WO2012009448A2 - Nanoparticules de silice mésoporeuses revêtues d'un polymère cationique et leurs utilisations - Google Patents

Nanoparticules de silice mésoporeuses revêtues d'un polymère cationique et leurs utilisations Download PDF

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WO2012009448A2
WO2012009448A2 PCT/US2011/043874 US2011043874W WO2012009448A2 WO 2012009448 A2 WO2012009448 A2 WO 2012009448A2 US 2011043874 W US2011043874 W US 2011043874W WO 2012009448 A2 WO2012009448 A2 WO 2012009448A2
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msnp
pei
submicron structure
sirna
cells
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WO2012009448A3 (fr
Inventor
Jeffrey I. Zink
Andre E. Nel
Tian Xia
Zhaoxia Ji
Huan MENG
Zongxi Li
Monty Liong
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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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Priority to US13/428,830 priority Critical patent/US20120207795A1/en
Publication of WO2012009448A3 publication Critical patent/WO2012009448A3/fr
Anticipated expiration legal-status Critical
Priority to US15/698,486 priority patent/US10343903B2/en
Priority to US16/427,253 priority patent/US20190382265A1/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • 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
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites

Definitions

  • the current invention relates to submicron structures having a silica body defining a plurality of pores and an outer surface between pore openings of said plurality of pores and a cationic polymer on the surface of said silica body.
  • Such submicron structures may be combined with oligonucleotides and therapeutic compounds for drug delivery, transfection, and cancer therapy.
  • MSNP mesoporous silica nanoparticles
  • silica materials are known to be safe, biodegradable and potentially biocompatible (Borm et al., Toxicol. Sci., vol. 90, pp. 23-32, 2006; Finnie et al., J. Sol-Gel. Sci. Techn., vol. 49, pp. 12-18, 2009).
  • This drug transport system is suitable for the delivery of anticancer drugs, including camptothecin, paclitaxel, and doxorubicin (Kim et al., Angew. Chem., Int. Ed, vol. 47, pp.
  • RNA interference describes natural processes that lead to gene silencing by siRNA (Moazed et al., Nature, vol. 457, pp. 413-420, 2009).
  • siRNA has been widely used as an experimental tool that is now also becoming the focus of the pharmaceutical industry (Blow et al., Nature, vol. 450, pp. 1117-1120, 2007).
  • Currently there are a number of clinical trials underway that include the use of siRNAs to treat various disease processes (Davis et al, Mol. Pharm., vol. 6, pp. 659-668, 2009; Judge et al, Mol. Ther., vol. 13, pp. 494-505, 2006).
  • Pgp P-glycoprotein
  • MDR-1 multiple drug resistance protein 1
  • Embodiments of the invention include a submicron structure having a silica body defining a plurality of pores and an outer surface between pore openings of said plurality of pores, and a cationic polymer on the surface of said silica body. Said submicron structure has a maximum dimension less than one micron.
  • the submicron structure also includes a cationic therapeutic compound. The cationic therapeutic compound may be, for example, in the interior or in the pores of the submicron structure.
  • the submicron structure also includes an oligonucleotide electrostatically bound to the cationic polymer. Some embodiments include both a cationic therapeutic compound and an oligonucleotide.
  • the oligonucleotide is an siR A that reduces translation of a protein that causes resistance in a cell.
  • the siRNA reduces translation of a protein that causes resistance to the therapeutic compound in the cell.
  • the siRNA reduces translation of p-glycoprotein.
  • the therapeutic compound is doxirubicin.
  • the cationic polymer is polyethyleneimine.
  • Some embodiments include an oligonucleotide electrostatically bound to the cationic polymer.
  • the oligonucleotide may be DNA or RNA.
  • RNA is may be a small inhibiting RNA (siRNA).
  • Some embodiments include a therapeutic compound within the silica body or pores of the silica body.
  • the therapeutic compound may be hydrophobic, neutral (i.e.
  • the silica body is mesoporous.
  • the pores are substantially cylindrical and have an ensemble average diameter between about lnm and about lOnm.
  • the silica body is substantially spherical and has a diameter between about 50nm and about lOOOnm. In some embodiments, the substantially spherical silica body has a diameter between about 100 nm and about 500 nm.
  • the submicron structure further includes a light-emitting compound, peptide, protein, oligonucleotide, sugar, oligosaccharide, or polysaccharide covalently bonded to the surface of the silica body.
  • a light emitting compound covalently bonded to the surface of the silica body.
  • the submicron structure further includes a core structure within the silica body.
  • the core structure is a superparamagnetic nanocrystal, silver nanocrystal or gold nanocrystal.
  • the core structure is a superparamagnetic iron oxide nanocrystal
  • Embodiments of the invention include pharmaceutical compositions having a submicron structure according to the invention and a pharmaceutically acceptable carrier or excipient.
  • Embodiments of the invention include use of the submicron structures according to the invention for the manufacture of a medicament or pharmaceutical composition for the treatment of a disease or disorder.
  • Embodiments of the invention include therapeutic methods have the step of administering an effective amount of a submicron structure according to the invention to a subject in need of treatment.
  • Embodiments include use of the submicron structures according to the invention for the treatment of a disease or disorder by administering the submicron structure to a subject in need of treatment.
  • Embodiments of the invention include methods of transfecting a cell by administering a submicron structure according to the invention having an oligonucleotide.
  • Embodiments of the invention include the use of a submicron structure according to the invention including an oligonucleotide to transfect a cell.
  • Figure 1 shows transmission electron microspopy (TEM) of the MSNP and cell viability detection by the MTS assay.
  • Figure 1 A shows TEM image shows the particle size and the ordered pore structure.
  • Figure IB shows cell viability after addition of appropriately dispersed MSNP exhibiting a range of surface modifications to pancreatic cancer cell lines at doses ranging from 12.5-50 ⁇ g/ml for 16 hrs, cells were incubated with the MTS reagent for 30 min and the absorbance was measured at 490 nm. All the MTS values were normalized according to the value of the control (no particle exposure) - this was regarded as 100% cell viability.
  • the IC 50 values of MSNP-PEI-25 KD in PANC-1 and BxPC3 cells were 37 ⁇ and 46 ⁇ g/ml, respectively. The results were reproduced 3 times.
  • Figure 2 shows cellular uptake of FITC-labeled MSNP in PANC-1 cells.
  • MSNP were labeled with FITC as described in Example 1.
  • Figure 2 A shows a representative histogram showing the shift in fluorescence intensity in PANC-1 cells treated with 25 ⁇ g/ml FITC-MSNP that contain different surface modifications (left panel). The fold-increase in MFI after 3 hr was calculated and used to generate the graph.
  • RITC-labeled MSNP-Phos served as a control particle to show that coating with PEI leads to enhanced uptake in the same particle type in the same cell (right panel).
  • Figure 2B shows confocal microscopy used to study the cellular uptake of FITC- MSNP in PANC-1 cells.
  • Figure 3 shows cellular uptake of FITC-labeled MSNP in BxPC3 cells.
  • BxPC3 cells were exposed to FITC-labeled MSNP and flow cytometry and confocal microscopy were conducted as in Figure 2.
  • Figure 3 A shows a representative histogram showing the shift in fluorescence intensity (left panel). The fold-increase in MFI after 3 hr was calculated and used to generate the graph.
  • RITC-labeled MSNP-Phos served as a control particle to show that coating with PEI leads to enhanced uptake in the same particle type in the same cell (right panel).
  • Figure 3B shows confocal microscopy used to study the cellular uptake of FITC-MSNP.
  • Cells were exposed to 25 ⁇ g/ml FITC-labeled particles for 3 hr. After cell membrane staining with 5 ⁇ g/ml red fluorescent wheat germ agglutinin (WGA), cells were visualized using a Confocal 1P/FCS Inverted microscope. Data are representative of 3 separate experiments. *p ⁇ 0.0l compared with control.
  • Figure 4 shows Cell viability detection by the MTS assay.
  • FIG. 5 shows Gel retardation and DNase I protection assays. Agarose gel electrophoresis of PEI-MSNP/plasmid DNA (pEGFP) ( Figure 5A) and PEI-MSNP/siRNA
  • phosphonate-coated MSNP was used as a control.
  • M MW marker.
  • Figure 5C shows DNase I protection assay.
  • M DNA marker.
  • ⁇ naked plasmid DNA (pEGFP), as negative control.
  • Lane 1 pDNA/PEI-1.2 KD complex.
  • Lane 2 pDNA/PEI 25 KD complex.
  • Lane 3 naked pDNA treated with DNase I, positive control.
  • Lane 4 pDNA/PEI 1.2 KD complex treated with DNase I before pDNA was released by 1% SDS.
  • Lane 5 pDNA/PEI 25 KD complex treated with DNase I before pDNA was released by 1% SDS.
  • Figure 6 shows GFP knockdown by siRNA in stable transfected GFP-HEPA cells. HEPA-1 cells with stable GFP expression were used for siRNA knockdown assays. MSNP coated with different size PEI polymers were used to transfect GFP-specific or scrambled siRNA and the results compared with Lipofectamine 2000 as transfection agent.
  • Figure 6A shows GFP knockdown assessed by flow cytometry in which GFP MFI was normalized to the value of control untransduced cells (100%).
  • Figure 6B shows confocal pictures showing GFP knockdown in GFP-HEPA cells. TEX 615-labeled siRNA was used to show the cellular localization of the nucleic acid bound particles (red dots). "X" represents scrambled siRNA. The experiment was reproduced 3 times.
  • FIG. 7 shows GFP plasmid DNA transfection into HEPA-1 cells.
  • HEPA-1 cells were used for GFP plasmid DNA transfection.
  • MSNP coated with different size PEI polymers were used to transfect GFP plasmid DNA and the results were compared with
  • FIG. 7A shows a representative histogram showing the shift in green fluorescence intensity in HEPA-1 cells after transfection with Lipofectamine 2000 or MSNP-PEI-10 KD.
  • Figure 7B shows confocal pictures showing GFP expression in transfected HEPA-1 cells. This demonstrates differences in the transfection efficiency as judged by fluorescent intensity and proportion of cells in the population showing GFP expression. The experiment was reproduced 3 times.
  • Figure 8 shows Drug delivery to PANC-1 and BxPC3 cells using PEI-MSNP.
  • MTS assay was conducted for the paclitaxel-loaded MSNP delivered to these cells at doses of 3- 50 ⁇ g/ml over a 48 hrs period in PANC-1 ( Figure 8A) and BxPC3 ( Figure 8B) cells.
  • the controls were cells treated with particles only and cells treated with paclitaxel suspended in culture medium with and without the addition of DMSO carrier. The experiment was reproduced 2 times.
  • FIG. 9 shows MSNP size distribution in aqueous solutions.
  • Dynamic light scattering (DLS) for MSNP exhibiting different surface modifications was performed in water, DMEM plus 10% FCS, BEGM or BEGM plus 2 mg/ml BSA. The presence of serum and BSA in the cell culture media improves MSNP dispersity.
  • DLS Dynamic light scattering
  • Figure 10 shows assessment of cell viability and mitochondrial membrane potential (MMP) in RAW 264.7 and BEAS-2B cells.
  • Figure 10A shows cell viability following treatment with MSNP displaying different surface modifications, wise determined by the MTS assay as described in Figure 1.
  • Cells were exposed to MSNP at doses of 12.5-50 ⁇ g/ml for 16 hrs. All the MTS values were normalized as outlined in Figure 1.
  • the IC 50 values for image NP- PEI-25 D in RAW 264.7 and BEAS-2B cells are 40.6 ⁇ and 9.7 ⁇ , respectively.
  • Figure 10B shows cell death and mitochondrial depolarization after treatment with MSNP- phosphonate and MSNP-PEI backspace-25kD was determined using PI and JC-1, respectively.
  • Figure 11 shows effect on cell viability after conversion of primary amines to
  • FIG. 11 A shows cell viability comparing non-modified with succinic anhydride treated particles in RAW 264.7 cells using MTS assay.
  • Figure 11B shows the conversion confirmed using fluorescamine, which yields green fluorescence when complexed to the primary NH 2 groups. The decline in fluorescence intensity was followed in a fluorometer.
