WO2025010407A2 - Structures organométalliques à échelle nanométrique avec promédicaments déclenchables par rayons x pour radiothérapie, chimiothérapie et immunothérapie combinées - Google Patents

Structures organométalliques à échelle nanométrique avec promédicaments déclenchables par rayons x pour radiothérapie, chimiothérapie et immunothérapie combinées Download PDF

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WO2025010407A2
WO2025010407A2 PCT/US2024/036893 US2024036893W WO2025010407A2 WO 2025010407 A2 WO2025010407 A2 WO 2025010407A2 US 2024036893 W US2024036893 W US 2024036893W WO 2025010407 A2 WO2025010407 A2 WO 2025010407A2
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dbp
mof
group
cells
ray
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WO2025010407A3 (fr
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Wenbin Lin
Ziwan XU
Wenyao ZHEN
Xiaomin Jiang
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University of Chicago
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/003—Compounds containing elements of Groups 4 or 14 of the Periodic Table without C-Metal linkages

Definitions

  • MOFs metal-organic frameworks
  • nMOFs nanoscale MOFs
  • the presently disclosed subject matter further relates to the use of the MOFs in treating diseases, such as cancer, e.g., via combinations of radiotherapy or immunotherapy with radiotherapy or radiotherapy-radiodynamic therapy.
  • APF aminophenyl fluorescein
  • BMDC bone marrow-derived dendritic cells
  • DBP bis(p-benzoato)porphyrin
  • DMRio% dose modifying ratio at 10% survival fraction
  • MOF metal-organic framework nm nanometer nMOF nanoscale metal-organic framework
  • TLR Toll-like receptor
  • MOFs Metal-organic frameworks
  • MOFs are of increasing interest for biomedical applications due to their tunable compositions, large porosity, ease of surface functionalization, and biodegradability. 1 ' 4 In particular, MOFs have found use as drug carriers. 5 ' 10
  • three approaches have been used to load drugs into MOFs: direct encapsulation of drugs in the pores, 11 ' 12 coordination of drugs to metal-cluster secondary building units (SBUs), 13 and covalent conjugation of drugs to the organic bridging ligands. 14
  • SBUs metal-cluster secondary building units
  • the first two approaches can result in premature drug release due to weak interactions between MOFs and drug molecules.
  • the third method can involve the use of an actionable trigger to release active drug from the prodrug-containing MOF. 16-17
  • MOF-based prodrug compositions comprising actionable triggers that can release drugs efficiently and on- demand, e.g., at a particular site in the body and/or at a particular time.
  • additional methods of using MOF-based compositions to treat diseases, such as cancer with improved activity and/or at lower doses and/or with reduced levels of side effects.
  • the presently disclosed subject matter provides a metal organic framework (MOF) comprising an X-ray sensitive prodrug, wherein the MOF comprises metal-containing secondary building units (SBUs) linked together via organic bridging ligands, wherein at least one of the SBUs comprises a metal cation capable of absorbing X- rays, and wherein at least one of the organic bridging ligands is substituted by a group having the formula: -L-D, wherein L is a bivalent linker group comprising one or more bonds capable of radical-promoted bond cleavage and wherein D is a monovalent moiety of a therapeutic agent.
  • MOF metal organic framework
  • the metal cation capable of absorbing X-rays is selected from the group comprising Hf, a lanthanide metal, Ta, W, Re, Os, Ir, Pt, Au, and Bi.
  • the metal cation capable of absorbing X-rays is an Hf cation, optionally wherein one or more of the SBUs comprise a Hf oxo cluster, optionally a Hfe oxo cluster or a Hfn oxo cluster.
  • L has the structure: wherein: X is -O-, -NR’-, -O-Xi-O-, or NH-Xi-NH-, wherein R’ is selected from H and alkyl, and Xi is alkylene, optionally wherein R’ is H or methyl and/or Xi is propylene or ethylene; and each R is hydroxyl, oxyalkyl, optionally methoxy, or dialkylamino, optionally dimethylamino.
  • the therapeutic agent is a chemotherapeutic agent or an immune modulator, optionally a TLR agonist or a STING agonist.
  • the therapeutic agent is selected from the group comprising SN38, R848, imiquimod, VTX-378, DSR-6434, ioxoribine, TLR7/8 agonist 1, Cu-712-9, neoseptin-3, and diABZI.
