control mice started to lose weight after day 3 and lost about 12% of their body weight by the end of the treatment period. In contrast, mice treated with free MXF or MSN-SS-MXF maintained their weight. Again this confirms that the nanoparticle was well tolerated by the mice (Figure 10).
  • FIG. 43 is a graph showing weight changes in infected mice. Percentage change in weight of infected mice that were sham-treated, treated with MXF administered as a free drug or treated with MSN-SS-MXF over the treatment period.
  • FIG. 44A-C shows disulfide snap-top MSN-SS-MXF treatment reduces bacterial burden in infected mice.
  • Mice were infected with F. tularensis LVS and either sham treated or treated with one of three different doses of free MXF or with one of two different doses of MSN- SS-MXF.
  • bacterial burden was assayed on the first day after infection (Day 0) and one day later (Day 1).
  • FIG. 44A is a graph showing bacterial burden in the lung.
  • FIG. 44B is a graph showing bacterial burden in the liver.
  • FIG. 44C is a graph showing bacterial burden in the spleen.
  • Benefits of the MSN-SS-MXF controlled drug release nanoparticle technology include a) it is more efficacious than an equivalent amount of free drug for treating tularemia and other infectious diseases; b) it preferentially targets macrophages, the host cells for F. tularensis and many other intracellular pathogens, thereby increasing the therapeutic index; c) it provides for controlled release of the drug intracellularly in the host cells for F.
  • NPs can be administered by a variety of routes including intravenously, subcutaneously, intramuscularly, orally, by inhalation, etc; and f) the MSNs are biodegraded and do not accumulate after administration.
  • Francisella tularensis is a facultative intracellular bacterial pathogen that causes tularemia, a serious and potentially fatal disease. Because Ft has extraordinarily high infectivity, causes serious morbidity and mortality, is relatively easily dispersed, is readily cultured on a large scale, and has previously been developed as a biological weapon, it is classified as a Tier 1 potential agent of bioterrorism. Pneumonic tularemia, the type of tularemia of greatest concern in a bioterrorist attack, has a very high morbidity with at least half the patients requiring
  • LTBI M. tuberculosis
  • ⁇ causes neurotoxicity and optic neuritis
  • MXF moxifloxacin
  • MXF moxifloxacin
  • Nanoparticle (NP) delivery platforms provide a more effective, less toxic, and shorter treatment for TB. Because host mononuclear phagocytes internalize particles more efficiently than other cells, intravenously (i.v.) injected NPs, or NPs delivered by other routes of administration, are preferentially taken up by macrophages of the mononuclear phagocyte (reticuloendothelial) system (MPS) and accumulate in liver, spleen, and lung.
  • MFS mononuclear phagocyte
  • NPs are ideally suited to treat Mtb, which infects macrophages in these organs.
  • Targeting antibiotic-loaded NPs to infected organs and tissues, selectively delivering the antibiotics into macrophages and releasing them at high concentrations intracellularly greatly increases their therapeutic index by achieving higher drug concentrations locally where Mtb replicate while limiting systemic toxicities.
  • by controlled release of the drug only after the NPs have been ingested protects the drug from hepatic metabolism and drug clearance before the drug has had the opportunity to attack target pathogens.
  • Increasing drug concentrations at the site of infection by orders of magnitude allows for a much shorter duration of therapy.
  • NP drug delivery vs. free drug a system that delivers high antibiotic concentrations to the site where bacteria divide facilitates sterilization of sites of infection and minimize emergence of drug resistance. Additional advantages of NP drug delivery vs. free drug are a) the drug is shielded from degradation or modification during delivery to infected tissues and b) the drug, by being targeted to macrophages rather than hepatocytes, will not impact hepatic cytochrome P450 metabolism of other drugs.
  • MSNs offer many advantages over previous delivery vehicles (e.g. liposomes, solid lipid particles, alginates) for TB drugs because of their stability, uniformity, inherent lack of toxicity, high internal surface area for drug binding, and versatility in incorporating additional design features. Because of their ultra-high internal surface area (-1000 m 2 /g), MSNs can encapsulate exceptionally high concentrations of different types of cargos. Loading capacities as high as 50 weight percent have been achieved, exceeding by several orders of magnitude that of conventional liposomal nanocarriers. MSNs can be synthesized with a variety of different internal and surface design features, including those that allow for specific targeting to infected host organs and tissues and those that enableakily controlled release of cargo under specific
  • concentrations of combined cargos with disparate physicochemical properties to be simultaneously delivered to overcome multidrug resistance achieve synergistic effects and/or enable combined therapy and diagnostics (theranostics).
  • MSN are degraded in the body over several days and the degradation products are excreted.
  • One embodiment of this invention is a composition for treating infectious diseases caused by pathogens sensitive to the antibiotic moxifloxacin (MXF). These diseases include those caused by intracellular pathogens including Tuberculosis (TB) and other
  • mycobacterial diseases e.g. Mycobacterium kansasii infection, Mycobacterium intracellular infection, disseminated BCG, etc.
  • tularemia etc.
  • diseases also include those caused by extracellular pathogens that are sensitive to the antibiotic moxifloxacin.
  • One embodiment of the invention comprises a mesoporous silica nanoparticle (MSN) with pores into which moxifloxacin is loaded and with valves on top of the pores that contain the moxifloxacin inside the pores until the nanoparticle encounters a low pH environment, e.g. the inside of a host cell for a pathogen, at which point the valves open and release the moxifloxacin.
  • Another embodiment is a method for treating infections caused by intracellular and extracellular pathogens sensitive to the antibiotic
  • MSN Mesoporous silica nanoparticles
  • amorphous silica which is highly porous and has large surface area. These materials have particulate sizes on the order of -100 nm and possess pore diameters of approximately 2 nm.
  • MSNs are synthesized by first dissolving a surfactant, cetyltrimethylammonium bromide (250 mg, CTAB) into a basic solution (120 mL, pH 12) and heating up to 80 °C. Once the solution is thermally stable, the silica precursor, tetraethyl orthosilicate (1.2 mL, TEOS), is added drop-wise into the solution and the solution slowly begins to become opaque.
  • FIG. 45 is a schematic of the pH sensitive nanovalve mechanism.
  • the pore orifice of MCM-41 are attached with organic molecule with pKa around 6 as stalk and modified nanoparticles are soaked in drug/dye solution for 12 hours, after which cyclodextrin is added as capping agent.
  • the outer ring of cyclodextrin is hydrophilic while the inner ring is hydrophobic which binds with stalk organic moiety.
  • tetrabutyammonium iodide (2 mg), benzimidazole (12 mg) and anhydrous triethylamine (150 ⁇ ) .
  • the solution was stirred and heated up to 70°C under N 2 for 24 hours.
  • the final product, pH sensitive nnaovalve modified MSN was washed with methanol, and then dispersed in 30 ml methanol and 1.15 ml 12 M HC1 and refluxed under nitrogen for 12 hours to extract surfactant template.
  • the MSN were washed with methanol and water to be prepared for loading.
  • FIG. 46 is a schematic of pH sensitive nanovalve (MBI) system synthesis.
  • MCM-41 (10 mg) with pH valve was dispersed in 1 ml ImM Moxifloaxcin PBS solution and rotating overnight, ⁇ cyclodextrin (40 mg) was added into the solution as capping agent to prevent drug from leaking out. After mixing the solution for another 12 hours, moxifloxacin loaded MCM-41 with pH nanovalve system was dried under vacuum overnight. In order to prove moxifloxacin can be release from MSN and detect moxiflxoacin fluorescence emission in supernatant, the dried powder was put at the corner of a glass vial containing 10 ml DI water.