  • Figure 12 shows determination of the stability of PEI coating on the MSNP surface. Rhodamine-B labeled PEI was used to coat the surface of FITC-labeled MSNP and the dual-labeled particles were added to RAW 264.7 cells prior to the performance of confocal microscopy. The composite overlay confirms that the polymer and the particle co-localize at the same intracellular site at 3 and 6 hrs.
  • Figure 13 shows cellular uptake of FITC-labeled MSNP in RAW 264.7 cells.
  • FIG. 13A shows a representative histogram showing the shift in fluorescence intensity in RAW 264.7 cells treated with 25 ⁇ g/ml FITC-MSNP exhibiting different surface modifications for 3 hrs (left panel). The fold-increase in MFI was calculated and used to generate the graph. RITC-labeled MSNP was used as a control as discussed in Figure 2 (right panel).
  • Figure 13B shows confocal microscopy to study the cellular uptake of FITC-MSNP in RAW 264.7 cells. Cells were exposed to 25 ⁇ FITC- labeled particles for 3 hr. After the cell membrane was stained with 5 ⁇ g/ml red fluorescent wheat germ agglutinin (WGA), cells were visualized in a Confocal 1P/FCS Inverted
  • Figure 14 shows Cell viability detection by the MTS assay in RAW 264.7
  • Figure 16 shows quantification of paclitaxel (Pac) loading capacity in MSNP.
  • MSNP with different surface modifications were loaded with paclitaxel.
  • Methanol was used for complete release of the drug from washed particles and the amount of paclitaxel in the supernatant was determined by UV absorbance at 230 nm.
  • Figure 17 shows animal weight and histology of major organs.
  • Figure 17A shows animal weight was monitored after particle injections.
  • Figure 17B shows histology of liver, kidney, spleen was performed by UCLA Division of Laboratory Animal Medicine (DLAM) diagnostic laboratory services. The sections were stained with hematoxylin-eosin and examined by light microscopy.
  • Figure 18 shows physicochemical characterization of PEI coated MSNP.
  • FIG. 18A shows TEM images of phosphonate-MSNP before and after coating with the 10 kD PEI polymer. The arrows indicate that the polymer decorates the MSNP surface but leaves the porous interior accessible to drug loading.
  • Figure 18B shows particle size and zeta potential in pure water, after stabilization with 1 mg/niL BSA in water, or in DMEM cell culture medium were measured. All of the size and zeta potential data do no significantly unchanged when the MSNP were loaded with Dox and siRNA (Table 3).
  • Figure 19 shows effective Pgp siRNA delivery and gene knockdown in KB- VI cells.
  • Figure 19A shows agarose gel electrophoresis of PEI-coated MSNP to which Pgp siRNA was complexed at various nanoparticle to nucleic acid (N/P) ratios. M is molecular weight marker.
  • the ⁇ lane contains Pgp siRNA only. Dox loading did not change the N/P ratio or the electrophoretic mobility. The results indicate that all siRNA was bound when the N/P ratio ratio >16 (PEI 1.8kD), >16 (PEI 10 kD), and >8 (PEI 25 kD).
  • Figure 19B shows confocal microscopy to demonstrate Texas red-labeled siRNA uptake in association with FITC-labeled PEI coated MSNP.
  • the cell membrane and nucleus were stained by WGA 633 and Hoechst 33342, respectively.
  • the panels on the right show merging of the images to show Pgp siRNA co-localization with FITC-MSNP.
  • Figure 19C shows quantitative comparison of labeled Pgp siRNA uptake by measuring fluorescent intensity of Texas red in various PEI groups, using Imaging J software. *p ⁇ 0.05.
  • Figure 19D shows detection of Pgp knockdown by siRNA-PEI- MSNP using western blotting. Lipofectamine 2000 was used as positive control. The relevant Pgp expression was calculated by the signal intensity of the protein bands.
  • "X" stands for cells treated by scrambled siRNA-PEI-MSNP.
  • Figure 20 shows that phosphonate-MSNP effectively binds Dox via a proton- sensitive mechanism.
  • Figure 20A shows modeling studies using positively and negatively charged MSNP under abiotic conditions. Loading yield of Dox in MSNP with various surface modifications. A photograph of the Dox-loaded MSNP (20 mg/ml) containing various surface modifications were taken. Consistent with loading yield, the phosphonate-MSNP was more intensively stained (red) than other particle types.
  • Figure 20B shows loading yield
  • FIG. 20C shows time-dependent release profile of Dox from drug loaded phosphonate MSNP in phenol red free DMEM acidified to pH 5.0. The effect of PBS or treatment with PBS containing 10% ethanol is shown for comparison. *P ⁇ 0.05.
  • I t is the fluorescent intensity of released Dox at certain time point; Io as the total Dox fluorescence signal intensity that can be recovered by repeated acid washing (considered as 100% release). The release percentage equals (I t Io) x 100%.
  • Figure 20D shows Dox release from phosphonate- MSNP coated with the 10 kD PEI polymer under similar acidification conditions; this demonstrates that the polymer does not interfere in drug release.
  • Figure 20E shows Confocal microscopy showing FITC-labeled MSNP uptake into the LAMP-1 + compartment in KB- VI cells. The yellow spots in the merged image show the co-localization. Calculation of co- localization ratio by Imaging J software indicates >55% co-localization of the green-labeled particles with the red-labeled lysosomes.
  • Figure 20F shows confocal microscopy showing Dox release from the MSNP to the nucleus in KB-V1 cells 72 hrs after the introduction the particles. The bottom panel shows that the lysosomal pH neutralizer, NH 4 C1, interferes in drug release.
  • Figure 21 shows simultaneous delivery of Dox and Pgp siRNA to the nucleus leads to a synergistic increase in cellular and nuclear Dox levels in KB-V1 cells.
  • Figure 21A shows quantitative comparison of Dox levels using a fluorescent readout of cellular drug levels 72 hrs after introduction of treatment, using 2 ⁇ g/ml free Dox or the equivalent amount of drug loaded into MSNP before or after PEI coating or PEI coating followed by the attachment of Pgp siRNA.
  • Figure 2 IB shows confocal images showing drug uptake in KB- VI cells that treated by 5 ⁇ g/ml free Dox or the equivalent amount of drug loaded into various MSNPs for 72 hrs.
  • Dox- MSNP Dox- MSNP
  • PEI-Dox-MSNP significantly enhanced particle uptake compared to the unmodified MSNP.
  • Pgp siRNA was added to this platform.
  • Pgp knockdown is likely effective at maintaining the Dox that is released from the particles long enough to allow the drug to find its way to the nucleus.
  • the cell membrane was stained by Alexa 633 -conjugated WGA (cyan color). Dox staining is in red.
  • Figure 21C shows quantitative analysis of the nuclear fluorescence signal in KB- VI nuclei was performed by the use of Image J software.
  • Figure 22 shows comparison of the cytotoxic effects of different delivery modalities of Dox in KB-Vl cells.
  • Figure 22A shows MTS cell viability assay showing that MSNP delivery of Dox concomitant with Pgp siRNA is capable of improving the induction of cytotoxicity by free Dox or Dox delivered by PEI-coated MSNP not attached to siRNA. The broken line is the cell killing curve of free Dox in parental cell line (KB-31, Dox sensitive).
  • Figure 22B shows annexin V- SYTOX staining showing enhanced apoptosis and cell death by siRNA-PEI-Dox compared to the other Dox modalities mentioned in Figure 22 A. The flow cytometry data was further confirmed by TUNEL staining assay (Figure 29).
  • Figure 23 shows identification of Dox sensitivity in KB-31 and KB-V1 cells.
  • Figure 23 A shows cytotoxicity profiles of Dox in KB-31 (parental line) and KB-Vl cells (resistant cell line).
  • Figure 23B shows immunoblotting showing Pgp expression in KB-31 and KB-Vl cell.
  • Figure 24 shows agarose gel electrophoresis of PEI 10 kD-coated MSNP to which Pgp siRNA was complexed at various nanoparticle to nucleic acid (N/P) ratios.
  • the ⁇ lane contains Pgp siRNA only.
  • Figure 25 shows Pgp expression was significantly knocked down (-80%) by siRNA-PEI 10 kD-Dox-MSNP treatment at the dose of 10 ⁇ g/ml for 72 hrs. Knockdown of Pgp expression by siRNA is not influenced by Dox loading.
  • Figure 26 shows assessment of PEI-MSNP safety in KB-Vl cells.
  • Figure 26A shows MTS assay assessment of the viability of the cells incubated with the polymer-coated MSNP.
  • Figure 26B shows MTS assay assessment of cell viability in response to 100 ⁇ g/ml MSNP coated with the 10 kD polymer. The cell viability began to decrease at 36 hrs time-point (* ⁇ 0.05)
  • Figure 27 shows loading and release profile of Hoechst 33342 loaded MSNP with various surface modifications.
  • Figure 27A shows loading yield of Hoechst 33342 in MSNP with different surface modifications.
  • H + induced Hoechst 33342 release is significantly higher than ethanol induced release (* ⁇ 0.05).
  • Figure 28 shows that the Dox release profile remains same with or without siRNA binding on PEI 10 kD-MSNP.
  • Figure 29 shows a TUNEL detection kit used according to the manufacturer's instructions to study Dox-induced apoptosis. Percentage of TUNEL positive cell showing enhanced apoptosis by siRNA-PEI-Dox compared to the other Dox modalities. The result shows the same trend of flow cytometry data ( Figure 22B)
  • Figure 30 shows cell viability analyzed after longer exposure periods (4 days) because Pgp knockdown by siRNA may need a certain amount of time, but did not observe a significant improvement in cytotoxicity.
  • Figure 31 shows that co-administration of free Dox and Tariquidar (50 nM) partially restores drug sensitivity in KB- VI cells for 72 hrs. This combination significantly improved cell killing capability in KB-V1 cells, but was not able to completely restore Dox sensitivity to the level seen in KB-31 cells.
  • Figure 32 shows loading and release profile of CPT loaded MSNP with various surface modifications.
  • Figure 32A shows loading yield of CPT in modified MSNP. As shown in Figure 32A, MSNP with different surface modifications were compared for their loading capacity to CPT. The loading yield of MSNP varied from 3.8% (w/w) to 6.2% (w/w) when different surface modifications were used. The amounts of CPT stored within the surface functionalized particles were similar, although storing lesser amounts than the silanol surface.
  • Figure 32B shows the release profile of CPT loaded into phosphonate-MSNP in response to acidification or induction of ethanol into the wash medium. PBS treatment was set as control. The ethanol-induced CPT release is significantly higher than acid-induced release.
  • hydrophilic cargo e.g. Dox
  • hydrophobic cargo e.g. CPT
  • Loading capacity is independent of the functional groups that are used, and the cargo can be quickly released by an organic solvent in which the cargo is dissolvable.
  • Embodiments of the invention include submicron structures of a silica body defining a plurality of pores and an outer surface between pore openings of said plurality of pores and a cationic polymer on the surface of said silica body.
  • the submicron structure has a maximum dimension less than one micron ( ⁇ ).
  • the submicron structure includes a silica body that defines a plurality of pores therein.
  • the silica body can be a mesoporous silica nanoparticle.
  • the fact that we refer to the body as a silica body does not preclude materials other than silica from also being incorporated within the silica body.
  • the silica body may be substantially spherical with a plurality of pore openings through the surface providing access to the pores.
  • the silica body can have shapes other than substantially spherical shapes in other embodiments of the current invention.
  • the silica body defines an outer surface between the pore openings, as well as side walls within the pores.
  • the pores can extend through the silica body to another pore opening, or can extend only partially through the silica body such that it has a bottom surface of the pore defined by the silica body.
  • the silica body is mesoporous. In other embodiments, the silica body is microporous.
  • “mesoporous” means having pores with a diameter between 2 nm and 50 nm, while “microporous” means having pores with a diameter smaller than 2 nm.
  • the pores may be of any size, but in some embodiments are large enough to contain one or more therapeutic compounds therein. In such embodiments, the pores allow small molecules, for example, therapeutic compound such as anticancer compounds to adhere or bind to the inside surface of the pores, and to be released from the silica body when used for therapeutic purposes.
  • the pores are substantially cylindrical.