  • the at least one organic bridging ligand substituted by a group having the formula -L-D is an organic bridging ligand comprising a terphenyl (TP) moiety or a quaterphenyl (QP) moiety and at least two groups, which can be the same or different, selected from the group comprising a carboxylate group, an aromatic or non-aromatic nitrogen-containing group, a phenol, an acetylacetonate, a phosphonate, and a phosphate; optionally wherein the at least one organic bridging ligand has a structure selected from:
  • the MOF further comprises at least one photosensitizer, optionally wherein the MOF comprises at least one organic bridging ligand selected from the group consisting of a porphyrin, a chlorin, a chlorophyll, a phthalocyanine, a ruthenium- bipyridine complex, an iridium-phenylpyridine complex, or an iridium-bipyridine complex.
  • the MOF comprises at least one organic bridging ligand comprising a porphyrin, optionally bis(p-benzoato)porphyrin (DBP).
  • the presently disclosed subject matter provides a pharmaceutical composition
  • a pharmaceutical composition comprising an MOF comprising an X-ray sensitive prodrug, wherein the MOF comprises metal-containing SBUs linked together via organic bridging ligands, wherein at least one of the SBUs comprises a metal cation capable of absorbing X- rays, and wherein at least one of the organic bridging ligands is substituted by a group having the formula: -L-D, wherein L is a bivalent linker group comprising one or more bonds capable of radical-promoted bond cleavage and wherein D is a monovalent moiety of a therapeutic agent.
  • the presently disclosed subject matter provides a method of treating a disease in a subject in need thereof, the method comprising: (a) administering to the subject a MOF comprising an X-ray sensitive prodrug, wherein the MOF comprises metal-containing SBUs linked together via organic bridging ligands, wherein at least one of the SBUs comprises a metal cation capable of absorbing X-rays, and wherein at least one of the organic bridging ligands is substituted by a group having the formula: -L-D, wherein L is a bivalent linker group comprising one or more bonds capable of radical- promoted bond cleavage and wherein D is a monovalent moiety of a therapeutic agent, or a pharmaceutical composition of said MOF; and (b) irradiating at least a portion of the subject with X-rays.
  • the disease is cancer, optionally selected from the group comprising a head tumor, a neck tumor, a head and neck tumor, a breast tumor, a gynecological tumor, a brain tumor, a colorectal cancer, a lung cancer, mesothelioma, a soft tissue sarcoma, and a pancreatic cancer.
  • D is a monovalent moiety of a chemotherapeutic agent, thereby providing to the subject, upon performing (b), a combination of (i) chemotherapy and (ii) radiotherapy (RT) or radiotherapy -radiodynamic therapy (RT-RDT).
  • the MOF comprises a photosensitizer, optionally DBP, thereby providing to the subject, upon performing (b), a combination of (i) chemotherapy and (ii) RT-RDT.
  • D is monovalent moiety of SN38 and a higher percentage of SN38 is released from the MOF upon performing (b) compared to release from a comparable amount of a homogenous SN38 prodrug, optionally wherein the homogenous SN38 prodrug is MeO-SN.
  • D is a monovalent moiety of an immune modulator, thereby providing to the subject, upon performing (b), a combination of (i) immunotherapy and (ii) RT or RT-RDT.
  • the MOF comprises a photosensitizer, optionally DBP, thereby providing to the subject, upon performing (b) a combination of immunotherapy and RT-RDT.
  • the method provides selective cytotoxicity and/or immune activation in a tumor.
  • the presently disclosed subject matter provides a use of an MOF comprising an X-ray sensitive prodrug, wherein the MOF comprises metal-containing SBUs linked together via organic bridging ligands, wherein at least one of the SBUs comprises a metal cation capable of absorbing X-rays, and wherein at least one of the organic bridging ligands is substituted by a group having the formula: -L-D, wherein L is a bivalent linker group comprising one or more bonds capable of radical-promoted bond cleavage and wherein D is a monovalent moiety of a therapeutic agent; or a pharmaceutical composition comprising said MOF, in treating a disease, optionally cancer, in a subject in need thereof.
  • the use provides selective cytotoxicity and/or immune activation in a tumor.
  • nanoscale metal-organic frameworks comprising X-ray sensitive prodrugs, as well as related pharmaceutical formulations, uses, and methods of treating disease.
  • Figure 1 A schematic drawing showing the synthesis of a nanoscale metal-organic framework (nMOF) comprising hafnium (Hf)-containing secondary building units (SBUs) and 2’ -((4-(hydroxymethyl)-2,6-dimethoxybenzamido)methyl)-[ 1 , 1’ :4’ , 1’ ’ -terphenyl]-4,4” - dicar-boxylic acid (H2TP-OH) organic bridging ligands.