  • TEM images of SMSNs and MSNs were obtained using a JEM1200-EX (JEOL) instrument (JEOL USA, Inc., Peabody, MA). Particle size was measured by ZetaSizer Nano (Malvern Insstruments Ltd, Worcestershire, UK) and MSN was dispersed in PBS in 50 ⁇ . 13 C-CPMS and 29 Si-CPMS NMR spectra were collected on a
  • UV-Vis spectra of moxifloxacin were collected by a Cary 500 UV-vis-NTR
  • the release profile was obtained by time-resolved fluorescence spectroscopy.
  • FIG. 47 is a TEM image of MSN with pH sensitive nanovalve that shows structural integrity preserved after all surface modifications and surfactant temple extraction
  • FIG. 48 shows dynamic light scattering (DLS) measurement of MSN with pH sensitive nanovalve. It shows that mean hydrodynamic diameter of the modified nanoparticle is around 100 nm
  • FIG. 49 A is a 13 C-CPMS NMR of MBI MSN.
  • the data illustrate that benzimidazole is bonded to the silica surface. The nanoparticle alone does not produce peaks in the aromatic region.
  • FIG. 49B is a 29 Si-CPMS NMR spectra of MBI MSN. It shows bulk silica band and attached thread containing a Si-C bond-band, proving the attachment of the MBI compound.
  • FIG. 50 is a UV-Vis spectrum of moxifloxacin under pH 1 and 7.4
  • FIG. 51 is a graph showing Moxifloxacin loaded MCM-41 with MBI pH sensitive nanovalve release profile. There is no leakage at pH 7 indicated as flat baseline. Drug release starts at pH 6 and release rate can be further increased by lowering pH down to 4.5
  • MXF was released from 100 nm pH-gated MSN1 -MXF or MSN2-MXF at neutral and acidic pH conditions and measured by spectrophotometry at 288 nm.
  • FIG. 52 is an example of a MXF standard curve used to calculate drug loading on nanoparticles eluted under neutral pH or acidic pH conditions. Table 11. MXF released under neutral pH or acidic pH from 1 mg of pH
  • MXF loading on pH-gated MSN-MXF also can be measured by sequentially eluting first at neutral pH and subsequently under acidic aqueous and organic conditions.
  • pH-gated MSN-MXF was suspended in 1 ml of 0.1 M FEPES, pH 7.4 at a concentration of 1 mg/ml, mixed by end-to-end rotation for about 1 hour at room temperature, and centrifuged at 10,000 g for 10 min. The supernate (Neutral Eluate) was collected for assay of MXF concentration.
  • nanoparticles were resuspended in 1 ml of 0.1 N HC1 (aqueous acid), mixed by end-to-end rotation for about 2 hours at room temperature, and centrifuged to pellet the nanoparticles.
  • the supernate (Aqueous Acid Eluate) was removed for assay of MXF release by aqueous acid.
  • the nanoparticles were resuspended in 1 ml of 0.1 N HC1 in DMSO (DMSO acid), mixed by end-to-end rotation overnight at room temperature, and centrifuged to pellet the nanoparticles.
  • the amount of MXF eluted under neutral pH, aqueous acid, and DMSO acid conditions was measured at 288 nm (Table 2) and calculated from the MXF standard curve ( Figure 2).
  • the amount of MXF eluted with 0.1 M FEPES was considered to be the residual MXF outside of the nanoparticle in that batch of pH-gated MSN-MXF.
  • the amount of MXF eluted by aqueous acid was considered to be the MXF released from the nanoparticle upon the opening of its pH-sensitive gates.
  • the additional amount of MXF released by acidified DMSO was considered to be the MXF absorbed on the nanoparticle through hydrophobic interaction.
  • the sum of MXF measured in neutral eluate, aqueous acid eluate, and DMSO acid eluate is the total MXF loaded on that batch of pH-gated MSN-MXF.
  • FIG. 53 is an example of a MXF standard curve used for calculating drug loading on nanoparticles after sequential elution under neutral and acidic pH conditions.
  • tularensis subsp. holarctica was grown from frozen stocks on GCII chocolate agar at 37°C for 3 days prior to use for infecting macrophaes.
  • Human monocytic THP-l cell line was differentiated with phorbol 12-myristate 13-acetate (PMA) for 3 days to mature the cells into a macrophage-like cell type and infected with F. tularensis LVS at a multiplicity of infection ratio of about 10 bacteria to 1 THP-1 cell for 90 min at 37°C, 5% C0 2 - 95% air atmosphere. Infected monolayers were washed to remove extracellular bacteria.
  • PMA phorbol 12-myristate 13-acetate
  • F. tularensis LVS were harvested from infected but not treated cultures at 3 hours and 1 day post infection to determine bacterial growth and from infected cultures that were treated at 1 day to assess the effect of treatment.
  • PBS phosphate buffered saline
  • Bacterial colony forming units (CFU) on agar plates were counted after incubation at 37°C, 5% CO2 - 95% air atmosphere for 3 days.
  • MSNl-MXF and MSN2-MXF also with serial two-fold increases in concentration from 0.0625 ⁇ g/ml to 8 ⁇ g/ml.
  • MSNl-MXF had a total drug loading measured as 2.64% wt/wt (0.00%) in Neutral Eluate + 2.64% in Aqueous Acid Eluate)
  • the amount of MXF that could potentially be released by acidic pH in the endo-lysosomal compartments of macrophages for MSNl-MXF over the range of concentrations tested was estimated to be from 1.7 to 211 ng/ml (Table 3).
  • the total drug loading on MSN2-MXF was 0.36%, therefore the acid releasable MXF for the nanoparticle over the range of nanoparticle concentrations tested was estimated to be from 0.23 to 29 ng/ml.
  • MSNl-MXF at 1 ⁇ g/ml reduced CFU by 3.37 logs compared with the level in the untreated control group, whereas the same concentration of MSN2-MXF reduced bacterial CFU by only by 0.17 logs compared with the untreated control group.
  • MSN2-MXF its neutral eluate had no impact on F. tularensis LVS growth in macrophages and the acid eluate reduced bacterial number by merely 0.2 logs (Figure 55B).
  • This study demonstrates that 1) the pH operative valves on MSNl-MXF are tightly closed at neutral pH and open at acidic pH, 2) MXF eluted under acidic pH retains biological activity, 3) MSNl-MXF and MSN2-MXF kill F. tularensis LVS in macrophages in a dose-dependent fashion, and 4) MSNl-MXF has greater efficacy than MSN2-MXF, most likely because of higher MXF loading on MSNl-MXF.
  • FIG. 54A is a graph showing that free MXF kill F. tularensis LVS in human macrophages in a dose dependent manner.
  • FIG. 54B is a graph showing that pH-gated MSNl-MXF kill F. tularensis LVS in human macrophages in a dose dependent manner.
  • FIG. 54C is a graph showing that MSN2-MXF kill F. tularensis LVS in human macrophages in a dose dependent manner.
  • THP-1 macrophages were infected with F. tularensis LVS, treated for one day, lysed, and the lysate serially diluted and plated to determine bacterial CFU. Infected but untreated
  • macrophages were lysed at 3 hours and one day to determine bacterial growth.
  • FIG. 55A is a graph showing that acid eluates of pH-gated MSN1-MXF reduce the number of F. tularensis LVS in macrophages.
  • FIG. 55B is a graph showing that MSN2-MXF do not reduce the number of F. tularensis LVS in macrophages.
  • tuberculosis Erdman strain for 90 min and not treated or treated with a) control MSN1 not loaded with MXF; b) various concentrations of pH- gated MSNl-MXF; or c) various concentrations of free MXF.