  • Some embodiments of the invention include nanoparticles having pore diameters between about 1 nm and about 10 nm in diameter. Other embodiments include nanoparticles having pore diameters between about 1 nm and about 5 nm. Other embodiments include particles having pore diameters less than 2.5 nm. In other embodiments, the pore diameters are between 1.5 and 2.5 nm. Silica nanoparticles having other pore sizes may be prepared, for example, by using different surfactants or swelling agents during the preparation of the silica nanoparticles. [0059] The submicron structures according to some embodiments of the current invention may be referred to as nanoparticles. The term nanoparticles as used herein is intended the include particles as large as 1000 nm.
  • colloidal suspensions may be formed using a plurality of submicron structures according to some embodiments of the invention. In that case, larger particles can tend to settle rather than remaining suspended in Brownian motion.
  • size of the submicron structure refers to the size of the primary particles, as measured by transmission electron microscopy (TEM) or similar visualization technique.
  • Particle size does not refer to agglomerates in solution or suspension.
  • Some embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 1000 nm.
  • Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 500 nm.
  • Other embodiments include nanoparticles having an average maximum dimension between about 50 nm and about 200 nm.
  • Other embodiments include nanoparticles having an average maximum dimension less than about 500 nm, less than about 300nm, less than about 200nm, or less than about lOOnm.
  • the surface of the submicron structure or nanoparticle is unmodified.
  • an "unmodified” nanoparticle has had no other functional groups added to the surface after formation of the nanoparticle.
  • Unmodified nanoparticles have an anionic charge due to free silyl hydroxide moieties present on the surface.
  • polymer is a macromolecule composed of repeating structural units, usually in a linear or branched sequence.
  • the cationic polymer may be any polymer bearing an overall positive charge, such as, for example, poly(ethyleneimine) (PEI), polyamidoamine, polylysine, poly(allylamine) or poly(diallyldimethylammonium chloride).
  • PEI poly(ethyleneimine)
  • polyamidoamine polylysine
  • poly(allylamine) poly(diallyldimethylammonium chloride).
  • Other cationic polymers will be apparent to those of skill in the art, and may be found, for example, in "Polymer Handbook, 4th Edition, Edited by: Brandrup et al.; John Wiley & Sons, 1999; and De Smedt et al., Pharmaceutical Research, vol. 17, no. 2, pp. 113-126).
  • Cationic polymer modified nanoparticles have a positive charge.
  • the cationic polymer is poly(ethyleneimine) (PEI).
  • the cationic polymer may be bound covalently or electrostatically to the surface of the silica body.
  • the cationic polymer is electrostatically bound to the surface of the silica body.
  • the cationic polymer may bind electrostatically to an unmodified silica body, which has an overall negative charge, or to a surface-modified silica body bearing a plurality of negatively charged surface modifying groups such that the silica body has an overall negative charge. Surface modification of the silica body is described in detail below.
  • the cationic polymer has a weight average molecular weight less than about 30,000, less than about 25,000, less than about 20,000, less than about 15,000, or less than about 10,000. In some embodiments, the cationic polymer has a weight average molecular weight greater than about 600, greater than about 1000, greater than about 1500, or greater than about 1800, greater than about 2000, greater than about 3000, or greater than about 4000, or greater than about 5000. The range of molecular weight may be between any recited endpoints.
  • the silica body may have a core structure.
  • a core structure is a metal crystal or nanocrystal in the interior of the silica body.
  • the silica body does not include a metal nanoparticle or metal nanocrystal as a core structure.
  • the core structure is superparamagnetic metal, silver, or gold.
  • Other embodiments include submicron structures having more than one core structure in the nanoparticle, such as, for example, a silver and gold nanocrystal in the core structure, or a silver nanocrystal and superparamagnetic iron nanocrystal.
  • the core structure is a superparamagnetic nanocrystal, such as, for example, an iron oxide nanocrystal.
  • a superparamagnetic nanocrystal core makes the particles visible using magnetic resonance imaging (MRI).
  • the nanoparticle may be used as MRI contrast agents in addition to any transfection or anti-cancer activity.
  • the superparamagnetic nanocrystal in the core of the nanoparticle also allows the particles to be manipulated or collected by a magnetic field, for example.
  • the core structure is a gold nanocrystal.
  • Other embodiments include submicron structures as described above, which further include a surface modification.
  • surface modification means attaching or appending molecules or other materials to the surface of the silica body, in addition to the cationic polymer. Surface modification also modifies the surface of the pores of the silica body.
  • the surface modification may be covalent, electrostatic or a combination of both.
  • the surface may include a covalent surface modification and an electrostatic surface modification on the same nanoparticle.
  • the surface modification may be further derivatized, for instance, by further covalent or electrostatic bonds.
  • Surface modifications, as described herein may be used on any silica body having an unreacted silica surface, including nanodevices having stoppers, impellers or valves, as described below.
  • the surface modification comprises a plurality of anionic or electrostatic molecules attached to an outer surface of said silica body, wherein the anionic or electrostatic molecules provide hydrophilicity or aqueous dispersability to the nanoparticle and are suitable to provide repulsion between other similar submicron structures.
  • anionic surface modified nanopartices are described, for example, in International Application No.
  • the plurality of anionic molecules include at least one phosphonate moiety. In some embodiments, the plurality of anionic molecules are
  • Trihydroxysilylpropyl methylphosphonate surface modifications are prepared, for example, by treating the silica body with trihydroxysilyl propyl methylphosphonate.
  • the surface modification is covalently bonded to the surface of the silica body.
  • the surface modification has a functional group covalently bonded to the surface.
  • the "functional group” defines a chemical moiety linked to the surface of the nanoparticle, either directly, or via a linker.
  • the functional group is a phosphonate, amine, sulfhydryl, disulfide, carboxylic acid, epoxide, halide (i.e. fluorine, chorine, bromine, or iodine), azide, alkyne, or hydrophobic moiety.
  • the functional group is a phosphonate or an amide.
  • the functional group may be further bonded, covalently or electrostatically to a further compound.
  • any reaction capable of reacting with the silyl hydroxide surface of the silica body may be used to covalently modify the surface.
  • the surface of the silica body may be treated with a trialkoxysilyl compound or trihydroxysilyl compound.
  • the compound reacts with the silyl hydroxide surface of the silica body, forming covalent silicon- oxygen bonds.
  • Trialkoxysilyl and trihydroxylsilyl compounds bearing various functional groups may be used to modify the surface of the nanoparticle.
  • the covalent surface modification comprises a
  • the functional group i.e. phosphonate, amine, sulfhydryl, disulfide, carboxylic acid, epoxide or hydrophobic organic moiety
  • the functional group may be separated from the silica surface by a linker.
  • the functional group is covalently bonded to the silica surface via a C ⁇ to Ci2 alkyl linker.
  • a Ci to C 12 alkyl group is present between the atom covalently bonded to the surface and the functional group (i.e. phosphonate, amine, sulfhydryl, disulfide, carboxylic acid, epoxide or hydrophobic organic moiety).
  • the functional group is covalently bonded to the silica surface via a Q to C 6 alkyl linker.
  • Nanoparticles bearing a surface modification are called surface-modified nanoparticles.
  • Ci to C 12 alkyl chain includes linear, branched and cyclic structures having 1 to 12 carbon atoms, and hybrids thereof, such as cycloalkylalkyl.
  • alkyl chains include methylene (CH 2 ), ethylene (CH 2 CH 2 ), propylene (CH2CH 2 CH 2 ), and so forth.
  • surface modification having a phosphonate also known as phosphonate-modified nanoparticles
  • the phosphonic or phosphinic acid may be charged or uncharged, depending on the pH. At physiological pH, phosphonic acids and phosphinic acids are negatively charged, or anionic.
  • Phosphonate modifications may be prepared, for example, by treating the silica body surface with a phosphonate bearing trialkylsiloxane compound or phosphonate-bearing trihydroxylsilyl compound, such as (trihydroxylsilyl)propyl methylphosphonate.
  • the surface modification has a phsophonate (i.e.
  • phosphonic acid or phosphinic acid group.
  • Functionalization of the particle surface with a phosphonate group provides electrostatic binding of positively charged (i.e. cationic) hydrophilic therapeutic compounds (e.g. doxorubicin) to the porous interior, from where the drug could be released by acidification of the medium under abiotic and biotic conditions.
  • phosphonate modification also improves exterior coating with the cationic polymer, PEI, which endows the MSNP with the ability to contemporaneously bind and deliver siRNA.
  • surface modifications having an amine will have at least one primary (-N3 ⁇ 4), secondary (-NHR), tertiary (- NR 2 ) or quaternary amine.
  • An amine-modified surface may be charged or uncharged, depending on the amine and pH.
  • Amine modifications may be prepared, for example, by treating the silica body surface with an amine bearing trialkoxysilane compound, such as aminopropyltriethoxysilane, 3-(2-aminoethylamino)propyl-trimethoxysilane, or 3- trimethoxysilylpropyl ethylenediamine.
  • An amine modified silica body may have an overall negative charge at certain pH, and when combined with anionic surface modifications, as discussed above.
  • surface modifications having a sulfhydryl (or thiol) group will have at least one -SH moiety.
  • Such a modification may be prepared, for example, by treating the surface of the nanoparticle with a sulfyhdryl bearing trialkoxysilane compound, such as 3- mercaptopropyltriethoxysilane.
  • surface modifications having a disulfide group will have at least one -S-S- moiety.
  • Such a modification may be prepared, for example, by treating the surface of the nanoparticle with a disulfide bearing trialkoxysilane compound, or by treating a sulfhydryl modified surface with 2,2'-dithiodipyridine or other disulfide.
  • Such a modification may be prepared, for example, by treating the surface with a carboxylic acid bearing trialkoxysilane compound, or by treating the surface with a trialkoxysilane compound bearing a functional group that may be converted chemically into a carboxylic acid.
  • the surface may be treated with 3-cyanopropyltriethoxysilane, followed by hydrolysis with sulfuric acid.
  • glysidoxypropyltriethoxysilane [0080] Surface modifications having a hydrophobic moiety will have at least one moiety intended to reduce the solubility in water, or increase the solubility in organic solvents.
  • hydrophobic moieties include long chain alkyl groups, fatty acid esters, and aromatic rings.
  • an anionic surface modification such as, for example, a phosphonate modification
  • an amine modification such as, for example, a phosphonate modification
  • thiol modification such as, for example, a phosphonate modification
  • hydrophobic modification if desired.
  • the modified silica body has an overall negative charge before being combined with a cationic polymer. If the silica body has an overal negative charge, a cationic polymer may bind electrostatically to the surface of the silica body.
  • any of the covalent surface modifications described above may be further derivatized, for example, by further covalent or electrostatic bonds.
  • the surface modification is further covalently bonded to another compound, such as a light-emitting molecule, targeting compound, polymer, peptide, protein, nucleic acid, sugar, oligosaccharide, or polysaccharide.
  • Light emitting molecules include compounds which emit light by either fluorescence or phosphorescence.
  • Light emitting molecules include dyes, such as fluorescent dyes. Examples of light emitting molecules include fluorescent dyes such as fluorescein, and rhodamine B.
  • Light emitting molecules may be covalently bonded to the surface modified silica body by any useable method.
  • amine-modified nanoparticles having a free NH 2 group may be reacted with fluorescent dyes bearing amine-reactive groups such as isocyanates, isothiocyanates, and activated esters, such as N-hydroxysuccinimide (NHS) esters.
  • fluorescent dyes bearing amine reactive groups include, for example, fluoresceine
  • Nanoparticles bearing light-emitting molecules may be used, for example, for fluorescence imaging, for instance when the nanoparticles interact with the surface of a microbe.
  • the surface modification is further bonded to a polymer, such as, for example, polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Polymers covalently bonded to the surface modification should be covalently bonded at only one location to prevent crosslinking.
  • the surface may be modified with poly(ethylene glycol) methyl ether, which has only one reactive end.
  • the surface modification is further bonded to a peptide or protein.
  • Peptides include polypeptides having at least 2 amino acids.
  • Various amino acid residues on peptides or proteins may form a covalent bond with surface-modified nanoparticles.
  • carboxylic acid residues from aspartic acid and glutamic acid
  • amine residues on proteins i.e. from lysine
  • Sulfhydryl surface modifications may react with disulfide bonds (e.g. from cystine residues) in the protein via thiol exchange.
  • Disulfide surface modifications such as 2-thiopyridine disulfides may react with free thiols (e.g. from cysteine residues) in the protein to form a covalent bond with the protein.