  • nMOF nanoscale metal-organic framework
  • Hf hafnium
  • SBUs secondary building units
  • This nMOF i.e., Hf-TP-OH
  • Hf-TP-OH is post- synthetically modified with a group comprising a monovalent moiety of 7-ethyl-10- hydroxycamptothecin (SN38) to afford Hf-TP-SN nMOF, an X-ray sensitive nMOF -based prodrug of SN38.
  • the proposed mechanism for X-ray triggered release of SN38 from Hf- TP-SN is shown on the bottom left.
  • Figures 2A-2C A series of micrograph images including ( Figure 2A) the transmission electron microscopy (TEM) micrograph image and ( Figure 2B) the high-resolution TEM (HRTEM) image and fast Fourier transform (FFT) pattern (inset) of a nanoscale metal- organic framework (nMOF) comprising hafnium-containing secondary building units (SBUs) and 2’-((4-(Hydroxymethyl)-2,6-dimethoxybenzamido)methyl)-[l,r :4’, 1 ”-terphenyl]-4,4”- dicarboxylic acid (H2TP-OH) organic bridging ligands, i.e., Hf-TP-OH.
  • nMOF nanoscale metal- organic framework
  • SBUs hafnium-containing secondary building units
  • H2TP-OH organic bridging ligands
  • Figure 2C is the TEM image of the 7-ethyl-10-hydroxycamptothecin (SN38) post-synthetically modified nMOF, i.e., Hf-TP-SN.
  • the scale bar in the lower left corner of each image represents 50 nanometers (nm).
  • Figures 3A-3D A series of graphs showing ( Figure 3A), the number averaged sizes (number expressed as a percentage (%) of the total population versus size in nanometers (nm)) of the nanoscale metal-organic frameworks (nMOFs) described for Figures 2A-2C, i.e., Hf-TP-OH and Hf-TP-SN, in ethanol; ( Figure 3B) the zeta (Q -potentials of Hf-TP-OH and Hf-TP-SN in water (H2O); (Figure 3C) the powder X-ray diffraction (PXRD) patterns (intensity versus 2 theta (20) of Hf-TP-OH, Hf-TP-SN, and the simulated pattern for Hfn-TP MOF; and (Figure 3D) the PXRD patterns of freshly prepared Hf-TP-SN or Hf-TP-SN dispersed in phosphate buffered saline (PBS) for 1, 2, 4, 8 and 24 hours.
  • Figures 4A-4E A series of graphs including ( Figures 4A, 4B, and 4D) graphs showing the ultraviolet-visible (UV-Vis) absorption spectra (absorbance versus wavelength (in nanometers (nm))) of ( Figure 4A) 7-ethyl-10-hydroxycamptothecin (SN38), a nanoscale metal-organic framework (nMOF) comprising hafnium-containing secondary building units (SBUs) and 2’-((4-(Hydroxymethyl)-2,6-dimethoxybenzamido)methyl)-[l, 1 ’ :4’, 1”- terphenyl]-4, 4” -dicarboxylic acid (H2TP-OH) organic bridging ligands, i.e., Hf-TP-OH, and a digested SN-38-modified nMOF, i.e., Hf-TP-SN; ( Figure 4B) H2TP-OH at different concentrations
  • hafnium (Hf) concentration was 40 micromolar (pM).
  • Figures 5C and 5D are graphs showing the concentration (pM) of 7-ethyl- 10-hydroxycamptothecin (SN38) released from the homogenous prodrug of SN38 (MeO-SN) or the nMOF prodrug, i.e., Hf-TP-SN, after 10 Gy X-ray irradiation ( Figure 5C) or after reaction with OH generated by the Fenton reaction ( Figure 5D).
  • Initial MeO-SN or Hf-TP- SN concentration was 100 pM.
  • Figures 6A-6D Figures 6A-6D are graphs showing the toxicity of the 2’-((4- (Hydroxymethyl)-2,6-dimethoxybenzamido)methyl)-[l,r :4’, 1 ”-terphenyl]-4,4”- dicarboxylic acid (i.e., H2TP-OH) ligand ( Figure 6A) and of the hafnium-containing nanoscale metal-organic framework (nMOF) prepared from the H2TP-OH ligand (i,e Hf-TP- OH, Figure 6B).
  • H2TP-OH hafnium-containing nanoscale metal-organic framework
  • Toxicity is assessed as cell viability of mouse colon carcinoma (CT26) cells treated by (Figure 6A) H2TP-OH without X-ray radiation (H2TP-OH(-)) or ( Figure 6B) Hf- TP-OH without X-ray radiation (Hf-TP-OH(-)) at the following concentrations: 0 micromolar (pM), 1.56 pM, 6.25 pM, 25 pM, or 100 pM.