  • the infected macrophages that were not treated were lysed at 3 hours and 3 days post infection, and all infected macrophages that were treated were lysed at 3 days post infection to determine bacterial CFU in the macrophages.
  • the infected macrophages were lysed with 0.1% SDS, and the lysates serially diluted and plated on 7H11 agar plates. Bacterial colonies on the plates were counted after two weeks of incubation at 37°C, 5% CO2 - 95% air atmosphere.
  • the amount of residual (neutral) and acidic pH releasable MXF from the nanoparticles was measured by spectrophotometry as described above.
  • the total amount of MXF on this batch of MSNl-MXF was determined to be 15.71% (wt/wt) with 5.23% (wt/wt) being in the neutral eluate.
  • the amount of MXF available from each concentration of MSNl-MXF tested in the experiment is calculated based on the total % wt/wt of MXF for the nanoparticle and is shown in Table 14.
  • MSNl-MXF at 3.1, 6.25, and 12.5 ⁇ g/ml killed 84%, 97%, and 99% of intracellular M. tuberculosis over 3 days. That the extent ofM tuberculosis killing achieved by any selected dose of MSNl-MXF is about the same as that of the corresponding amount of MXF available from that dose of nanoparticle suggests that MXF delivered by pH-gated MSNl-MXF has the same potency in killing M. tuberculosis in macrophages as the equivalent amount of MXF by itself. Thus, the efficacy ratio of MSNl-MXF to MXF in macrophages (the amount of killing by MSNl-MXF/amount of killing by an equivalent amount of free MXF) was close to 1.
  • FIG. 56A-56C shows killing of M. tuberculosis by pH-gated MSN1-MXF in human macrophages.
  • FIG. 56A shows THP-1 macrophages infected with M tuberculosis and treated with various doses of MXF.
  • FIG. 56B shows THP-1 macrophages infected with M tuberculosis and treated with various doses of MSN 1 -MXF.
  • FIG. 56C shows THP-1 macrophages infected with M tuberculosis and treated with various doses of eluates prepared from MSN1-MXF in acidified DMSO.
  • mice per group were then either sham treated or treated with 100, 200 or 400 ⁇ g of MXF or 2 mg of pH-gated MSN1- MXF by tail vein injection every other day (Friday, Sunday, and Tuesday) for a week.
  • the 2 mg of MSN1-MXF had about 140 ⁇ g of MXF.
  • Mice were euthanized one day after the last treatment (day 6) and lungs spleens, and livers assayed for CFU of F. tularensis.
  • FIG. 57 is a chart showing the percentage change in weight of the F. tularensis- infected mice that were sham-treated, treated with the broad spectrum antibiotic MXF administered as a free drug, or treat with pH-gated MSNl-MXF were monitored over the course of treatment.
  • Lungs, livers, and spleens from the infected mice with or without treatment were homogenized.
  • the organ homogenates were serially diluted and plated on GCII chocolate agar containing sulfamethoxazole (40 ⁇ g/ml), trimethoprim (8 ⁇ g/ml), and erythromycin (50 ⁇ g/ml).
  • the agar plates were incubated at 37°C for 3 days at which time bacterial colonies on each plate were counted.
  • Treatment with MSNl-MXF reduced bacterial burden in the lung and spleen by 4.0-, and 4.3-logs, respectively, more so than treatment with 400 ⁇ g of free MXF (Table 15).
  • pH-gated MSNl-MXF kills more F. tularensis LVS than about 2.8-fold its equivalent amount of free MXF in infected mice.
  • Mice were infected with F. tularensis LVS and either sham treated or treated with MXF or MSNl-MXF.
  • FIG. 58B is a graph showing bacterial burdens in the lung.
  • FIG. 58A is a graph showing bacterial burdens in the liver.
  • FIG. 58C is a graph showing bacterial burdens in the spleen. All were monitored throughout the course of infection.
  • mice were euthanized to determine the initial bacterial burden in the lung.
  • days 1 two mice were euthanized to determine the initial bacterial burden in the lung.
  • day 2 three additional mice were euthanized to determine bacterial growth at the onset of the treatment period.
  • Three mice per group were then either sham treated or treated with various concentrations of MSNl-MXF or free MXF by tail vein injection every other day, 3 days a week (Friday, Sunday and Tuesday) for one week. Mice were euthanized one day (day 6) after the last treatment.
  • MXF at each dose of MSNl-MXF used for treatment was calculated according to the drug release capacity and shown in Table 16.
  • FIG. 59 is a graph showing percentage change in weight of the F. tularensis- infected mice that were sham-treated, treated with the broad spectrum antibiotic MXF administered as a free drug, or treated with pH-gated MSNl-MXF was monitored over the treatment period.
  • FIG. 60A is a graph showing that pH-gated MSNl-MXF treatment reduces bacterial burden in the lung of F. tularensis-m ' iected mice.
  • FIG. 60B is a graph showing that pH- gated MSNl-MXF treatment reduces bacterial burden in the liver of F. tularensis-miected mice.
  • FIG. 60C is a graph showing that pH-gated MSNl-MXF treatment reduces bacterial burden in the spleen of F. tularensis-m ' iected mice. Mice were infected with F.
  • tularensis LVS tularensis LVS and either sham treated or treated with one of four different doses of free MXF or MSNl-MXF.
  • bacterial burden was assayed on the first day after infection (Day 0) and one day later (Day 1).
  • Bacterial burden in lung (A), liver (B), and spleen (C) was determined day 6, one day after the last treatment (Day 6) by assaying bacterial CFU.
  • mice were infected with about 4000 CFU of F. tularensis LVS by the intranasal route.
  • mice were sham treated or treated with one of the three doses of MXF (50, 100, and 200 ⁇ g) or with 640 ⁇ g of MSNl-MXF (with about 50 ⁇ g of releasable MXF) every other day (Friday, Sunday and Tuesday) for a week.
  • the three doses of MXF were equal to lx, 2x, and 4x the amount of the releasable MXF from 640 ⁇ g of MSNl-MXF by acidic DMSO (Table 17).
  • mice suffered substantial weight loss but mice treated with free
  • MXF or MSNl-MXF did not ( Figure 61).
  • MSNl-MXF treatment reduced bacterial burden by 2.8 logs in the lung, 3.2 logs in the liver, and 3.3 logs in the spleen to a level close to that achieved by 100 ⁇ g MXF ( Figure 62).
  • This experiment demonstrates that mice showed no toxicity from pH- gated MSNl-MXF.
  • MSNl-MXF had an efficacy twice the equivalent amount of free MXF in the lung, spleen and liver.
  • FIG. 61 is a graph showing percentage change in weight of the F. tularensis- infected mice that were sham-treated, treated with MXF administered as a free drug, or treated with pH-gated MSNl-MXF was monitored over the treatment period.
  • FIG. 62A-C are graphs showing that pH-gated MSNl-MXF treatment reduced bacterial burden in F. tularensis-m ' iected mice. Mice were infected with F. tularensis LVS and either sham treated or treated with MSNl-MXF or lx, 2x or 4x the equivalent amount of free MXF. In sham-treated mice, bacterial burden was assayed on the first day after infection (Day 0) and one day later (Day 1).
  • FIG. 62A is a graph showing bacterial burden in lung.
  • FIG. 62B is a graph showing bacterial burden in the liver.
  • FIG. 62C is a graph showing bacterial burden in the spleen. All were determined on day 6, one day after the last treatment by assaying bacterial CFU.
  • Benefits of the MSNl-MXF controlled drug release nanoparticle technology include a) it is more efficacious than an equivalent amount of free drug for treating tuberculosis and tularemia and other infectious diseases; b) it preferentially targets macrophages, the host cells for M. tuberculosis, F. tularensis and many other intracellular pathogens, thereby increasing the therapeutic index; c) it provides for controlled release of the drug intracellularly in the host cells for M. tuberculosis and F.