  • free thiols e.g. from cysteine residues
  • Other suitable methods for conjugating the proteins to the surface-modified nanoparticles will be evident to those of skill in the art.
  • the polymer, protein, peptide, oligonucleotide, sugar, oligosaccharide, or polysaccharide is covalently attached to the surface modifying group via a linker.
  • a linker Various bifunctional crosslinkers are known to those in the art for covalently bonding to proteins, any of which may be used to covalently link a surface modified nanoparticle to a protein.
  • heterodifunctional crosslinkers such as succinimidyl-4-[N- maleimidomethyl]cyclohexane-l-carboxylate (SMCC) and melaimidobutyryloxysuccinimide ester (GMBS) may be used to react with amine-modified nanoparticles (via the succinimide esters), and then form a covalent bond with a free thiol in the protein (via the maleimide).
  • SMCC succinimidyl-4-[N- maleimidomethyl]cyclohexane-l-carboxylate
  • GMBS melaimidobutyryloxysuccinimide ester
  • crosslinkers such as succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) may react with amine- modified nanoparticles (via the succinimide ester), and form a covalent bond with a free thiol in the protein via thiol exchange.
  • Other difunctional crosslinkers include suberic acid bis(N- hydrosuccinimide ester), or disuccinimidyl carbonate which can react with amine-modified, hydroxyl-modified, or unmodified nanoparticles, and free amines or hydroxyl groups on the polymer (such as poly(ethyleneglycol) methyl ether) or protein (e.g. from lysine residues).
  • Other bifunctional and heterobifunctional crosslinkers useable with various surface
  • the surface modifying material is a polymer, protein, peptide, nucleic acid, sugar, oligosaccharide, polysaccharide, or combination thereof.
  • Surface-modified nanoparticles bearing a protein are also called protein-modified nanoparticles.
  • the protein may be bonded covalently (directly to the surface modification or via a linker) or may be electrostatically bonded to the modified or unmodified nanoparticles as discussed above.
  • the protein may be a targeting protein or an antibody.
  • a "targeting protein" as used herein, means a protein which binds to a particular surface feature of a cell.
  • Antibodies and peptides are also used to bind to particular surface features of cells, and may be used to modify the nanoparticles of the invention. Protein-modified nanoparticles may be used to selectively target specific cells, by interacting specifically or selectively to a cell of interest.
  • the surface modification is electrostatically bonded to the surface.
  • electrostatically bonded means bonded based on the attraction of opposite charges.
  • An unmodified nanoparticle has a negative charge, due to the presence of free silyl hydroxide residues on the surface of the nanoparticle.
  • the particle may also bear a surface modification having a negative charge (such as a phosphonate modification), such that the overall charge of the surface is negative.
  • the surface may be modified with material bearing a positive charge, which will bind to the surface electrostatically.
  • a protein in addition to the cationic polymer, may bind to the surface electrostatically.
  • a protein having a net positive charge will bind electrostatically to unmodified nanoparticles or surface modified nanoparticles bearing a negative charge.
  • proteins such as Bovine Serum Albumin (BSA) and protein solutions such as Fetal Bovine Serum (FBS) bind electrostatically to unmodified or negatively charged nanoparticles.
  • BSA Bovine Serum Albumin
  • FBS Fetal Bovine Serum
  • a protein having a net negative charge will bind electrostatically to modified nanoparticles having a positive charge, such as amine-modified nanoparticles, or nanoparticles modified by cationic polymers.
  • modified nanoparticles having a positive charge such as amine-modified nanoparticles, or nanoparticles modified by cationic polymers.
  • negatively charged proteins bind electrostatically to nanoparticles bearing a cationic polymer, as described herein.
  • the submicron structure further includes an
  • the oligonucleotide binds electrostatically to the cationic polymer and outside surface of the silica body.
  • an oligonucleotide is a nucleic acid polymer, and may be a ribonucleic acid polymer (RNA) or deoxyribonucleic acid polymer (DNA).
  • DNA may be double stranded, and may be, for example a plasmid.
  • RNA may be single stranded or double stranded and may be, for example a siRNA.
  • Other oligonucleotides include, for example, microRNA (miRNA) and small hairpin RNA (shRNA).
  • the cationic polymer envelopes and protects the oligonucleotide from degradation.
  • Oligonucleotide-containing structures according to the invention may, for example, deliver oligonucleotides to the interior of a cell when the submicron structure enters the cell. Genes or therapeutic oligonucleotides (such as siRNA) may thus be successfully delivered into a cell. In other words, oligonucleotide-containing submicron structures may be used for transfection.
  • the submicron structure further includes a therapeutic compound within the silica body or pores of the silica body.
  • a therapeutic compound is a small molecule used to treat a disease or disorder. In principle any type of therapeutic compound may be incorporated into the pores of the silica body.
  • the therapeutic compound is an anticancer compound (i.e. used to treat cancer).
  • the therapeutic compound is hydrophobic. Examples of hydrophobic therapeutic compounds include, for example paclitaxel and camptothecin and related compounds.
  • the therapeutic compound is cationic. Cationic compounds electrostatically bind to the surface of the silica body.
  • cationic compounds may bind to the interior of the silica body or within the pores of the silica body.
  • cationic therapeutic compounds include, for example, daunomycin, doxorubicin or related compounds.
  • the therapeutic compound is anionic.
  • Anionic compounds may bind in pores or surface of the silica body, for example, by binding electrostatically to amine-modified surfaces or to cationic- polymer bound silica bodies.
  • the submicron structure includes both an oligonucleotide and therapeutic compound.
  • the benefits of both the oligonucleotide and therapeutic compound may be realized.
  • the oligonucleotide may be a DNA plasmid.
  • the oligonucleotide may be an RNA polymer.
  • the oligonucleotide may be a small interfering RNA (siRNA).
  • the oligonucleotide may be a siRNA that decreases translation of a gene that produces drug resistance.
  • the siRNA decreases translation of a gene that produces drug resistance in a cancer cell, and the therapeutic compound is an anticancer compound.
  • the siRNA may decrease the resistance of the cell to the therapeutic compound.
  • the siRNA may reduce translation of the p-glycoprotein (pgp), implicated in multiple drug resistance (MDR).
  • MDR multiple drug resistance
  • Other resistance genes that may be supressed by siRNA include, for example, MRPl (ABC
  • BCRP breast cancer resistance protein
  • BCRP ABC transporters
  • GCS glucosylceramide synthase
  • c-Myc oncogene regulating MDR1 expression.
  • Other resistance genes will be apparent to one of ordinary skill in the art.
  • the therapeutic compound may be, for example doxirubicin.
  • doxirubicin a cancer cell that is normally resistant to doxirubicin may become susceptible to doxirubicin activity. In this way, multiple drug resistant cancers may be treated.
  • the submicron structure described above further includes a stopper assembly attached to the silica body.
  • the stopper assembly has a blocking unit arranged proximate at least one pore and has a structure suitable to substantially prevent material from entering or being released when the blocking unit is arranged in a blocking configuration.
  • the stopper assembly is responsive to the presence of a predetermined stimulus such that the blocking unit is released in the presence of the predetermined stimulus to allow material to enter or be released.
  • the predetermined stimulus is a predetermined catalytic activity that is suitable to cleave, hydrolyze, oxidize, or reduce a portion of the stopper assembly. Examples of stopper assemblies are described, for example, in International Application No. PCT/US2009/031891, filed January 23, 2009, now published as WO 2009/094580 and incorporated herein by reference in its entirety.
  • the stopper assembly can include a thread onto which the blocking unit can be threaded.
  • the thread has a longitudinal length that is long relative to a transverse length and is suitable to be attached at one longitudinal end to the silica body.
  • the stopper assembly can also have a stopper attached to a second longitudinal end of the thread in some embodiments.
  • the stopper can be selected among a wide range of possible stoppers based on the type of environment [0099]
  • a synthetic strategy can involve the use of a snap-top "precursor". The assembly of the snap-top precursors can be performed step-wise from the silica nanoparticle surfaces outward.
  • the silica nanoparticles are treated with aminopropyltriethoxysilane (APTES) to achieve an amine- modified nanoparticle surface.
  • APTES aminopropyltriethoxysilane
  • An azide terminated tri(ethylene)glycol thread is attached to the amine-modified nanoparticles.
  • the precursor is completed through the addition of a- cyclodextrin as the blocking unit at 5°C, which complexes with the threads at the low
  • stoppers can be selected that respond to enzymes (for example, ester linked or peptide linked), pH (for example, vinyl ether linked), and redox (for example disulfide linked) stimulation.
  • enzymes for example, ester linked or peptide linked
  • pH for example, vinyl ether linked
  • redox for example disulfide linked
  • the broad concepts of the current invention are not limited to only these specific examples. There are a wide range of possible stoppers that may be selected according to the particular application.
  • inventions include submicron structures further including an impeller attached to the silica body.
  • Silica bodies modified by impellers are described, for example in International Application No. PCT/US2009/031871, filed January 23, 2009, published as WO 2009/094568, the contents of which are incorporated herein in their entirety.
  • impeller as used herein is intended to have a broad meaning to include structures which can be caused to move and which can in turn cause molecules located proximate the impeller to move in response to the motion of the impeller.
  • the impellers are driven by an energy transfer process.
  • the energy transfer process can be, but is not limited to, absorption and/or emission of electromagnetic energy.
  • illuminating with light at an appropriate wavelength can cause the plurality of impellers to wag back and forth between two molecular shapes.
  • the motion of the plurality of impellers causes motion of molecules (for example, peptides, proteins, ions, drugs or antibiotics) of interest into and/or out of the silica body.
  • the plurality of impellers can remain substantially static, at least for time periods long enough for the desired application, to act as impediments to block molecules from exiting and/or entering the storage chamber.
  • the impellers can be, but are not limited to, azobenzenes according to some embodiments of the current invention.
  • the azobenzenes can include the following: 1) One phenyl ring derivatized with a functional group that enables attachment directly to the silica surface or to a modified silica surface as described later.
  • suitable functional groups contains but is not limited to: alcohols, (-ROH), anilinium amines (-NH 2 ) primary amines (-RNH 2 ), secondary amines (-R R 2 NH), azides (N 3 ), alkynes (RC ⁇ CH), isocyanates (-RNCO), isothiocyanates (-RNCS), acid halides (RCOX), alkyl halides (RX) and succinimidyl esters. 2) other functional groups on the other phenyl ring (which is the moving end of the machine).
  • the list of these functional groups includes but is not limited to: -H (here the phenyl ring is underivatized), esters (-OR), primary and secondary amines, alkyl group, polycyclic aromatics, and various generations of dendrimers.
  • -H here the phenyl ring is underivatized
  • esters -OR
  • primary and secondary amines alkyl group
  • polycyclic aromatics and various generations of dendrimers.
  • the bulkiness of these functional groups can be designed for specific systems. For example, large dendritic functionalities might be required when very large pore openings or very small guest molecules are employed.
  • impellers are based on redox of copper complexes.
  • the copper complexes can include bifunctional bidentate stators that contain diphosphine and/or diimine bidentate metal chelators on one end of the stator, while at the other end functionalities such as alkoxysilanes (for immobilization on silica and silicon substrates) and thiols (for immobilization on gold substrates) are present.
  • the copper complexes can contain a rotator that is a rigid bidentate diimine metal chelator, which rotates and changes the shape of the overall molecule upon redox or photons. These copper complexes exist in two oxidation states, each of which corresponds to a specific shape.
  • Copper (I) is tetrahedral while copper (II) is square planar.
  • the different oxidation states, and hence different shapes that are caused by a 90° rotation of the rotator, can be generated in three ways: Reduction and oxidation (1) using electrodes and an electric current (2) by use of chemical reducing and oxidizing agents, and (3) by the photo- excitation of light of the appropriate wavelength.
  • Some embodiments include submicron structure further including a valve assembly attached to the silica body.
  • Porous nanoparticles having valves are described, for example, in International Application No. PCT/US2009/032451, filed January 29, 2009, published as WO 2009/097439, the contents of which are incorporated herein in their entirety.
  • the valve assembly is operable in an aqueous environment.
  • the valve assembly has a valve arranged proximate the at least one pore and has a structure suitable to substantially prevent material from entering or being released while the valve is arranged in a blocking configuration.