  • Figure 6C is a graph showing the survival fractions of CT26 cells after incubation with phosphate buffered saline (PBS), Hf- TP-OH, or Hf-TP-SN under different doses of X-ray irradiation (0 Gray (Gy), 2 Gy, 4 Gy, or 6 Gy).
  • Figure 6D is a series of confocal laser scanning microscopy (CLSM) images of CT26 cells stained by hydroxyphenyl fluorescein (HPF, green) and Hoechst 33342 (blue, cell nucleus) for detecting the generation of OH.
  • CLSM confocal laser scanning microscopy
  • Figures 7A-7C are graphs showing the cell viability curves of mouse colon carcinoma (CT26) cells treated by ( Figure 7A) 7-ethyl-10-hydroxycamptothecin (SN38) without X-ray radiation (SN38(-)) or ( Figure 7B) the dimethyl ester of a SN38- modified terphenyl bridging ligand without X-ray radiation (Me2TP-SN38(-)).
  • Graphs show cell viability (expressed as a percentage (%)) versus SN38 or Me2TP-SN concentration (micromolar (pM)).
  • Figure 7C is a graph showing the time-dependent cellular uptake of a hafnium-containing nanoscale metal-organic framework (nMOF) comprising an X-ray sensitive SN38 prodrug (Hf-TP-SN) quantified by inductively coupled plasma mass spectroscopy (ICP-MS).
  • Hafnium (Hf) uptake (measured in nanomoles per 10 6 cells (nmol/10 6 cells)) is measured at 0, 1, 2, and 6 hours (h).
  • n 3.
  • Figures 8A-8D ROS generation in vitro.
  • Figure 8A is a series of confocal laser scanning microscopy (CLSM) images of mouse colon carcinoma (CT26) cells stained by 2’,7’-dichlorofluorescein diacetate (DCFH-DA, top and bottom rows) and/or Hoechst (middle and bottom row) for detecting the generation of reactive oxygen species (ROS).
  • CLSM confocal laser scanning microscopy
  • Figure 8B is a graph showing the relative fluorescence intensity (ROS signal) from the CLSM images of Figure 8A as analyzed by Image J.
  • Figure 8C is a graph showing the quantification of histograms of intracellular ROS signals of cells treated as described in Figure 8A by flow cytometry.
  • Figure 8D is a graph showing the percentage of dichlorofluorescein-positive (DCF + ) CT26 cells after Hf-TP-OH(+) or Hf-TP-SN(+) treatment.
  • X-ray dose was 3 Gray (Gy).
  • FIG. 9 Confocal laser scanning microscopy (CLSM) images of mouse colon carcinoma (CT26) cells showing results of the phosphorylation of histone variant H2AX (y- H2AX) assay and treated with phosphate buffered saline (PBS) with (+) or without (-) X-ray radiation; a hafnium (Hf) and terphenyl (TP)-ligand containing nanoscale metal organic framework nMOF without a prodrug (Hf-TP-OH) with (+) or without (-) X-ray radiation, or a HF and TP-containing nMOF comprising an X-ray sensitive 7-ethyl-10- hydroxycamptothecin prodrug (Hf-TP-SN) with (+) or without (-) X-ray radiation.
  • the scale bar in the lower right comer of the image on the bottom right represents 10 micrometers (pm). X-ray dose was 3 Gray (Gy).
  • FIG. 10 Immunogenic cell death. Representative flow cytometry dot plots showing cell apoptosis/death stained by fluorescein isothiocyanate (FITC)-annexin-V and propidium iodide (PI) in different treatment groups: phosphate buffered saline (PBS) with (+) or without (-) X-ray radiation; hafnium (Hf) and terphenyl (TP)-ligand containing nanoscale metal organic framework (nMOF) without a prodrug (Hf-TP-OH) with (+) or without (-) X- ray radiation; and Hf and TP-containing nMOF with an X-ray sensitive prodrug (Hf-TP-SN) with (+) and without (-) X-ray radiation.
  • PBS phosphate buffered saline
  • Hf hafnium
  • TP terphenyl
  • nMOF nanoscale metal organic framework
  • Hf-TP-OH prodrug
  • Figures 11A-11D In vivo anticancer efficacy.
  • Figure 11A is a graph showing growth curves of subcutaneous mouse colon carcinoma (CT26) tumors implanted in BALB/c mice and after treatment with phosphate buffered saline (PBS), irinotecan, a hafnium (Hf) and terphenyl (TP)-ligand containing nanoscale metal organic framework (nMOF) without a prodrug (Hf-TP-OH), or a Hf and TP-containing nMOF with an X-ray sensitive 7-ethyl-10- hydroxycamptothecin prodrug (Hf-TP-SN) followed by X-ray irradiation (+).