  • tularensis and other intracellular pathogens thereby avoiding off-target effects and premature metabolism of the drug; d) it allows for improved treatment of both active pulmonary and extra-pulmonary tuberculosis (TB) and other mycobacterial diseases (e.g.
  • NPs can be administered by a variety of routes including intravenously, subcutaneously, intramuscularly, orally, by inhalation, etc; and g) the MSNs are biodegraded and do not accumulate after
  • LTBI M. tuberculosis
  • F H causes neurotoxicity and optic neuritis
  • MXF moxifloxacin
  • MXF moxifloxacin
  • Nanoparticle (NP) delivery platforms provide a more effective, less toxic, and shorter treatment for TB. Because host mononuclear phagocytes internalize particles more efficiently than other cells, intravenously (i.v.) injected NPs, or NPs delivered by other routes of administration, are preferentially taken up by macrophages of the mononuclear phagocyte (reticuloendothelial) system (MPS) and accumulate in liver, spleen, and lung.
  • MFS mononuclear phagocyte
  • NPs are ideally suited to treat Mtb, which infects macrophages in these organs.
  • Targeting antibiotic-loaded NPs to infected organs and tissues, selectively delivering the antibiotics into macrophages and releasing them at high concentrations intracellularl greatly increases their therapeutic index by achieving higher drug concentrations locally where Mtb replicate while limiting systemic toxicities.
  • by controlled release of the drug only after the NPs have been ingested protects the drug from hepatic metabolism and drug clearance before the drug has had the opportunity to attack target pathogens.
  • Increasing drug concentrations at the site of infection by orders of magnitude allows for a much shorter duration of therapy.
  • Francisella tularensis is a facultative intracellular bacterial pathogen that causes tularemia, a serious and potentially fatal disease. Because Ft has extraordinarily high infectivity, causes serious morbidity and mortality, is relatively easily dispersed, is readily cultured on a large scale, and has previously been developed as a biological weapon, it is classified as a Tier 1 potential agent of bioterrorism. Pneumonic tularemia, the type of tularemia of greatest concern in a bioterrorist attack, has a very high morbidity with at least half the patients requiring
  • MSNs offer many advantages over previous delivery vehicles (e.g. liposomes, solid lipid particles, alginates) for TB drugs because of their stability, uniformity, inherent lack of toxicity, high internal surface area for drug binding, and versatility in incorporating additional design features. Because of their ultra-high internal surface area (-1000 m 2 /g), MSNs can encapsulate exceptionally high concentrations of different types of cargos. Loading capacities as high as 50 weight percent have been achieved, exceeding by several orders of magnitude that of conventional liposomal nanocarriers. MSNs can be synthesized with a variety of different internal and surface design features, including those that allow for specific targeting to infected host organs and tissues and those that enableakily controlled release of cargo under specific
  • concentrations of combined cargos with disparate physicochemical properties to be simultaneously delivered to overcome multidrug resistance achieve synergistic effects and/or enable combined therapy and diagnostics (theranostics).
  • MSN are degraded in the body over several days and the degradation products are excreted.
  • One embodiment of the invention is a composition for treating Tuberculosis (TB) and other mycobacterial diseases (e.g. Mycobacterium kansasii infection, Mycobacterium
  • intracellular infection disseminated BCG, etc.
  • an anti-TB drug e.g. isoniazid (INH)
  • INF isoniazid
  • MSN mesoporous silica nanoparticle
  • Another embodiment of the invention is a method for treating TB, other mycobacterial diseases, and infections caused by intracellular pathogens in general utilizing this technology.
  • Another embodiment of the invention is a method for targeting nanoparticles preferentially to the lung for the treatment of lung diseases of all kinds.
  • Another embodiment of the invention is a method for loading a nanoparticle with a prodrug by directly binding the prodrug to the nanoparticle in such a way that it is released in a controlled way as an active drug under low pH conditions; as such the technology is broadly applicable to the delivery of prodrugs by nanoparticles for the treatment of many diseases, infectious and otherwise.
  • MSN Mesoporous silica nanoparticles
  • amorphous silica which is highly porous and has large surface area. These materials have particulate sizes on the order of -100 nm and possess pore diameters of approximately 2 nm.
  • MSNs are synthesized by first dissolving a surfactant, cetyltrimethylammonium bromide (250 mg, CTAB) into a basic solution (120 mL, pH 12) and heating up to 80 °C. Once the solution is thermally stable, the silica precursor, tetraethyl orthosilicate (1.2 mL, TEOS), is added drop-wise into the solution and the solution slowly begins to become opaque.
  • a coating of 3-(trihydroxysilyl)propyl methylphosphonate, (300 ⁇ ⁇ , HTMP) is added to the solution, and the solution is further aged for 90 minutes.
  • the nanoparticles are collected by centrifugation and washed with methanol.
  • the as-synthesized nanoparticles are suspended in methanol, and hydrochloric acid is added, and refluxed overnight to remove the templating surfactant.
  • Nanoparticles are collected by centrifugation and washed extensively with methanol and water.
  • SMSNs mesoporous silica nanoparticles
  • a co-surfactant we made nanoparticles with diameters of 50 nm and pore diameters of approximately 2 nm.
  • SMSNs are synthesized in a similar method as the MSNs (see above). Briefly, cetyltrimethylammonium bromide (250 mg, CTAB) and Pluronic F127 (200 mg) are dissolved into aqueous solution (120 mL, pH 12) and heated to 80 °C. Tetraethyl orthosilicate (1.2 mL, TEOS), is added drop-wise into the solution.
  • Nanoparticles can be labeled with fluorescent dye molecules for imaging purposes using two different methods: co-condensation and post-synthetic grafting.
  • the dye is co- condensed with the silica precursor to ensure labeling throughout the silica matrix.
  • Rhodamine B isothiocyanate (RITC, 2 mg) is dissolved in dry ethanol (1.5 mL), 3-aminopropyltrimethoxysilane (6 ⁇ ⁇ , APTES) is added, and the molecules are left to react under nitrogen for 2 hours.
  • TEOS is then added to the solution, and the solution is added in the same manner as the silica precursor mentioned in the previous procedure.
  • the dye can be post-synthetically grafted by condensing amines (6 ⁇ ., APTES) on the surface of nanoparticles (100 mg) and refluxing overnight in toluene (10 mg). The particles are washed with toluene, methanol, water, and finally suspended in DMF (10 mL). The amine-reactive DyLight 680 (N-hydroxysuccinimide ester-activated, 10 mg) is then added and left to react for 12 hours. The near-IR labeled particles are washed with water.
  • the hydroxyl group on m-PEG was replaced with an NHS-ester to react with the amines groups on PEI [1].
  • PEG was attached to the amine groups of the PEI coating by suspending the PEI-coated MSNs (10 mg) in dry DMF (1.5 mL), adding 50 mg of activated m-PEG, and stirring for 24 hours. The nanoparticles were washed with DMF, water, and resuspended in PBS.
  • Antibodies were used to direct nanoparticles to the lung.
  • Protein G was first covalently attached to the nanoparticles through amine-carboxyl coupling to immobilize the APP2 antibody onto the surface of MSNs.
  • INH-PEI-PEG-MSNs (10 mg) were suspended in PBS (1 mL, pH 7.4) and slowly added dropwise to a solution of Protein G (5 mg/mL, PBS). Finally a solution of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, 50 ⁇ ., 50 mg/200 ⁇ . PBS) was added to the solution and left to react for 24 hours. The nanoparticles were carefully washed with PBS.