  • the valve assembly is responsive to a change in pH such that the valve moves in the presence of the change in pH to allow the material to enter or be released from the silica body.
  • the pH-responsive valve assembly relies on the ion-dipole interaction between cucurbit[6]uril (CB[6]) and bisammonium stalks, and that can operate in water.
  • CB[6] a pumpkin-shaped polymacrocycle with D 6h symmetry consisting of six glycouril units strapped together by pairs of bridging methylene groups between nitrogen atoms has received considerable attention because of its highly distinctive range of physical and chemical properties.
  • [2]pseudorotaxanes having bisammonium stalks and CB[6] rings may be constructed on the surface of the mesoporous silica nanoparticles, and the pH-dependent binding of CB[6] with the bisammonium stalks is exploited to control the entry or release of molecules from the silica nanoparticles.
  • the CB[6] rings encircle the bisammonium stalks tightly, blocking the nanopores efficiently when employing suitable lengths of tethers. Deprotonation of the stalks upon addition of base results in spontaneous dethreading of the CB[6] rings and unblocking of the pores.
  • Embodiments include pharmaceutical compositions comprising any of the submicron structures according to the invention.
  • the composition may be in any suitable form such as a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • the compositions may be in a form suitable for administration by sterile injection.
  • a parenterally acceptable liquid vehicle water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p- hydroxybenzoate).
  • the carrier will usually comprise sterile water, though other ingredients, for example, ingredients that aid solubility or for preservation, may be included. Injectable solutions may also be prepared in which case appropriate stabilizing agents may be employed.
  • Formulations suitable for parenteral administration usually comprise a sterile aqueous preparation, which may be, for example, isotonic with the blood of the recipient (e.g., physiological saline solution).
  • a sterile aqueous preparation which may be, for example, isotonic with the blood of the recipient (e.g., physiological saline solution).
  • Such formulations may include suspending agents and thickening agents and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose form.
  • Parenteral administration may comprise any suitable form of systemic delivery or localized delivery.
  • Administration may for example be intravenous, intratumoral, intra-arterial, intrathecal, intramuscular, subcutaneous, intramuscular, intra-abdominal (e.g., intraperitoneal), etc., and may be effected by infusion pumps (external or implantable) or any other suitable means appropriate to the desired administration modality.
  • Embodiments include methods for using the submicron structures according to any embodiment of the invention for treatment of a disease or disorder by administering to a subject in need of treatment an effective amount of a submicron structure of the invention.
  • the submicron structures may be administered, for example, as a pharmaceutical composition. Administration may be achieved by any suitable means.
  • the compositions may be administered systemically, for example, formulated in a
  • pharmaceutically-acceptable buffer such as physiological saline.
  • routes of administration include, for example, subcutaneous, intravenous, intraperitoneal, intramuscular, or intradermal injections that provide continuous, sustained levels in the patient.
  • Administration to human patients or other animals is generally carried out using a physiologically effective amount of a compound of the invention in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W.
  • compositions may be administered parenterally by injection, infusion or implantation in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and/or adjuvants.
  • the compositions are added to a retained physiological fluid, such as cerebrospinal fluid, blood, or synovial fluid.
  • the compositions of the invention can be amenable to intravenous (i.v.) injection and direct injection (i.e. intratumoral injection), application or infusion at a site of disease or injury.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, organ or subject.
  • an effective amount is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated subject.
  • the effective amount of an active therapeutic agent for the treatment of a disease or injury varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending clinician will decide the appropriate amount and dosage regimen.
  • a subject is meant an animal.
  • a subject may be a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
  • treat refers to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • Embodiments of the invention include methods of treating drug resistant cancer by administering to a patient in need a submicronstructure according to the invention having an siRNA that reduces the translation of a gene responsible for the drug resistance, and an anticancer compound.
  • the anticancer compound is a compound to which the cancer is resistant but becomes susceptible to treatment upon co-administration with an siRNA that reduces the translation of a resistance gene.
  • MSNP In addition to being used for the delivery of chemical therapeutic agents, MSNP have the potential as hybrid organic-inorganic materials that can act as carriers for nucleic acids and therefore potentially useful for the delivery of small interfering RNAs (siRNAs) and other forms of gene therapy (Park et al., Int. J. Pharm., vol. 359, pp. 280-284, 2008; Radu et al., J Am. Chem. Soc, vol. 126, pp. 13216-13217, 2004; Torney et al., Nat. Nanotechnol, vol. 2, pp. 295-300, 2007).
  • siRNAs small interfering RNAs
  • the porous structure of MSNP allows both the binding of nucleotides on the surface as well as the encapsulation of small molecules within the particles. It is even possible to combine these modules to achieve dual delivery of drugs and nucleic acids (Torney et al., Nat. Nanotechnol, vol. 2, pp. 295-300, 2007).
  • the silica surface may be converted into positive charge in order to bind DNA and siRNA.
  • PEI polyethyleneimine
  • PEI was chosen as the polymer coating to enhance the particle uptake into cells and facilitate endosomal escape for the nucleotide delivery (Duan et al., J Am. Chem. Soc, vol. 129, pp. 3333-3338, 2006). It is documented that while low MW PEI is not cytotoxic, these polymers are ineffective at transfecting nucleotides in contrast to the high MW PEI. In this regard, it has been demonstrated that the size (MW), compactness and chemical modification of PEI affect the efficacy and toxicity of this polymer (Florea et al., AAPS PharmSci., vol. 4, p. E12, 2002; Neu et al., J Gene. Med., vol. 7, pp.
  • PEI polymer sizes ranging from MW of 0.6 to 25 KD were investigated in order to balance the efficiency of nucleic acid delivery and cellular toxicity, which proceed by the proton sponge effect that involves proton sequestration on the polymer surface, heightened activity of the proton pump, osmotic swelling lysosomal injury, intracellular Ca 2+ flux and mitochondrial damage (Xia et al., Nano Lett. Vol. 6, pp. 1794-1807, 2006; Xia et al, ACS Nano, vol. 2, pp. 85-96, 2008).
  • the large number of amine groups of PEI also allows the polymer-coated particles to move across cell membranes through rapid endocytosis.
  • Cellular uptake of PEI-coated MSNP may be enhanced by at least two orders of magnitude compared to that of the MSNP which are unmodified (silanol surface) or coated with phosphonate or poly(ethylene gylcol) groups. Furthermore, with coating of PEI MW 10 KD, it is possible to achieve both increased cellular uptake and high transfection efficacy while minimizing the toxicity normally observed with higher molecular weight PEI.
  • surface functionalization with polyethyleneimine (PEI) polymers may enhance oligonucleotide (i.e. DNA, RNA, shRNA or siRNA) binding.
  • PEI polyethyleneimine
  • the tight complexing between PEI and nucleic acids on the particle surface protects these cargo molecules from enzymatic degradation.
  • the positive charge of PEI-coated nanoparticles may also lead to strong electrostatic interaction with the negatively charged cell surface membrane, which may facilitate particle cellular uptake and lysosomal release via a proton sponge mechanism.
  • the molecular weight of PEI polymer may be carefully selected in order to reduce possible cytotoxicity.
  • cationic MSNP with therapeutically useful nucleic acid delivery properties that include high binding avidity of DNA and siRNA as well as a high rate of cellular uptake.
  • siRNA complexed to the MSNP-PEI surface is quite effective to achieve green fluorescent protein (GFP) knockdown in transduced HEPA-1 cells, while plasmid DNA delivery is comparable to a commercially available transfection agent.
  • GFP green fluorescent protein
  • the facilitated cellular uptake of cationic particles may enhance the ability of MSNP to deliver hydrophobic chemotherapeutic agents, such as paclitaxel, to pancreatic cancer cells.
  • a potential downside of cationic functionalization to achieve drug or nucleic delivery is induction of cytotoxicity, best demonstrated by the use of MSNP-PEI-25 KD. However, this toxicity could be reduced or eliminated by attaching shorter length polymers that retain nucleic acid and drug delivery capabilities.
  • the therapeutic use of the MSNP platform can be extended to delivery of DNA and siRNA constructs.
  • Packaging siRNA on the surface of cationic MSNP may provide several benefits.
  • the MSNP surface can be functionalized to enhance siRNA binding through the attachment of PEI polymers, for example, which in their own right have been used as effective siRNA compacting and transducing agents.
  • PEI polymers for example, which in their own right have been used as effective siRNA compacting and transducing agents.
  • the tight complexing between PEI and nucleic acids on the particle surface protects these molecules from enzymatic degradation as
  • PEI can be used in various ways to make siRNA delivery complexes, coating it onto the surface of MSNP utilizes this therapeutic platform as a carrier with large surface area, which is inexpensive and simple to synthesize, and which can be decorated with functional groups and fluorescent tags and can be used for magnetic resonance imaging through the inclusion of superparamagnetic iron oxide nanocrystals (Liong et al., ACSNano, vol. 2, pp. 889- 896, 2008).
  • PEI coating enhance siRNA delivery, but the particles also are capable of anticancer compound such as paclitaxel delivery, which constitutes a significant advance for the use of MSNP as a therapeutic platform.
  • siRNA and drug delivery simultaneously, e.g., delivery of siRNA that knocks down the expression of the P- glycoprotein (Pgp) drug exporter at the same time as delivering a chemotherapeutic agent that is exported by Pgp (Ludwig et al., Cancer Res., vol. 66, pp. 4808-4815, 2006). This may be an effective strategy for the treatment of cancers that have developed a drug resistance due to the activity of this exporter.
  • Pgp P- glycoprotein
  • intermediate length polymers may provide effective cellular uptake and siRNA/plasmid DNA delivery. Moreover, this approach may produce transfection of > 70% cells in the population, which may increase effectiveness from the perspective of gene therapy.
  • PEI toxicity has been reduced include neutralizing the cationic charge by anhydride, differential ketalization, altering PEI crosslinking through adjustment of disulfide content and using shorter polymers (Xia et al., ACSNano, vol. 2, pp. 85-96, 2008; Shim et al., J Control. Release., vol. 133, pp. 206-213, 2009; Veiseh et al., Biomaterials, vol. 30, pp. 649-657, 2009; Hobel et al, Eur. J. Pharm. Biopharm., vol. 70, pp. 29-41, 2008 Peng et al., Bioconjugate Chem., vol. 20, pp. 340-346, 2009).
  • the first step is to conduct toxicity test in mice.
  • Results show that i.v. injection of MSNP in mice has no obvious toxic effects to the major organs or systems of the animals.
  • MSNP-PEI 25 KD showed toxicity to cultured cells while no toxicity in vivo. This may be because of the dilution of the MSNP in the blood system, the huge number of cells in the body, and strong defense/clearance capability in living organisms.
  • MSNP mesoporous silica nanoparticles for in vivo imaging
  • MSNP were in the blood vessel 30 min after administration, then MSNP tended to accumulate more in the RES systems such as liver and spleen tissue than in kidney, lung, and heart; in fact MSNP appeared to have cleared kidney after 2 h (Kim et al., Angew. Chem., Int. Ed, vol. 47, pp. 8438-8441, 2008; Wu et al., ChemBioChem, vol. 9, pp. 53- 57, 2008).
  • mesoporous silica nanoparticles constitute a multi-functional platform that can be used for drug (Lu et al., Small, vol. 4, pp. 421-426, 2008; Coti et al., Nanosacle, vol. 1, pp. 16-39, 2009; Slowing et al., Adv. Drug Deliv. Rev., vol. 60, pp. 1278-1288, 2008; Liong et al., ACS Nano, vol. 2, pp. 889-896, 2008) and nucleic acid delivery (Xia et al., ACS Nano, vol. 3, pp. 3273-3286, 2009).
  • nanoparticles that contain a phosphonate surface coating may be beneficial because of the ease of particle dispersal, good bio-safety index as well as the ability to adsorb cationic polyethylenimine (PEI) polymers for complexing and delivery of DNA and siRNA (Xia et al., ACS Nano, vol. 3, pp. 3273-3286, 2009). Since the polymer attachment leaves the porous interior free for drug binding and delivery, it establishes the potential to achieve simultaneous drug and nucleic acid delivery (Astrid et al., J Am. Ceram. Soc, vol. 84, pp. 806-812, 2001). It was envisaged that there some disease conditions such as multidrug resistant cancer might benefit from the delivery of both agents. Dual delivery might, therefore achieve simultaneous drug and siRNA delivery, leading to a synergistic therapeutic outcome.