  • CT26 subcutaneous mouse colon carcinoma
  • PBS phosphate buffered saline
  • TP terphenyl
  • nMOF nanoscale metal organic framework
  • Hf-TP-OH X-ray sensitive 7-ethyl-10- hydroxycamptothec
  • FIG. 1 IB is a graph showing the weights (in grams (g)) of the excised tumors at the endpoint (day 19) of mice treated as described for Figure 11 A, while Figure 11C is a photographic image showing the excised tumors.
  • Figure 1 ID is a graph showing relative body weights (expressed as a percentage) of CT26 bearing BALB/c mice after different treatments as a function of time (days) during the treatment regimen described for Figure 11 A.
  • FIG. 12 Microscopy images showing pathological changes of tumors from the treatments described for Figures 11A-11D. Top row shows detection of phosphorylation of histone variant H2AX (y-H2AX); row second from top shows detection for antigen Kiel 67 (Ki67), a marker of proliferation; row second from the bottom shows detection for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL); and the bottom row shows hematoxylin and eosin (H&E) staining of excised mouse colon carcinoma (CT26) tumors after the treatments described for Figures 11 A-l ID.
  • the scale bar in the bottom right corner of the image on the bottom right represents 50 micrometers (pm).
  • FIG 13 A series of images of hematoxylin and eosin (H&E) staining of hearts, livers, spleens, lungs, and kidneys of mouse colon carcinoma (CT26) tumor-bearing BALB/c mice in different treatment groups as described for Figures 11 A-l ID.
  • the scale bar in the lower right comer of the image on the bottom right represents 200 micrometers (pm).
  • Figures 14A-14B Synthesis of a Hf-DBP-QP-SN nanoscale metal-organic framework (nMOF) and proposed mechanism of action.
  • Figure 14A is a schematic diagram showing the synthesis of a dicarboxylic acid quaterphenyl (H2QP) bridging ligand containing a dimethoxy benzyl alcohol derivative.
  • H2QP dicarboxylic acid quaterphenyl
  • Figure 14B is a schematic diagram showing the synthesis of a hafnium (Hf)-bis-(p-benzoato)porphyrin (DBP)-quaterphenyl (QP) nMOF (i.e., Hf-DBP-QP) and its post-synthetic modification with 7-ethyl-10-hydroxycamptothecin (SN38) to afford a prodrug nMOF, i.e., Hf-DBP-QP-SN along with the proposed mechanism for X-ray triggered release of SN38 from Hf-DBP-QP-SN for synergistic radiotherapy-radiodynamic therapy (RT-RDT) and chemotherapy.
  • Hf hafnium
  • DBP bis-(p-benzoato)porphyrin
  • QP quadterphenyl
  • nMOF i.e., Hf-DBP-QP
  • SN38 7-ethyl-10-hydroxycamptothecin
  • Figures 15A-15D Transmission electron microscopy (TEM) and dynamic light scattering (DLS) characterization.
  • Figures 15A and 15B show TEM images of ( Figure 15 A) nanoscale metal organic frameworks (nMOFs) containing hafnium (Hf) secondary building units and both bis(p-benzoato)porphyrin (DBP) and quaterphenyl (QP) bridging ligands (i.e., Hf-DBP-QP) and (Figure 15B) a Hf-DBP-QP nMOF post-synthetically modified as a prodrug of 7-ethyl-10-hydroxycamptothecin (Hf-DBP-QP-SN).
  • nMOFs nanoscale metal organic frameworks
  • Hf hafnium
  • QP quaterphenyl
  • Figure 15B a Hf-DBP-QP nMOF post-synthetically modified as a prodrug of 7-ethy
  • FIG. 15C is a graph showing the number-averaged sizes (percentage (%) versus size in nm) of the Hf-DBP-QP and HF-DBP- SN nMOFs.
  • Figure 15D is a graph showing the zeta ( -potentials of the Hf-DBP-QP and Hf-DBP-QP-SN nMOFs.
  • Figures 16A-16B Crystallinity and stability of Hf-DBP-QP-SN.
  • Figure 16A shows the powder x-ray diffraction (PXRD) patterns (intensity versus 2 theta (20)) of (bottom) a hafnium-bis(p-benzoato)porphyrin (Hf-DBP) nanoscale metal-organic framework (nMOF), (middle) a hafnium-bis(p-benzoato)porphyrin-quaterphenyl (Hf-DBP-QP) nMOF, and (top) a 7-ethyl-10-hydroxycamptothecin prodrug modified Hf-DBP-QP nMOF, i.e., Hf-DBP-QP-SN nMOF.