  • EDAC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • TEM images of SMSNs and MSNs were obtained using a JEM1200-EX (JEOL) instrument ( Figure 1).
  • UV-vis spectra of INH loading were collected by a Cary 500 UV-vis-NTR spectrophotometer.
  • DLS and zeta potentials were measured by ZetaSizer Nano (Malvern Instruments Ltd., Worcestershire, U.K.).
  • ICP-OES ICP-OES
  • FIG. 63 is a diagram of isoniazid ( ⁇ ) attaching to the surface of the aldehyde- modified nanoparticles to form the 'pro-drug' MSN.
  • FIG. 64A is a TEM image of INH-CHO-PEI-PEG- SMSNs.
  • FIG. 64B is a TEM image of INH-CHO-PEI-PEG- MSNs.
  • FIG. 65 is a graph showing Isoniazid standard curve measured at 262 nm to measure loading and release.
  • FIG. 66A is a UV-vis spectra of supernatant after washing INH-loaded nanoparticles (black trace) and the release of INH (red trace) for SMSN.
  • FIG. 66B is a UV-vis spectra of supernatant after washing INH-loaded nanoparticles (black trace) and the release of INH (red trace) for SMSN.
  • FIG. 67A-67D are graphs showing distribution of injected silica determined by
  • FIG. 67A is a graph showing that after 24 hours INH-CHO-MSNs are primarily in the liver.
  • FIG. 67B is a graph showing that after 24 hours INH-CHO-SMSNs are well distributed throughout the body.
  • FIG. 67C is a graph showing that after two weeks of accumulation, INH- MSNs are still primarily in the liver.
  • FIG. 67D is a graph showing that after two weeks of accumulation, INH-SMSNs have higher quantities of silica in the lung, liver, and spleen.
  • Trans-cinnamaldehyde reagent (0.04%).
  • a 1 ml/100 ml stock solution of trans-cinnamaldehyde is prepared in absolute EtOH and stored for up to 3 weeks at 4°C. Before use in INH determinations, the stock solution is diluted 25-fold with absolute ethanol to give a final concentration of 0.04 ml of trans-cinnamaldehyde per 100 ml.
  • An INH standard curve is generated using 0 ⁇ g/ml, 2 ⁇ g/ml, 4 ⁇ g/ml, 8 ⁇ g/ml, and 10 ⁇ g/ml INH in 0.1 N HCl. Each tube has 1 ml of 0.1 N HCl.
  • transcinnamaldehyde reagent 0.15 ml of transcinnamaldehyde reagent is added to each of the tubes containing sample or a standard, and the tube vortexed and incubated at Room Temperature for 15 min.
  • MSN-CHO-INH or SMSN-CHO-INH in 1 ml of phosphate buffered saline (PBS) or in 0.1 N HCl, mixed the suspension on a nutator for 1 hour at room temperature, and centrifuged at 10,000 g for 10 min to pellet the nanoparticles.
  • the supernate was diluted 1 :25 to 1 :200 in 0.1 N HCl to a final volume of 1 ml and mixed with trans-cinnamldehyde reagent.
  • standards with known amounts of INH were prepared in tubes with 0.1 N HCl at a final volume of 1 ml.
  • FIG. 68 is an example of an INH standard curve used to calculate drug loading on nanoparticles eluted under neutral pH or acidic pH conditions.
  • INH on MSN-CHO-INH and SMSM-CHO-INH also can be measured by sequentially eluting first at neutral pH and then at acidic pH.
  • MSN-CHO-INH and SMSM-CHO- INH were suspended in PBS or 1% BSA in PBS, pH 7.4, at a concentration of 1 mg/ml, mixed by end-to-end rotation for about 30 min at room temperature, and centrifuged at 10,000 g for 10 min. The supernate was collected for assay of INH concentration.
  • the nanoparticles were resuspended in 1 ml of 0.1 N HC1, mixed by end-to-end rotation for about 30 min at room temperature, and centrifuged to pellet the nanoparticles.
  • the amount of INH eluted under neutral pH or acidic pH conditions was measured using the trans-cinnamaldehyde assay and a standard curve for INH (Figure 69) as described above (Table 19).
  • FIG. 69 is an example of an INH standard curve used for calculating drug loading on nanoparticles after sequential elution under neutral and acidic pH conditions.
  • MSN-CHO-INH The efficacy of MSN-CHO-INH was assessed in a macrophage infection model ofM tuberculosis.
  • Human monocytic THP-1 cell line was differentiated with phorbol 12-myristate 13 -acetate (PMA) for 3 days to mature the cells into a macrophage-like cell type and infected with single-cell bacterial suspension of virulent M. tuberculosis Erdman strain at a multiplicity of infection ratio of about 10 bacteria to 1 THP-1 cell for 90 min at 37°C, 5% C0 2 - 95% air atmosphere.
  • Infected monolayers were washed to remove extracellular bacteria. Fresh medium with or without FNH and MSN-CHO-FNH was added to the infected monolayer.
  • M. tuberculosis were harvested from infected but not treated cultures at 2 hours and 3 days post infection to assess bacterial growth over the three days of the assay and from infected cultures that were treated at 3 days to assess the effect of treatment.
  • SDS serum-derived sulfate
  • CFU Bacterial colony forming units
  • MSN-CHO-INH INH killed M. tuberculosis in infected macrophages in a dose dependent manner (Figure 70).
  • MSN-CHO-INH at the two lowest concentrations tested i.e. 0.625 and 1.25 ⁇ g/ml
  • Total drug loading of MSN-CHO-INH was measured as 9.61% wt/wt (1.09% in Ethanol Wash + 0.23% in 1% BSA Neutral Eluate + 8.29% in 0.1 N HCl).
  • the amount of INH that could potentially be released from 0.625 and 1.25 ⁇ g/ml of MSN-CHO-INH by acidic pH in the endo-lysosomal compartments of macrophages was estimated to be 0.06 and 0.12 ⁇ g/ml, respectively.
  • INH treatment at a concentration of 0.05 and 0.1 ⁇ g/ml yielded a 1.4- and 2.1-log reduction in CFU in infected THP-1 macrophages, respectively, a level close to that achieved by the MSN-CHO-INH (Table 20).
  • This study demonstrates that INH delivered by the nanoparticle MSN-CHO-INH is as effective as an equivalent amount of free INH in killing M. tuberculosis in infected human macrophages.
  • FIG. 70 A is a graph showing that INH kills M. tuberculosis in human
  • FIG. 70B is a graph showing that MSN-CHO-INH kill M. tuberculosis in human macrophages in a dose dependent manner.
  • THP-1 macrophages were infected with M. tuberculosis and treated for 3 days before lysing and plating for bacterial CFU. Infected but untreated macrophages were lysed at 2 hours and 3 days to determine bacterial growth.
  • tuberculosis in infected human macrophages.
  • M. tuberculosis grew about 0.8 logs in macrophages over 3 days with no treatment or when treated with the eluate from 5 ⁇ g/ml of MSN-CHO-INH under neutral pH (Neutral Eluate).
  • This result indicates that negligible INH activity is present in the Neutral Eluate.
  • the eluates prepared from 1.25 and 5 ⁇ g/ml of MSN-CHO-INH under acidic pH (Acid Eluate) reduced bacterial numbers in macrophages by 2.4 and 3.3 logs, respectively, to a level close to the 2.6 and 3.5 logs of reduction achieved by an equivalent amount of MSN-CHO-INH.
  • FIG. 71 is a graph showing that acid eluates of MSN-CHO-INH kill M
  • tuberculosis in macrophages to a similar extent as the nanoparticle.