  • PEI polyethylenimine
  • a cationic polymer such as PEI, will not interfere in drug binding to the porous interior.
  • cationic therapeutic compounds such as doxirubicin can be loaded into the silica body, but only after a cationic polymer, such as PEI is coated on the surface of the silica body. Furthermore, loading therapeutic compounds, such as doxorubicin or paclitaxel did not disturb siR A binding, as demonstrated herein. Finally, as demonstrated herein, the dual delivery system could be preferentially taken up by drug resistant cancer cells and was capable of acting synergistically to overcome drug resistance. In other words, the cationic polymer did not prevent release of the therapeutic compound in the cell.
  • Dox can be stably attached to the porous interior by a proton-sensitive electrostatic binding interaction that allows effective drug release from the acidifying LAMP- 1 -positive compartment.
  • Pgp siRNA co-delivery increases intracellular Dox concentrations with improved cytotoxic killing.
  • the improvement of Dox resistance provides proof-of-principal testing that MSNP can be engineered to provide contemporaneous drug and siRNA delivery by effective use of charge and the state of protonation or deprotonation at the particle surface.
  • MSNP can be functionalized to deliver a chemotherapeutic agent as well as Pgp siRNA to a drug-resistant cancer cell line.
  • the functionalization of the particle surface with a phosphonate group allows electrostatic binding of Dox to the porous interior, from where the drug could be released by acidification of the medium under abiotic and biotic conditions.
  • phosphonate modification also allows exterior coating with the cationic polymer, PEI, which endows the MSNP with the ability to contemporaneously bind and deliver Pgp siRNA.
  • Nanoparticle drug delivery is capable of overcoming both problems through tumor cell targeting as well as the capability to overcome drug resistance (Jabr-Milane et al., Cancer Treat. Rev., vol. 34, pp. 592-602, 2008; Ferrari et al., Nat. Rev. Cancer, vol. 5, pp. 161-171, 2005).
  • MDR can basically be divided into two distinct categories, namely pump and non-pump resistance (Jabr-Milane et al., Cancer Treat. Rev., vol. 34, pp. 592- 602, 2008; Saad et al., Nanomedicine, vol. 3, pp. 761-776, 2008).
  • Pump resistance refers to the inducible formation of membrane-bound channels or pores that actively expel a series of structural and functionally distinct chemotherapeutic agents from the cell. Drug efflux significantly decreases the intracellular concentration that limits their cytotoxic potential.
  • the key proteins involved in pump resistance are Pgp and MRP-1, while the major mechanism in non-pump resistance is activation of cellular anti-apoptotic defense pathways, including drug- induced expression of Bcl-2 protein (Saad et al., Nanomedicine, vol. 3, pp. 761-776, 2008).
  • the pump and non-pump resistance mechanisms could be mutually interactive (Jabr- Milane et al., Cancer Treat. Rev., vol. 34, pp. 592-602, 2008). Given this background, a number of nanomaterial design strategies can be used to overcome drug resistance:
  • the first is co-delivery of the chemotherapeutic drug with a pharmaceutical agent that interferes in pump activity or in non-pump pathways.
  • a pharmaceutical agent that interferes in pump activity or in non-pump pathways.
  • verapamil as a Pgp inhibitor that have been combined with Dox, aimed at reducing cardiotoxicity of Dox as well as overcoming Pgp-mediated MDR (Wu et al., J Pharm. Pharmaceut. Set, vol. 10, pp. 350-357, 2007).
  • IC 50 11.4 ⁇
  • a second strategy is to use nanoparticles to deliver the chemotherapeutic together with siRNA that interfere in key protein expression in pump-dependent or independent drug resistance pathways, as demonstrated herein.
  • paclitaxel that was delivered by poly(d,l-lactide-co- glycolide) nanoparticles along with Pgp siRNA to the MDR murine mammary cancer cell line, JC.
  • the dual delivery system showed significantly higher cytotoxicity in vitro and significantly greater inhibition of tumor growth in vivo than nanoparticles loaded with paclitaxel alone (Patil et al., Biomaterials, vol. 31, pp. 358-365, 2009).
  • the third strategy is to deliver the drug together with a combination of siRNA's that interfere in both pump ⁇ e.g. Pgp) and non-pump (e.g. Bcl-2) mechanisms. Such an approach may be necessitated by the co-existence of pump and non-pump resistance
  • a cationic liposome carrier system was developed to deliver Dox contemporaneously with two species of siRNA targeting of MRP- 1 as well as Bcl-2 (Saad et al, Nanomedicine, vol. 3, pp. 761-776, 2008).
  • This triple component (Dox, MRP-1 siRNA, and Bcl- 2 siRNA) delivery system demonstrated enhanced Dox cytotoxicity in human MDR H69AR lung cancer cells which showed >100 fold enhancement in cytotoxicity (Saad et al.,
  • phosphonate-MSNP exhibit good particle dispersibility and biocompatibility.
  • the role of the lysosomal proton pump is supported by the finding that NH 4 C1 interferes in Dox release to the nucleus ( Figure 20F, lower panel).
  • Phosphonate attachment also facilitates the binding of cationic PEI to the particle exterior ( Figure 18, right panel). This binding interaction is sufficiently strong to allow the polymer and attached siRNA to stay on the particle surface until entry into the lysosomal compartment.
  • PEI may play a role in firm cellular attachment and selection of the initial endosomal compartment.
  • the polymer is attached to the particle surface to leave the pores accessible to Dox binding ( Figure 20B).
  • 25 kD PEI polymer as well as high doses of the 10 kD polymer can render the MSNP toxic as a result of the proton sponge effect in the lysosome (Xia et al., ACS Nano, vol. 3, pp. 3273-3286, 2009).
  • the particle dose and exposure time may be limited to within safe limits to conduct Dox and siRNA delivery with PEI 10 kD polymer
  • siRNA delivery to the cytosol is also dependent on the proton sponge effect of the PEI-coated particle and in this case, the lysosome appears to be a key organelle in the dual drug delivery paradigm.
  • Pgp overexpression is one of the major mechanisms of multiple drug resistance (MDR) in cancer cells
  • knockdown of Pgp gene expression by nanoparticle siRNA delivery could help to restore the intracellular drug levels to the concentrations required for induction of apoptosis and cytotoxicity.
  • dual drug and siRNA delivery by nanoparticles can be used to overcome drug resistance in MDR cancer cells.
  • the feasibility of the MSNP platform to improve the cytotoxicity of Dox by co-delivery of Pgp siRNA is demonstrated as proof-of-principle.
  • Downregulation of Pgp expression allowed the intranuclear Dox levels to increase above the threshold required for inducing apoptosis and cell death.
  • the MDR phenotype is quite complex and often involve a combination of drug resistance mechanisms such as increased efflux, blocked apoptosis, decreased drug influx, increased drug metabolism, and increased DNA repair (Gottesman et al., Annu. Rev. Med., vol. 53, pp. 614-627, 2002; Jabr-Milane et al., Cancer Treat. Rev., vol. 34, pp. 592-602, 2008).
  • MSNP can be functionalized to act as a dual delivery vehicle for Dox as well as Pgp siRNA in a drug-resistant cancer cell line.
  • phosphonate attachment was used to deliver the therapeutic compound as well as the siRNA via a lysosomal processing pathway.
  • This dual delivery system increased the intracellular Dox levels to the extent that it improves cytotoxic killing in this KB-V1 MDR cell line. This strategy could be an effective new approach for the treatment of cancers that develop multiple drug resistance.
  • Polyethyleneimine (MW 0.6, 1.8, and 10 KD) were from Alfa Aesar (Ward Hill, MA).
  • the MTS assay kit was from Promega (Madison, WI).
  • Dulbecco's Modified Eagle's medium (DMEM), penicillin/streptomycin, and L-glutamine were purchased from Invitrogen (Carlsbad, CA).
  • Fetal calf serum (FCS) was from Atlanta Biologicals, Inc
  • siRNA for GFP knock down was purchased from IDT Technologies (Coralville, IA).
  • water was de-ionized and filtered with a 0.45 ⁇ pore size polycarbonate syringe filter (Millipore, Billerica, MA). All chemicals were reagent grade and used without further purification or modification.
  • the stock solution in water was sonicated (Tekmar Sonic Disrupter probe) for 15 sec prior to aliquoting.
  • Tekmar Sonic Disrupter probe Tekmar Sonic Disrupter probe
  • the aliquoted NP suspension ⁇ 10 ⁇ was mixed with an equal volume of 4% BSA.
  • Tissue culture media (1 ml) was added to the BSA coated MSNP suspension.
  • Cell culture media deprived of serum e.g. BEGM
  • the cell culture media containing MSNP at the desired concentration was sonicated for 15 sec and characterized as described before.
  • Cells were analyzed in a LSR flow cytometer using mean FL-2 and FL-1 to assess RITC and FITC fluorescence, respectively. Data are reported as fold increase above control (cells without MSNP).
  • Cellular uptake of MSNP was performed by adding 25 ⁇ of the various MSNP to 8-well chamber slides (Nunc) in which 5 x 10 4 cells were cultured in each well containing 0.4 ml culture medium. Cell membranes were stained with 5 ⁇ g/ml wheat germ agglutinin (WGA) Alexa Fluor ® 594 conjugate in PBS for 30 min.
  • WGA wheat germ agglutinin
  • the basic synthesis of MSNP was conducted by mixing the silicate source tetraethylorthosilicate (TEOS) with the templating surfactants cetyltrimethylammonium bromide (CTAB) in basic aqueous solution (pH 11).
  • TEOS silicate source tetraethylorthosilicate
  • CTAB cetyltrimethylammonium bromide
  • pH 11 basic aqueous solution
  • 100 mg CTAB was dissolved in a round-bottom flask containing solution of 48 ml distilled water and 1.75 ml sodium hydroxide (2 M). The solution was heated to 80°C and stirred vigorously. After the temperature had stabilized, 0.5 ml TEOS was added slowly into the heated CTAB solution. After 15 min, 0.23 mmol of the organosilane solution was added into the mixture.
  • 3- trihydroxysilylpropyl methylphosphonate was used for phosphonate surface modification, and aminopropyltriethoxy silane (APTS) was used for amine surface modification.
  • APTS aminopropyltriethoxy silane
  • the solution was cooled to room temperature and the materials were washed with methanol using centrifugation.
  • fluorescein-modified silane was first synthesized and then mixed with TEOS.
  • 2.4 APTS was mixed with 1 mg fluorescein isothiocyanate (FITC) in 0.6 ml absolute ethanol, and stirred for 2 hr under inert atmosphere.
  • FITC fluorescein isothiocyanate
  • rhodamine B isothiocyanate (RITC) was used instead of FITC to synthesize rhodamine B-modified APTS.
  • the dye-modified silane was then mixed with TEOS before adding the mixture into the heated CTAB solution.
  • the surfactants were removed from the pores by refluxing the particles in a mixture of 20 ml methanol and 1 ml hydrochloric acid (12.1 M) for 24 hr. The materials were then centrifuged and washed with methanol.
  • poly(ethylene glycol) modification 1 g of poly(ethylene glycol) methyl ether (MW 5 KD, mPEG) was dried under vacuum for 30 min and dissolved in 5 ml dioxane (with slight heating). mPEG has only one reactive end that can be attached to the particle surface and limits the coupling process only to that end, whereas normal PEG has two reactive ends and may cause particle cross-linking. 307.4 mg disuccinimidyl carbonate (DSC) was dissolved in 2 ml anhydrous DMF (with slight heating) and mixed with the mPEG solution.
  • DSC disuccinimidyl carbonate
  • the activated mPEG was dried under vacuum. 60 mg of amine-modified MSNP was washed and resuspended in 2 ml anhydrous DMF. 300 mg of the activated mPEG was dissolved in 9 ml DMF and mixed with the particles. The mixture was stirred for 12 hr and washed thoroughly with DMF and PBS.