  • PXRD powder x-ray diffraction
  • Figure 16B is a graph showing the PXRD patterns (intensity versus 20) of (from top to bottom) Hf-DBP-QP-SN nMOF freshly prepared or after dispersion in phosphate buffered saline (PBS) for 1, 2, 4, 8, or 24 hours.
  • FIG 17 Ultraviolet- visible (UV-Vis) spectra (absorbance versus wavelength in nanometers (nm)) of 7-ethyl-10-hydroxycamptothecin (SN38), dicarboxyl-quaterphenyl (H2QP) bridging ligand, bis(p-benzoato)porphyrin (H2DBP) bridging ligand, and digested hafnium (Hf)-containing nanoscale metal-organic frameworks (nMOFs), including a nMOF containing both bis(p-benzoato)porphyrin and quaterphenyl bridging ligands (i.e., Hf-DBP- QP) and the same nMOF post-synthetically modified with SN38 (i.e., Hf-DBP-QP-SN) in dimethyl sulfoxide (DMSO).
  • UV-Vis UV-visible
  • Figures 18A-18D Ultraviolet-visible (UV-Vis) standard curves for quantification.
  • Figures 18A and 18C are the UV-Vis absorption spectra (absorbance versus wavelength in nanometers (nm)) of ( Figure 18 A) a dicarboxyl-quaterphenyl (H2QP) bridging ligand and (Figure 18C) a bis(p-benzoato)porphyrin (H2DBP) bridging ligand.
  • Figures 18B and 18D are the fitted standard curves (absorbance versus concentration (in milligrams per liter (mg/L))) of ( Figure 18B) H2QP and (Figure 18D) H2DBP.
  • Figures 19A-19C Release mechanism and reactive oxygen species (ROS) generation in test tubes.
  • Figure 19A is a schematic diagram showing a mechanism for X-ray triggered release of 7-ethyl-10-hydroxycamptothecin (SN38) from a hafnium (Hf)-containing nanoscale metal-organic framework (nMOF) comprising bis(p-benzoato)porphyrin and SN38-prodrug-contianing quarterphenyl bridging ligands, (Hf-DBP-QP-SN) via radiotherapy-radiodynamic therapy (RT-RDT).
  • Hf-DBP-QP-SN hafnium
  • RT-RDT radiotherapy-radiodynamic therapy
  • Figures 20A-20B are graphs showing the concentration of 7- ethyl-10-hydroxycamptothecin (SN38, micromolar (pM)) released from a homogenous methoxy-prodrug of SN38 (MeO-SN) or a hafnium (Hf)-containing metal organic framework (nMOF) prodrug comprising bis(p-benzoato)porphyrin and SN38-modifed quaterphenyl bridging ligands (i.e., Hf-DBP-QP-SN) ( Figure 20A) after 10 Gray (Gy) X-ray irradiation or ( Figure 20B) after reaction with hydroxy radical ( OH) generated by the Fenton reaction.
  • Initial MeO-SN or Hf-DBP-QP-SN concentration was 100 pM.
  • Figure 21 A graph showing cell viability of mouse colon carcinoma (CT26) cells after incubation with a hafnium-containing metal organic framework (nMOF) comprising bis(p-benzoato)porphyrin and quaterphenyl bridging ligands (Hf-DBP-QP).
  • CT26 mouse colon carcinoma
  • nMOF hafnium-containing metal organic framework
  • Hf-DBP-QP quaterphenyl bridging ligands
  • Figures 22A-22C Cytotoxicity of 7-ethyl-10-hydroxycamptothecin (SN38) and prodrugs.
  • Figures 22A-22C are graphs showing the cell viability of mouse colon carcinoma (CT26) cells after incubation with different concentrations (micromolar (pm) of ( Figure 22A) SN38 ( Figure 22B) a SN38 modified quaterphenyl homogenous compound (Me2QP-SN), and (Figure 22C) a SN38-prodrug nanoscale metal-organic framework (nMOG) comprising hafnium (Hf)-containing secondary building units and both bis(p-benzoato)porphyrin (DBP) and SN-38-modified quaterphenyl organic bridging ligands (Hf-DBP-QP-SN).