  • MSN-CHO-INH was washed with ethanol and incubated sequentially with 1% bovine serum albumin (BSA) in PBS, pH 7.4, and 0.1 N HC1 at room temperature for about 30 min for each step. Between each wash and incubation step, the sample was centrifuged to bring down the nanoparticles. The sequential supernates (referred to as "Ethanol Wash”, “1% BSA Neutral Eluate”, or “Acid Eluate”) were collected, and the amount of INH present in the supernate was assayed by the trans-cinnamldehyde assay as described above.
  • BSA bovine serum albumin
  • FIG. 72 is a graph showing that MSN-CHO-INH is stable at 4°C for at least one month. INH was eluted from MSN-CHO-INH after storage for one month in refrigerator and measured by the trans-cinnamldehyde assay. [00483] In the second macrophage experiment, the efficacies of MSN-CHO-INH nanoparticles of two different sizes (50 nm and 100 nm) were studied. PMA differentiated THP-1 macrophages were infected with M.
  • tuberculosis for 90 min and not treated or treated with a) control MSN not loaded with INH; b) 100 nm MSN-CHO-INH; c) 50 nm SMSN-CHO-INH; or d) various concentrations of free INH.
  • the infected macrophages that were not treated were lysed at 2 h and 3 days post infection, and all infected macrophages that were treated were lysed at 3 days post infection to determine bacterial CFU in the macrophages.
  • M. tuberculosis grew similarly in macrophages that were untreated or treated with control nanoparticles (no INH loaded) indicating that the nanoparticle carrier by itself has no inhibitory effect on the bacterium.
  • INH delivered by MSN or SMSN killed the bacteria in macrophages in a dose-dependent fashion ( Figure 73).
  • We determined the drug loading on the nanoparticles by incubating MSN-CHO-INH and SMSN-CHO-INH sequentially with 1% BSA at neutral pH and then with 0.1 N HCl. The amount of residual (neutral) and acidic pH releasable INH from the nanoparticles was measured by spectrophotometry as described above.
  • the amount of INH was determined to be 0.28% (wt/wt) in the neutral eluate and 5.45% (wt/wt) in the acid eluate, and thus a total of 5.73% INH (wt/wt) was loaded on this batch of nanoparticles.
  • the amount of INH was determined to be 0.45% (wt/wt) in the neutral eluate and 2.81% (wt/wt) in the acid eluate, and thus a total of 3.26% INH (wt/wt) was loaded on the nanoparticles.
  • the amount of INH available from each concentration of MSN-CHO- INH and SMSN-CHO-INH tested in the experiment is calculated based on the total % wt/wt of INH for the nanoparticles and is shown in Table 21. That the extent ofM tuberculosis killing achieved by any selected dose of MSN-CHO-INH or SMSN-CHO-INH is about the same as that of the corresponding amount of INH available from that dose of nanoparticle suggests that INH delivered by MSN-CHO-INH and SMSN-CHO-INH has the same potency in killing M. tuberculosis in macrophages as the equivalent amount of INH by itself.
  • SMSN-CHO-INH (0.125 0.0041 ⁇ g/ml 3 days 4.47
  • SMSN-CHO-INH (0.25 ⁇ g/ml) 0.0082 ⁇ g/ml 3 days 4.50
  • SMSN-CHO-INH (0.5 ⁇ g/ml) 0.0163 ⁇ / ⁇ 1 3 days 4.49
  • SMSN-CHO-INH (1 ⁇ ) 0.0326 ⁇ g/ml 3 days 4.23
  • FIG. 73 A is a graph showing killing of M tuberculosis by INH.
  • FIG. 73B is a graph showing killing ofM tuberculosis by MSN-CHO-INH.
  • FIG. 73C is a graph showing killing ofM tuberculosis by SMSN-CHO-INH.
  • FIG. 73D is a graph showing killing ofM tuberculosis by MSN-CHO-INH under neutral or acidic pH conditions.
  • FIG. 73E is a graph showing killing of M tuberculosis by SMSN-CHO-INH under neutral or acidic pH conditions.
  • Nanoparticles carrying INH in human macrophages THP-1 macrophages were infected with M tuberculosis and treated with various doses of INH (A), MSN-CHO-INH (B), or SMSN-CHO-INH (C), or with eluates prepared from MSN-CHO-INH (D) or SMSN-CHO-INH (E) under neutral or acidic pH conditions.
  • mice in the sham control group had the worst pathology with numerous tubercle lesions visible on the surface of their lungs and enlarged livers and spleens.
  • Organs of mice in the INH treated group showed less pathology than organs of sham treated mice, and organs of mice in the MSN-CHO-INH treated group showed less pathology than organs of INH treated mice.
  • Lungs, livers, and spleens from mice with or without treatment were homogenized.
  • the organ homogenates were serially diluted and plated on 7H11 agar containing ampicillin (12.5 ⁇ g/ml), amphotericin B (5 ⁇ g/ml), and polymyxin B (20 U/ml).
  • the agar plates were incubated at 37°C, 5% C0 2 -95% air atmosphere for two and half weeks at which time bacterial colonies on each plate were counted.
  • FIG. 74A-74D show that MSN-CHO-INH kills more M. tuberculosis than an equivalent amount of free INH in infected mice. Mice were infected with M. tuberculosis and either sham treated or treated with INH or MSN-CHO-INH.
  • FIG. 74A is a graph showing bacterial burdens in the lung throughout the course of infection.
  • FIG. 74B is a graph showing the effect of the treatments on M tuberculosis burden in the lung.
  • FIG. 74C is a graph showing the effect of the treatments on M tuberculosis burden in the liver.
  • FIG. 74D is a graph showing the effect of the treatments on M tuberculosis burden in the spleen. All were determined by assaying bacterial CFU three days after the final treatment.
  • mice treated with MSN-CHO-INH maintained their weights indicating that the nanoparticle was well tolerated by the mice ( Figure 75A).
  • PEI polyethylenimine coated mesoporous silica nanoparticle carrying anti-TB drug rifampicin
  • mice treated with MSN-CHO-INH lost 23% of their body weight after a single treatment dose and had to be euthanized (Figure 75B).
  • This result shows that efficacy of a nanoparticle in killing M. tuberculosis macrophages and non-toxicity in vitro does not ensure that the nanoparticle is safe and effective in vivo.
  • FIG. 75 A is a chart showing weights of infected mice that were sham-treated, treated with the anti-TB drug INH administered as a free drug or delivered by MSN-CHO-INH.
  • FIG. 75B is a chart showing weights of infected mice that were sham-treated, treated with the anti- TB drug rifampin (RIF) as free drug or delivered by MSN-PEI-RIF. All were monitored over the course of treatment.
  • RIF anti- TB drug rifampin
  • MSN-CHO-INH Since the efficacy of MSN-CHO-INH in the first mouse experiment was greater than an equivalent amount of free INH, in the second mouse experiment, we tested the efficacy of MSN-CHO-INH against an equivalent amount and twice the equivalent amount of free INH. With 9.01% drug release capacity, 2 mg of MSN-CHO-INH has 180 ⁇ g of INH. Mice were infected with 500 CFU ofM tuberculosis Erdman strain by aerosol. One day later, two mice were euthanized to determine the initial bacterial burden in the lung. Two weeks later, three additional mice were euthanized to determine bacterial growth over the previous two weeks.
  • mice were then either sham treated or treated with 180 ⁇ g of free INH; 360 ⁇ g of free INH; or 2 mg of MSN-CHO-INH (180 ⁇ g releasable INH) by tail vein injection every other day, 3 days a week (Monday, Wednesday and Friday) for three weeks. Mice were euthanized three days after the last treatment. As observed in the first mouse experiment, mice in the sham control group had the worst lung pathology and many lung tubercle lesions. Mice in the INH treated groups had less lung pathology than mice in the sham treated group, and mice in the MSN-CHO-INH treated group had the least pathology.