  • PEI polyethyleneimine
  • MSNP were synthesized according to a modified procedure previously described (Radu et al., J Am. Chem. Soc, vol. 126, pp. 13216-13217, 2004; Cai et al., Chem. Mater., vol. 13, no. 2, pp. 258-263, 2001). All MSNP were characterized for size, size distribution, shape, and charge (Table 1). Table 1. Size distribution of MSNPs in aqueous solutions.
  • DMEM Complete Dulbecco's Modified Eagle Media, which contains 10% fetal calf serum (FCS).
  • BEGM Bronchial Epithelial Growth Medium, which includes growth factors, cytokines, and supplements (no serum).
  • the primary particle size is in the 100-130 nm range with a uniform pore size -2.5 nm as shown by TEM ( Figure 1).
  • particle size and zeta potential were measured in water as well as tissue culture media (Xia et al., ACS Nano, vol. 2, pp. 2121-2134, 2008).
  • DMEM fetal calf serum
  • BEGM was used as is or supplemented with 2 mg/ml BSA (Table 1).
  • the addition of protein leads to improved particle dispersal by countering the colloidal forces that promote particle aggregation in salt containing media (Figure 9).
  • the MTS assay was used, which reflects dehydrogenase activity in healthy cells (Xia et al., ACS Nano, vol. 2, pp. 85-96, 2008). While most of the MSNP did not interfere in MTS activity in the PANC-1 and BxPC3 pancreatic cancer cell lines, particles coated with the 25 KD PEI polymer showed decreased cellular viability ( Figure 1). The particles coated with the 25 KD polymer also induced toxicity in macrophage (RAW 264.7) and bronchial epithelial (BEAS-2B) cell lines staining ( Figure 10).
  • MSNP were coated with 0.6, 1.2, 1.8, 10 and 25 KD polymers and their cytotoxic potential assessed in various cell types. This included the use of HEP A- 1 cells for which there is a commercial variant expressing green fluorescent protein (GFP) for the purposes of assessing siRNA knockdown. No toxicity was seen with particles coated with 0.6, 1.2 and 1.8 KD PEI polymers ( Figure 4 and 14).
  • GFP green fluorescent protein
  • MSNP-PEI-10 KD exerted toxic effects at the highest dose (50 ⁇ g/ml) tested
  • MSNP-PEI-25 KD was responsible for the decline in MTS activity at doses > 12.5 ⁇ g/ml ( Figure 4 and 14). This demonstrates that it is possible to adjust MSNP toxicity according to the polymer length used and is the first demonstration that the choice of the PEI polymer length can be used to modify the toxicity of MSNP while still maintaining a useful function.
  • Agarose gel retardation assay was used to determine the DNA/siRNA binding ability of PEI-coated MSNP.
  • 0.1 ⁇ g plasmid DNA (pEGFP) or siRNA in aqueous solution was used to mix with PEI-coated nanoparticles to obtain particle to pDNA ratios (N P) ratios of 5- 600.
  • the mixture was incubated at room temperature for 30 min for complex formation.
  • 10 L of the polyplex solution mixed with 2 ih of 6* loading buffer was electrophoresed on 1 % agarose gel containing 0.5 ⁇ g/ml ethidium bromide (EB) with Tris-boric acid (TBE) running buffer (pH 8) at 100 V for 30 min.
  • EB ⁇ g/ml ethidium bromide
  • TBE Tris-boric acid
  • DNA/RNA bands were visualized by an UV (254 nm) illuminator and photographed with a Bio-Rad imaging system (Hercules, CA). The binding capacity was expressed by the N/P ratio that shows total retardation of DNA or siRNA migration (as reflected by the disappearance of DNA/RNA bands on the gel).
  • Particle/pDNA complexes were prepared at a MSNP/pDNA ratio of 100 with 100 ng pDNA in 10 ⁇ total volume.
  • the complex solutions were incubated with 1 ⁇ DNase I (2.7 U/ ⁇ ) in 50 mM Tris-Cl, 10 mM MgCl 2 , pH 7.4. at 37°C for 30 min.
  • the DNase I was inactivated by adding 1 ⁇ of 100 mM ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • the pDNA was then released from the complex by adding 1% sodium dodecyl sulfate (SDS), and analyzed by 1% agarose gel electrophoresis.
  • Plasmid DNA (pDNA) containing a GFP insert was used to transfect HEP A- 1 cells cultured either in Nunc chamber slides for performance of confocal microscopy or in 48- well plates for assessment by flow cytometry. Cells were plated at a density of 2 x 10 4 cells per well in 0.4 ml medium. pDNA/MSNP complexes were prepared by mixing 100 ng/ml DNA with 25 ⁇ g/ml MSNP for 30 min prior to cellular incubation for 24 hr. Cells were fixed for confocal microscopy as described above. Harvested cells were used to conduct flow cytometry on FL-1 channel.
  • HEPA-1 cells were prior transfected with a GFP plasmid and then sorted in the FL-1 channel to select stable GFP expressing cells.
  • the sorted cells were plated at a density of 5 x 10 4 cells per well containing 0.4 ml culture medium in chamber slides for performance of confocal microscopy and in 48-well plates for performance of flow cytometry.
  • MSNP/siRNA complexes were prepared by incubating 500 ng/ml siRNA with 25 ⁇ g/ml of MSNP-PEI for 30 min in serum-free DMEM. Cells were then exposed to the complexes for 3 hr. DMEM + 10% FCS was then added to bring the final volume to 400 ⁇ for 48 hr. Cells were fixed and prepared for confocal microscopy as described above. For flow cytometry cells were harvested and analyzed for fold decrease in GFP expression (FL-1 channel).
  • MSNP are capable of delivering water-insoluble drugs to cancer cells (Liong et al., ACSNano, vol. 2, pp. 889-896, 2008).
  • the polymer attachment still allows effective delivery of the hydrophobic cancer drug, paclitaxel, to PANC-1 and BxPC3 cells.
  • Paclitaxel was loaded into MSNP-PEI- 1.2 KD and -25 KD in DMSO, followed by washing in an aqueous buffer to entrap the hydrophobic drug in the particle pores. Briefly, the modified materials were loaded with paclitaxel by incubating 10 mg of the nanoparticles in a solution of 1 mg of paclitaxel and 0.25 ml of DMSO for 6 hours. After the drug-laden nanoparticles were removed from the suspension by centrifugation and the supernatant removed completely, the materials were dried under vacuum. The drug-laden nanoparticles were washed and sonicated with PBS.
  • PANC-1 and BxPC3 cells were plated at 2 x 10 4 cells per well in a 96 well plate.
  • MSNP particles, loaded with paclitaxel were incubated with the cells at doses of 10-50 ⁇ g/ml for 48-72 hrs.
  • Free paclitaxel corresponding to the amount loaded into MSNP particles was suspended or dissolved in PBS and DMSO to serve as controls.
  • MTS assays were performed after 48 hours to determine the cell viability after treatment.
  • mice were randomly divided into three groups: MSNP-phosphonate, MSNP-PEI 25 KD and the control group. Six mice were used per group, since this number has enough statistical power to discern differences in the toxic responses. Forty mg/kg particles were used for i.v. injection through tail vein once a week for two weeks. Animal weight was monitored after particle injections. Animals were sacrificed later to obtain blood and organs.
  • the serum was obtained by centrifugation of the whole blood at 3000 rpm for 15 min.
  • the biochemical parameters were assayed by UCLA Division of Laboratory Animal Medicine (DLAM) diagnostic laboratory services.
  • liver, kidney, spleen, lung, heart, and brain was fixed by 10% formalin and then embedded into paraffin, sectioned for 5 ⁇ thick, and mounted on the glass microscope slides by UCLA Division of Laboratory Animal Medicine (DLAM) diagnostic laboratory services. The sections were stained with hematoxylin-eosin and examined by light microscopy.
  • DLAM Laboratory Animal Medicine
  • MSNP nucleic acid and drug delivery
  • drug carriers i.v. injection is the most used route.
  • In vivo toxicity test of MSNP were conducted in mice by i.v. injection of 20 mg/kg nanoparticles once a week for two weeks. Before the injection, the particle size was characterized in saline, which is commonly used in in vivo assays.
  • the average size of MSNP-phosphonate and MSNP- PEI 25 KD is 984 and 842 nm in saline, respectively.
  • protein corona the protein coating on the particle surface, can stabilize nanoparticle suspension and serum is high in proteins that can be used as a dispersing agent for nanoparticles.
  • 2% mouse serum was added to saline and used to suspend the MSNP.
  • the serum-containing saline substantially decreased the average size of MSNP-phosphonate and MSNP-PEI 25 KD to 249 and 278 nm, respectively.
  • Biochemical parameters includes Cholesterol (CHOL), Creatine Kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), Alkaline phosphatase (ALP), total bilirubin (TBILI), total protein (TPROT), Glucose (GLU), Inorganic phosphorus (PHOS), Calcium (CA), Carbon Dioxide (C02_LC), Blood Urea Nitrogen (BUN), Creatinine (CREAT), Direct bilirubin (DBILI), Albumin (ALB), Albumin-globulin ratio (AGR), Blood Creatinine (B_CREA), Amylase (AMYL), Lactate dehydrogenase (LDH), Magnesium (MG), Triglycerides (TRIG).
  • Cholesterol CHOL
  • Creatine Kinase CK
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP Alkaline
  • Tetraethylorthosilicate (TOES, 98%), cetyltrimethylammonium bromide (CTAB, 95%), fluorescein isothiocyanate (FITC, 90%), doxorubicin hydrochloride (Dox, >98%), camptothecin (CPT, 95%), vinblastine, polyethylenimine (PEI, branched, MW 25 kD), bafilomycin A (>95%), ammonium chloride, ⁇ -actin antibody, and bovine serum albumin (BSA) were from Sigma (St. Louis, MO). Polyethylenimine (branched, MW 1.8 and 10 kD) was purchased from Alfa Aesar. 3-trihydroxysilylpropyl methylphosphonate,
  • cyanoethyltriethoxysilane, and aminopropyltriethoxysilane were purchased from Gelest.
  • DMEM Dulbecco's Modified Eagle's medium
  • penicillin/streptomycin penicillin/streptomycin
  • L-glutamine purchased from Invitrogen (Carlsbad, CA).
  • Fetal calf serum (FCS) was from Atlanta
  • LAMP-1 antibody was obtained from Abeam
  • siRNA for Pgp knockdown was purchased from IDT Technologies
  • Tariquidar (>97%) was purchased from MedKoo Biosciences, Inc.
  • water was de-ionized and filtered with a 0.45 ⁇ pore size polycarbonate syringe filter (Millipore, Billerica, MA). All chemicals were reagent grade and used without further purification or modification.
  • KB-31 and KB-V1 cells lines were confirmed as Dox-sensitive and Dox-resistant cell lines, with ICso values of 0.21 and 53.0 ⁇ g/ml, respectively ( Figure 23). Immunoblotting analysis confirmed that the Pgp expression in KB-Vl was > 1,000 times that of KB-31 cells ( Figure 23B).
  • KB-31 and KB-Vl cells were plated at 1 x 10 4 cells per well in 96 well plate.
  • the cells were treated with free Dox in solution in doses ranging from 0.05-3.2 ⁇ g/ml (KB-31) and 0.1-64 ⁇ g/ml (KB-Vl) for 72 hrs.
  • the cell viability was determined by the MTS assay.
  • the treated cells were incubated with MTS working solution for 2-3 hrs before measurement.
  • the mean absorbance of non-exposed cells served as the reference value for calculating 100% cellular viability.
  • the cells were washed in PBS and the pellets lysed in a buffer containing Triton X-100 and protease inhibitors after centrifugation.
  • the protein content of the supernatants was determined by the Bradford method. 100 ⁇ g total protein was electrophoresed by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking, the membranes were incubated with 1 : 1000 dilution of primary monoclonal antibody to MDR1 (anti-Pgp C219, Abeam). The membranes were overlayed with goat anti-mouse secondary antibody (1 :1000 dilution) before the addition of the HRP-conjugated streptavidin-biotin complex. The proteins were detected using ECL reagent according to the manufacturer's instructions.
  • Data represent the mean ⁇ SD for duplicate or triplicate measurements in each experiment, which was repeated at least 3 times. Differences between the mean values were analyzed by two-sided Student's t test or one way ANVOA and results were considered statistically significant dXp ⁇ 0.05.