  • CT26 mouse colon carcinoma
  • PMP SN38 modified quaterphenyl homogenous compound
  • Figure 22C a SN38-prodrug nanoscale metal-organic framework
  • Figure 23 A graph showing the time-dependent cellular uptake of 7-ethyl-10- hydroxycamptothecin (SN38)-prodrug nanoscale metal-organic frameworks (nMOFs) comprising hafnium (Hf)-containing secondary building units and both bis(p- benzoato)porphyrin (DBP) and SN-38-modified quaterphenyl organic bridging ligands (Hf- DBP-QP-SN) as quantified by flow cytometry.
  • nMOFs 7-ethyl-10- hydroxycamptothecin
  • nMOFs nanoscale metal-organic frameworks
  • Hf hafnium
  • DBP bis(p- benzoato)porphyrin
  • Hf- DBP-QP-SN SN-38-modified quaterphenyl organic bridging ligands
  • FIG 24 A graph showing the total reactive oxygen species (ROS) generation in mouse colon carcinoma (CT26) cancer cells after different treatments (phosphate buffered saline (PBS), a hafnium (Hf)-containing nanoscale metal-organic framework (nMOF) with both bis(p-benzoato)porphyrin (DBP) and quaterphenyl organic bridging ligands (Hf-DBP- QP), or the Hf-DBP-QP nMOF modified as a prodrug of 7-ethyl-10-hydroxycamptothecin (SN38), i.e., Hf-DBP-QP-SN, all with (+) or without (-) X-ray irradiation), as probed by dichlorofluorescein-diacetate (DCFH-DA) and quantified by flow cytometry.
  • the X-ray dose is used, was 3 Gray (Gy).
  • FIG. 25 A series of confocal light scanning microscopy (CLSM) images showing mouse colon carcinoma (CT26) cells after different treatments (phosphate buffered saline (PBS), a hafnium (Hf)-containing nanoscale metal-organic framework (nMOF) with both bis(p-benzoato)porphyrin (DBP) and quaterphenyl organic bridging ligands (Hf-DBP-QP), or the Hf-DBP-QP nMOF modified as a prodrug of 7-ethyl-10-hydroxycamptothecin (SN38), i.e., Hf-DBP-QP-SN, all with (+) or without (-) X-ray irradiation) stained by dichloroflurescein-diacetate (DCFH-DA, top and bottom rows) and Hoechst (middle and bottom rows) for reactive oxygen species (ROS) detection.
  • PBS phosphate buffered saline
  • Figure 26 A series of confocal light scanning microscopy (CLSM) images showing mouse colon carcinoma (CT26) cells after different treatments (phosphate buffered saline (PBS), a hafnium (Hf)-containing nanoscale metal-organic framework (nMOF) with both bis(p-benzoato)porphyrin (DBP) and quaterphenyl organic bridging ligands (Hf-DBP-QP), or the Hf-DBP-QP nMOF modified as a prodrug of 7-ethyl-10-hydroxycamptothecin (SN38), i.e., Hf-DBP-QP-SN, all with (+) or without (-) X-ray irradiation) stained by hydroxyphenyl fluorescein (HPF, top and bottom rows) and Hoechst (middle and bottom rows) for hydroxy radical ( OH) detection.
  • PBS phosphate buffered saline
  • nMOF ha
  • Figure 27A is a pair of flow cytometric analysis of mouse colon carcinoma (CT26) cells treated with (right) or without (left) 7-ethyl-10-hydroxycamptothecin (SN38).
  • Figure 27B is a series of confocal laser scanning microscopy (CLSM) images showing reactive oxygen species (ROS) generation (probed by dichlorofluorescein-diacetate (DCFH-DA)) in CT26 cells after incubation with SN38.
  • the scale bar in the lower right of the image on the lower right represents 50 micrometers (pm).
  • Figure 27C is a series of CLSM images showing hydroxy radical (OH) generation (probed by hydroxyphenyl fluorescein (HPF)) in CT26 cells after incubation with SN38.
  • the scale bar in the lower right of the image on the lower right represents 50 pm.
  • Figure 27D is a series of CLSM images showing mitochondria membrane potentials (probed by 5,5,6,6’-tetrachloro-l,r,3,3’- tetraethylbenzimidazolyl carbocyanine iodide dye (JC-1)).
  • the scale bar in the lower right of the image on the lower right represents 10 pm.
  • Figures 28A-28C Clonogenic assay and immunogenetic cell death.
  • Figure 28A is a graph showing the results of clonogenic assays showing radioenhancement of a hafnium (Hf)-containing nanoscale metal-organic framework (nMOF) containing bis(p- benzoato)porphyrin (DBP) and quaterphenyl (QP) organic bridging ligands (Hf-DBP-QP) and of the same nMOF modified as a prodrug of 7-ethyl-10-hydroxycamptothecin (Hf-DBP- QP-SN) on mouse colon carcinoma (CT26) cells upon radiation with different doses of X- rays (0 to 8 Gray (Gy).