  • INH at 180 ⁇ g per dose over three weeks was reduced by 0.9 logs in the lung, 2.1 logs in the liver, and 3.4 logs in the spleen (Table 23).
  • MSN-CHO-INH killed 0.45 logs more M tuberculosis in the lung, 0.36 logs more in the liver, and 0.34 logs more in the spleen than an equivalent amount of free INH (180 ⁇ g).
  • MSN-CHO-INH was more efficacious than twice the equivalent dose of INH (360 ⁇ g) in liver and lung, but not in spleen, where the effectiveness of MSN-CHO-INH was comparable to twice the equivalent dose of free INH (Table 23 & Figure 76).
  • FIG. 76A is a graph showing in sham-treated mice, bacterial burden in the lung was assayed on the first day after infection (Day 1) and bacterial burden in all organs was assayed two weeks later at the start of the treatment period (Day 14) and three weeks and 3 days later (Day 38), 3 days after the conclusion of the three week treatment period.
  • FIG. 76B is a graph showing the effect of various treatments on M tuberculosis burden in the lung.
  • FIG. 76C is a graph showing the effect of various treatments on M tuberculosis burden in the liver.
  • FIG. 76D is a graph showing the effect of various treatments on M tuberculosis burden in the spleen. All were determined in all treatment groups at the end of the experiment (Day 38) by assaying bacterial CFU.
  • SMSN-CHO-INH nanoparticles that had been shown to be effective in killing M tuberculosis in our macrophage assay in vitro.
  • SMSN-CHO-INH delivered by two different routes, namely by the intravenous or subcutaneous route.
  • This batch of 50 nm SMSN-CHO-INH had 10.6% (wt/wt) releasable INH under acidic pH conditions.
  • Mice were infected with 500 CFU ofM tuberculosis Erdman strain by aerosol. One day later, two mice were euthanized to determine the initial number of bacteria in the lung. Two weeks later, three additional mice were euthanized to determine bacterial growth over the previous two week period.
  • mice per group were then sham treated or treated with one of the three doses of free INH (15, 106, 212 ⁇ g) by tail vein injection; 1 mg of SMSN-CHO-INH (106 ⁇ g INH that is releasable) by tail vein injection; or 1 mg of SMSN-CHO-INH by subcutaneous injection every other day, 3 days a week (Monday, Wednesday and Friday) for two weeks. Mice were euthanized three days after the last treatment.
  • mice treated with 1 mg SMSN-CHO-INH either by intravenous or subcutaneous injection had fewer surface lesions than those of mice treated with an equivalent amount (106 ⁇ g) or twice the equivalent amount of INH (212 ⁇ g) by intravenous injection.
  • SMSN-CHO-INH delivered either by intravenous or subcutaneous injection killed more M tuberculosis than twice the equivalent amount (212 ⁇ g) of free INH in the lung, liver and spleen (Table 24 and Figure 78).
  • FIG. 77 is a graph showing lung tubercle lesion counts.
  • *i.v. indicates drug delivered by tail vein injection.
  • ⁇ SQ indicates drug delivered by subcutaneous injection.
  • SMSN-CHO-INH reduced bacterial burden in organs of M. tuberculosis infected mice to a much greater extent than an equivalent amount or twice the equivalent amount of free INH.
  • Mice were infected with M. tuberculosis and either sham treated or treated with one of three different doses of INH or with SMSN-CHO-FNH by either intravenous or subcutaneous (SQ) injection.
  • FIG. 78 A shows bacterial burdens in the lung throughout the course of infection.
  • FIG. 78B is a graph showing the effect of the various treatments on M tuberculosis burden in lung.
  • FIG. 78C is a graph showing the effect of the various treatments on M tuberculosis burden in liver.
  • mice treated with 50 nm SMSN-CHO-INH either by tail vein injection (Group G) or by subcutaneous injection (Group J) maintained stable body weight with a net gain of about 2% at the end of the two weeks of treatment ( Figure 79A). This result indicated that 50 nm SMSN-CHO-INH is well tolerated by mice and by both the i.v. and SQ routes of administration.
  • FIG. 79A is a chart showing weights of infected mice that were sham-treated or treated with the anti-TB drug INH as free drug or delivered by SMSN-CHO-INH.
  • FIG. 79B is a chart showing weights of infected mice that were sham-treated or treated with the anti-TB drug RIF as free drug or delivered by MSN-Z-RIF. All were monitored over the course of treatment.
  • mice were aerosol infected with 250 CFU ofM tuberculosis as described. Two weeks later, mice were sham treated or treated with one of the three doses of INH (164, 328, and 656 ⁇ g) or with 2 mg of MSN-CHO-INH (with 164 ⁇ g of releasable INH) every other day, 3 days a week (Monday, Wednesday and Friday) for a total of two weeks.
  • the three doses of INH were equal to lx, 2x, and 4x the amount of the releasable INH from 2 mg of MSN-CHO-INH by acidic pH (Table 26).
  • MSN-CHO-INH reduced bacterial burden by 1.3 logs to a level equivalent to that of 4x free INH.
  • both the MSN-CHO-INH and the 4x INH lowered bacterial CFU to a level below the experimental limit of detection.
  • MSN-CHO-INH reduces bacterial burden in organs ofM tuberculosis infected mice. Mice were infected with M. tuberculosis and either sham treated or treated with one of three different doses of INH, as indicated, or with 2 mg of MSN-CHO-INH (164 ⁇ g of acid releasable INH) by tail vein injection.
  • FIG. 80A is a graph showing bacterial burden in the lung throughout the course of infection.
  • FIG. 80B is a graph showing the effect of the treatments on M tuberculosis burden in lung.
  • FIG. 80C is a graph showing the effect of the treatments on M tuberculosis burden in liver.
  • FIG. 80D is a graph showing the effect of the treatments on M tuberculosis burden in spleen. All were determined by assaying bacterial CFU. The limit of detection is indicated by the dashed line.
  • Antibody APP2 was used to target nanoparticles to the lung, as these antibodies are known to bind to antigen on lung endothelial cells and thereafter to enter lung tissue. Particles of two sizes (50, 100 nm diameter) were made with and without the targeting antibody and delivered intravenously into mice. Mice were sacrificed and the organs were homogenized for CFU plating and ICP-OES elemental analysis. To analyze the efficacy of the anti-APP2, the organs were analyzed by ICP-OES for Si content to provide an organ distribution. In both cases, the SMSNs and MSNs with anti-APP2 targeting resulted in higher silica content in the lung than SMSNs and MSNs that were not targeted (Figure 81).
  • FIG. 81 A is a graph showing that after 24 hours, INH-CHO-SMSNs lacking a targeting molecule are located primarily in the spleen, followed by the lung.
  • FIG. 8 IB is a graph showing that targeted
  • nanoparticles APP2-INH-CHO- SMSNs show much greater localization to the lung.
  • FIG. 81C show that after 2 weeks of dosing, non-targeted INH-CHO-SMSNs are primarily localized in the liver with negligible amounts in the lung.
  • FIG. 8 ID shows that targeted APP2-INH-CHO- SMSNs show greatly increased localization in the lung compared with the non-targeted nanoparticles.
  • mice were given DyLight 680 near-infrared labeled MSN (NIR-NP) or
  • NIR-NP coated with anti-aminopeptidase 2 antibody by tail vein injection. Two days later, mice were euthanized and their organs were collected for ex vivo imaging using the IVIS Imaging System. NIR-NP preferentially distributed to liver over lungs. APP2 antibody coating changes the dynamic of bio-distribution and targets the nanoparticle (Anti-APP2-NIR-NP) preferentially to the lung ( Figure 82).