  • PEI potential cytotoxicity
  • the polymer size plays an important role in the cytotoxicity that results from proton sequestration by unsaturated PEI amines in the lysosomal compartment (Xia et al., ACSNano, vol. 3, pp. 3273-3286, 2009).
  • the cells were treated with 100 ⁇ g/ml PEI-MSNP for 1, 4, 8, 16, 24, and 48 hrs, and the old medium was replenished by fresh medium for another 71, 68, 56, 48, and 24 hrs culture.
  • the cytotoxicity assay was checked at 72 hrs after particle treatment.
  • MSNPs were synthesized according to previously published sol-gel procedure (Xia et al., ACS Nemo, vol. 3, pp. 3273-3286, 2009; Jie et al, Small, vol. 3, pp. 1341- 1346, 2007). Briefly, for the synthesis of unmodified MSNP (OH-MSNP), 100 mg of CTAB was dissolved in a solution of 48 mL water and 0.35 mL sodium hydroxide (2 M) and heated to 80 °C. One half mL of TEOS was added into the aqueous solution containing CTAB surfactants.
  • phosphonate-modified MSNP were dispersed in a solution containing 2.5 mg PEI (1.8 kD, 10 kD, 25 kD) in 1 ml absolute ethanol. After sonication and stirring for 30 min the PEI coated particles were washed with PBS. The amount of polymer coated onto the particle surface was approximately 5 weight percentage.
  • organoalkoxysilanes made up in ethanol with TEOS before adding the mixture into the CTAB solution (Lim et al., J. Am. Chem. Soc, vol. 119, pp. 4090-4091, 1997).
  • carboxylate modification 50 ⁇ L cyanoethyltriethoxysilane was mixed with 500 ethanol and 500 ⁇ TEOS, then added into the surfactant solution. After the surfactant removal process, the particles were further heated in a solution of 50% sulfuric acid to hydro lyze the cyanide groups into carboxylic groups.
  • For amine modification 50 of aminopropyltriethoxysilane was first mixed with 500 ⁇ ethanol and 500 ⁇ . TEOS before adding to the surfactant solution. After 2 hrs, the solution was cooled to room temperature and the materials were washed with methanol before the surfactant removal process.
  • PEI-coated phosphonate MSNP were characterized for size, zeta potential, and shape, respectively.
  • the shape and porous structure were characterized using transmission electron microscopy (JEOL JEM 2010, JEOL USA, Inc., Peabody, MA).
  • Microfilms for TEM imaging were made by placing a drop of the respective MSNP suspensions onto a 200-mesh copper TEM grid (Electron Microscopy Sciences, Washington, PA) and then drying at room- temperature overnight. A minimum of 5 images for each sample was captured and representative images included in Figure 18.
  • Particle size and zeta potential in pure water, after stabilization with 1 mg/mL BSA in water, or in cell culture medium were measured by ZetaSizer Nano (Malvern Instruments Ltd., Worcestershire, UK). All the measurements were performed in 40 ⁇ g/ml MSNP suspensions in filtered water or filtered complete cell culture media at pH 7.4. The analysis was also studied on Dox loaded particles. Similar analysis was also performed on cargo (Dox and siRNA) loaded PEI-MSNP samples.
  • PEI polymers in the size range 1.8-25 kD were electrostatically bound to the phosphonate-MSNP surface. Polymer binding to 100-120 nm size MSNP, exhibiting uniform pore sizes of 2-2.5 nm was confirmed by TEM ( Figure 18A, left). The TEM image of the particles decorated with the 10 kD polymer shows that the surface coating (arrows) did not occupy the porous interior ( Figure 18A, right). To optimize the particle dispersal for biological experimentation, the PEI-coated particles were further treated with 1 mg/ml BSA before transfer to the complete cell culture medium.
  • agarose gel retardation assay was used to determine the siRNA binding to PEI-MSNP.
  • 0.1 ⁇ g siRNA was mixed with 0.4-6.4 ⁇ g amount PEI-MSNP in aqueous solution to obtain particle/nuclei acid (N/P) ratios of 4-64.
  • Nucleic acid bands were detected by UV light (254 nm) and the photos were captured in a Bio-Rad imaging system (Hercules, CA). The results were used to calculate the threshold N/P ratios for subsequent experiments.
  • the threshold is defined as the lowest N/P ratio value that prevents free siRNA from entering the gel.
  • the gel electrophoresis assay was also performed.
  • siRNA-PEI-MSNP complexes were freshly prepared as described above.
  • the siRNA duplex consists of 5'-r(CGGAAGGCCUAAUGCCGAA) dTdT (sense) and 5'- r(UUCGGCAUUAGGCCUUCCG) dTdG (antisense) strands.
  • KB-31 and KB-V1 cells were plated at 1 x 10 4 cells per well in 96 well plate.
  • the cells were treated with free Dox in solution in doses ranging from 0.05-3.2 ⁇ ⁇ 1 (KB-31) and 0.1-64 ⁇ g/ml (KB-V1) for 72 hrs.
  • the cell viability was determined by the MTS assay.
  • the treated cells were incubated with MTS working solution for 2-3 hrs before measurement.
  • the mean absorbance of non-exposed cells served as the reference value for calculating 100% cellular viability.
  • the cells were washed in PBS and the pellets lysed in a buffer containing Triton X-100 and protease inhibitors after centrifugation.
  • the protein content of the supernatants was determined by the Bradford method. 100 ⁇ g total protein was electrophoresed by 10% SDS-PAGE and transferred to a PVDF membrane. After blocking, the membranes were incubated with 1 :1000 dilution of primary monoclonal antibody to MDR1 (anti-Pgp C219, Abeam). The membranes were overlayed with goat anti -mouse secondary antibody (1 : 1000 dilution) before the addition of the HRP-conjugated streptavidin- biotin complex. The proteins were detected using ECL reagent according to the manufacturer's instructions.
  • a commercially available cationic liposomal transfection agent (Lipofectamine 2000) was used as a positive control. Protein abundance was quantified by densitometric scanning using a laser Personal Densitometer SI and Image Quant software (Amersham
  • Dox-loaded MSNP was suspended into 3 ml phenol red-free DMEM medium acidified to pH 5.0 or replenished with 10% (v/v) ethanol at 37 °C. The supernatants were collected at various time points and cleared by centrifugation for
  • MSNP are capable of loading and releasing water- insoluble drugs (paclitaxel and camptothecin) by a phase transfer mechanism that can be reversed by ethanol washing of the particles to demonstrate the role of hydrophobicity in MSNP drug entrapment (Xia et al., ACS Nano, vol. 3, pp. 3273-3286, 2009; Jie et al., Small, vol. 3, pp. 1341-1346, 2007).
  • One conceivable approach is electrostatic attachment to the negatively charged MSNP surface.
  • Dox could be released in a time-dependent manner from the phosphonate-MSNP or PEI-phosphonate-MSNP surface by lowering of the solution pH ( Figure 20C and 20D).
  • the Dox release profile was not be affected by siRNA binding to PEI on exterior surface of the particle ( Figure 28). This establishes the possibility that intracellular drug release should be possible if the particles are capable of gaining entrance to acidifying cellular compartments.
  • KB- VI cells grown on chamber slides were fixed, permeabilized, and labeled with standard immunocytochemistry protocol (Xia et al, ACSNano, vol. 3, pp. 3273-3286, 2009).
  • LAMP-1 staining was performed by using a 1 :500 dilution of mouse-anti-human mAb (H4A3, Abeam, USA) for 16 hrs at 4 °C. This was followed by a 1 :500 diluted TRITC- conjugated goat-anti-mouse secondary antibody (Santa Cruz, USA) for 1 hr at room
  • Dox uptake in KB- VI cells was quantitatively evaluated in a microplate reader at 72 hrs. 5 x 10 4 cells were placed into a 96 wells plate and treated with 2 g/ml free Dox or the equivalent amount of drug loaded into MSNP before or after PEI coating, with or without attachment of Pgp siRNA. Following the washing of the cells in cold PBS, the intracellular Dox fluorescence was detected at excitation and emission wavelength of 485/550 nm in a microplate reader (SpectraMax M5 Microplate Reader, Molecular Device, USA). Moreover, confocal images were captured at the end of experiment. Image J software (version 1.37c, NIH) was used to analyze the nuclear fluorescence.
  • the relative inefficiency of free Dox to induce KB-V1 cytotoxicity may be a result of the rapid rate by which the drug was being exported by the overexpressed Pgp.
  • Intracellular Dox concentration can be determined by measuring cellular Dox fluorescence intensity in a microplate reader ( Figure 21 A). The comparatively low drug uptake after treatment with free Dox was slightly improved by delivering the drug via the phosphonate- MSNP. While the total amount of intracellular drug increased when being delivered by particles coated with the 10 kD PEI polymer ( Figure 21 A), little of the drug reached the nucleus as determined by confocal imaging ( Figure 21B). Interestingly, the intracellular Dox concentration increased significantly in the presence of siRNA (Figure 21 A) so that there was also a significant increase in nuclear Dox staining by 72 hr ( Figure 21B).
  • Dox delivered by PEI- MSNP in the presence of siRNA significantly enhance intranuclear Dox concentration when compared to free Dox or Dox delivered by MSNP or PEI-MSNP without siRNA.
  • KB-V1 cells were treated with free Dox, Dox-MSNP, PEI-Dox-MSNP and siRNA-PEI-Dox-MSNP, respectively.
  • incubation time was for 16 hrs before replenishment of the old medium with fresh complete DMEM and performance of a MTS assay at 72 hrs.
  • the IC 50 of free and Dox-loaded MSNP were calculated.
  • the induction of apoptosis at 72 hours was assessed through the use of Annexin V-SYTOX Blue.
  • FITC -Annexin V- SYTOX Blue working solution (Annexin V, Trevigen; SYTOX Blue, Invitrogen) at room temperature for 15 min. The cells were washed in binding buffer before performance of flow cytometry (Becton Dickinson, Mountain View, CA). Date analysis was performed by BD CellQuest. To confirm the flow data in which there may be a minor overlap of Dox with FITC-Annexin V, a TUNEL detection kit was used according to the manufacturer's instructions to confirm the induction of apoptosis.
  • TUNEL-positive cells were washed, fixed, and permeabilized before TUNEL staining. The number of TUNEL-positive cells was assessed under a fluorescent microscope (200x). At least 3 fields were counted by the same investigator to calculate the percentage of TUNEL positive cells.
  • the MTS assay was used to compare KB-V1 cytotoxicity under incremental Dox concentrations ( Figure 22A). Based on the calculated IC50 values of the various formulations, it was possible to rank the killing efficiency as follows: siRNA-PEI-Dox-MSNP > PEI-Dox-MSNP « Dox-MSNP > free Dox. Moreover, the IC 50 value of the siRNA-delivering MSNP was approximately 2.5 times lower than the IC 50 of free Dox or other Dox-loaded particles. This suggests an additive effect between the drug and the siRNA that are being delivered by MSNP.
  • KB- VI cells were treated by co-administration of Dox and Tariquidar, which is a potent and effective Pgp inhibitor in human clinical trial, to see if KB- VI sensitivity can be restored to that seen in KB-31 cells. While this combination significantly improves cell killing capability in KB- VI cells, it is incapable of completely restoring Dox sensitivity to the level seen in KB-31 cells ( Figure 31).

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Abstract

Cette invention concerne une structure sous-micronique ayant un corps en silice définissant une pluralité de pores et un polymère cationique à la surface dudit corps en silice. La structure sous-micronique peut, en outre, comprendre un oligonucléotide et servir à administrer ledit oligonucléotide à une cellule. La structure sous-micronique peut, en outre, comprendre un agent thérapeutique et servir à administrer ledit agent thérapeutique à une cellule. Un oligonucléotide et un agent thérapeutique peuvent être utilisés ensemble. Par exemple, quand l'oligonucléotide est un ARNsi, la composition peut être utilisée pour réduire la résistance cellulaire à l'agent thérapeutique par réduction de la traduction d'un gène de résistance.
PCT/US2011/043874 2010-07-13 2011-07-13 Nanoparticules de silice mésoporeuses revêtues d'un polymère cationique et leurs utilisations Ceased WO2012009448A2 (fr)

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US9993437B2 (en) 2007-12-06 2018-06-12 The Regents Of The University Of California Mesoporous silica nanoparticles for biomedical applications
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