  • FIG. 82A shows animal organs (liver, spleen, heart, lungs, and kidneys, as indicated) photographed under normal light.
  • FIG. 82B shows animal organs (liver, spleen, heart, lungs, and kidneys, as indicated) imaged for near infra-red emission using the IVIS Imaging System.
  • mice had the fewest lung tubercles of all groups ( Figure 77). They also had the lowest burden ofM tuberculosis in their liver and spleen, although the M. tuberculosis burden in the lung was comparable to that of mice treated with the same nanoparticle without anti-APP2 targeting (Table 24).
  • Nanoparticles (NPs) with INH of two sizes were analyzed, and SMSNs with targeting (SMSN-INH-APP2) were also analyzed.
  • the silica distribution was the fraction of silica measured from the ICP-OES analysis divided by the total quantity of silica injected during each experiment. In the short-term studies, one dose of 2 mg nanoparticles was injected into each mouse. In the long term, study six doses of up to 12 mg nanoparticles were injected. Organs were digested and analyzed for Si content via ICP-OES elemental analysis. Figure 83 shows the distribution of nanoparticles can vary, depending on surface chemistry, size, and time.
  • SMSN-INH the larger NPs
  • SMSN-INH the smaller NPs
  • a much greater amount ( ⁇ 4-fold greater) of SMSNs were found in the lung than MSNs.
  • the targeted SMSNs (SMSN-INH- APP2) like the non-targeted SMSNs also preferentially localized to the spleen followed by the lung, but a much greater amount ( ⁇ 5-fold greater) localized to the lung than in the case of the non-targeted NPs.
  • the total quantity of silica recovered in the organs after 24 hours was 15% (0.3 mg of 2 mg) of the total injected silica; for SMSN-INH, the total quantity of silica recovered in the organs after 24 hours was 5% of the total injected silica (0.1 of 2 mg); for SMSN-INH-APP2, the total quantity of silica recovered in the organs after 24 hours was 9.1% of the total injected silica (0.182 of 2 mg).
  • SMSNs showed a similar localization pattern as in the short-term study, but the amounts present were much lower, indicating that most of the silica had been cleared by that time point.
  • the SMSNs showed a different localization pattern with the liver now the primary location of silica. With targeting, the SMSNs also showed a different localization pattern, with the liver now the primary location of the silica. Again, a much higher amount ( ⁇ 4-fold greater) of silica was present in the lung with targeting than without targeting.
  • FIG. 83 is a graph showing silica nanoparticle distribution over a period of 24 hours (1 dose). The majority of particles are distributed to the liver, spleen, and lung (blue bars). Three days after a full regimen (2 weeks, 6 doses total), particles remain in the liver, spleen and lung (pink bars). The majority of the silica has been excreted, likely through the urine or feces.
  • Benefits of the MSN-CHO-INH controlled drug release nanoparticle technology include a) it is more efficacious than an equivalent amount of free drug for treating tuberculosis; b) it preferentially targets macrophages, the host cells for M. tuberculosis and many other intracellular pathogens, thereby increasing the therapeutic index; c) it provides for controlled release of the drug intracellularly in the host cells for M. tuberculosis, thereby avoiding off-target effects and premature metabolism of the drug; d) it provides improved treatment of both active pulmonary and extrapulmonary tuberculosis (TB) and other mycobacterial diseases (e.g.
  • Mycobacterium kansasii infection Mycobacterium intracellular infection, disseminated BCG, etc.
  • it can be used to treat latent TB infection (LTBI), which also requires a prolonged treatment regimen, more rapidly and effectively
  • LTBI latent TB infection
  • NPs can be administered by a variety of routes including intravenously, subcutaneously, intramuscularly, orally, by inhalation, etc; and g) the MSNs are biodegraded and do not accumulate after administration.
  • One benefit of the lung targeting technology is that it allows for improved treatment of lung diseases while avoiding off-target effects.
  • This lung targeting technology has broad applicability for nanoparticle delivery of numerous drugs for the treatment of lung diseases of all kinds including infectious diseases and other types of diseases that affect the lungs.
  • This technology also comprises a method for attaching a prodrug to a
  • nanoparticle in such a way that the drug is released as an active drug in a controlled fashion under low pH conditions.
  • This technology has broad applicability for nanoparticle delivery of numerous drugs for treatment of infectious diseases as well as other types of diseases including cancers, heart diseases, liver diseases, renal diseases, neurological diseases arthritis, etc.
  • LTBI M. tuberculosis
  • NP nanoparticle delivery platforms provide a more effective, less toxic, and shorter treatment for TB. Because host mononuclear phagocytes internalize particles more efficiently than other cells, intravenously (i.v.) injected NPs, or NPs delivered by other routes of administration, are preferentially taken up by macrophages of the mononuclear phagocyte (reticuloendothelial) system (MPS) and accumulate in liver, spleen, and lung.
  • MXF moxifloxacin
  • NPs are ideally suited to treat Mtb, which infects macrophages in these organs.
  • Targeting antibiotic-loaded NPs to infected organs and tissues, selectively delivering the antibiotics into macrophages and releasing them at high concentrations intracellularly greatly increases their therapeutic index by achieving higher drug concentrations locally where Mtb replicate while limiting systemic toxicities.
  • by controlled release of the drug only after the NPs have been ingested protects the drug from hepatic metabolism and drug clearance before the drug has had the opportunity to attack target pathogens.
  • Increasing drug concentrations at the site of infection by orders of magnitude allows for a much shorter duration of therapy.
  • hepatocytes will not impact hepatic cytochrome P450 metabolism of other drugs.
  • MSNs offer many advantages over previous delivery vehicles (e.g. liposomes, solid lipid particles, alginates) for TB drugs because of their stability, uniformity, inherent lack of toxicity, high internal surface area for drug binding, and versatility in incorporating additional design features. Because of their ultra-high internal surface area (-1000 m 2 /g), MSNs can encapsulate exceptionally high concentrations of different types of cargos. Loading capacities as high as 50 weight percent have been achieved, exceeding by several orders of magnitude that of conventional liposomal nanocarriers. MSNs can be synthesized with a variety of different internal and surface design features, including those that allow for specific targeting to infected host organs and tissues and those that enableakily controlled release of cargo under specific
  • concentrations of combined cargos with disparate physicochemical properties to be simultaneously delivered to overcome multidrug resistance achieve synergistic effects and/or enable combined therapy and diagnostics (theranostics).
  • MSN are degraded in the body over several days and the degradation products are excreted.
  • the antibiotics are distributed throughout the body rather than being targeted to a specific organ or tissue.
  • the major site of infection for TB for many other mycobacterial diseases, for many other infectious diseases, and for many non-infectious diseases, higher drug concentrations can be delivered at the site of disease, improving therapeutic efficacy while decreasing off-target effects.

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Abstract

Le domaine des modes de réalisation revendiqués de l'invention concerne des compositions comprenant une pluralité de particules de silice mésoporeuse définissant des pores et des méthodes d'utilisation de telles compositions pour le traitement de maladies infectieuses provoquées par des pathogènes intracellulaires dans des cellules hôtes.
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CN108743958A (zh) * 2018-05-31 2018-11-06 四川大学 药物分子与阀门分子联合作用的gsh响应型介孔硅纳米载药颗粒及其制备方法
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JP2023516111A (ja) * 2020-10-27 2023-04-18 エルシダ オンコロジー， インコーポレイテッド ナノ粒子を官能化する方法
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