  • n 3.
  • Figure 28B is a series of respective images of colony formation of CT26 cells after treatment with Hf-DBP-QP or Hf-DBP-QP-SN followed by 250 peak kilovoltage (kVp) X-ray irradiation.
  • Figure 28C is a graph showing the quantification (as a percentage) of early apoptotic (top portion of each bar), late apoptotic (middle portion of bars with three sections or bottom portion of bars with two sections), and necrotic (bottom portion of bars with three portions) CT26 cells after incubation with Hf-DBP-QP or Hf-DBP-QP-SN with (+) or without (-) X-ray radiation (X-ray dose: 3 Gy).
  • Figure 29 A series of confocal light scanning microscopy (CLSM) images of mouse colon carcinoma (CT26) cells using the phosphorylation of histone variant H2AX (y-H2AX) assay after different treatments (phosphate buffered saline (PBS), a hafnium (Hf)-containing nanoscale metal-organic framework (nMOF) with both bis(p-benzoato)porphyrin (DBP) and quaterphenyl organic bridging ligands (Hf-DBP-QP), or the Hf-DBP-QP nMOF modified as a prodrug of 7-ethyl-10-hydroxycamptothecin (SN38), i.e., Hf-DBP-QP-SN, all with (+) or without (-) X-ray irradiation).
  • the scale bar in the lower left corner of the image on the lower right represents 10 micrometers (pm).
  • PBS phosphate buffered saline
  • Hf hafnium
  • nMOF nanoscale metal-organic framework
  • DBP bis
  • Figure 30B is a graph showing the relative body weights of CT26 tumor-bearing BALB/c mice being treated with the different treatments as a function of time (day) after tumor.
  • FIGs 31 Microscopy images showing pathological changes of excised mouse colon carcinoma (CT26) tumors from the study described for Figures 30A and 30B at day 1 after the last X-ray radiation dose.
  • the top row shows hematoxylin and eosin (H&E) staining of excised tumors; the row second from the top shows detection for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in the excised tumors; the row second from the bottom shows detection of phosphorylation of histone variant H2AX (y- H2AX) in the excised tumors; the bottom row shows detection for antigen Kiel 67 (Ki67), a marker of proliferation, in the excised tumors.
  • the scale bar in the bottom right corner of the image on the bottom right represents 50 micrometers (pm).
  • Figure 32 A series of images of hematoxylin and eosin (H&E) staining of hearts, livers, spleens, lungs, and kidneys of mouse colon carcinoma (CT26) tumor-bearing BALB/c mice in different treatment groups as described for Figures 30A and 30B.
  • the scale bar in the lower right corner of the image on the bottom right represents 200 micrometers (pm).
  • Figure 33 A graph showing the survival curves of mouse colon carcinoma (CT26) tumor-bearing BALB/c mice after different treatments as described for Figures 30A and 30B.
  • Figure 34 is a graph showing the interleukin-6 (IL-6) secretion (in picograms per milliliter (pg/mL)) of bone marrow-derived dendritic cells (BMDCs) after treatment with various concentrations (125 nanomolar (nM), 250 nM, 500 nM, or 1000 nM) of a nanoscale metal-organic framework (nMOF) comprising hafnium (Hf) secondary building units (SBUs), bis(p-benzoato)porphyrin (DBP) bridging ligands and quarterphenyl (QP) bridging ligands modified with an X-ray sensitive prodrug of resiquimod (R848) under various doses of X-ray irradiation (0 Gray (Gy), 1 Gy, 5 Gy, 10 Gy, or 50 Gy, bars from left to right for each concentration). The concentration refers to the conjugated R848.
  • nMOF nanoscale metal-organic framework
  • SBUs ha
  • FIGS 35A-35C are a series of schematic drawings showing the chemical structures of metal-organic frameworks (MOFs) comprising exemplary prodrugs of immune modulators, including (Figure 35A) Toll-like receptor (TLR) 7/8 agonists; ( Figure 35B) other TLR agonists; and ( Figure 35C) stimulator of interferon genes (STING) agonists.
  • MOFs metal-organic frameworks
  • TLR Toll-like receptor
  • Figure 35B other TLR agonists
  • STING interferon genes
  • MOFs Heavy metal -based metal-organic frameworks
  • nMOFs nanoscale heavy metalbased metal-organic frameworks
  • ROS reactive oxygen species
  • MOFs e.g., nMOFs
  • X-ray triggerable prodrugs that can harness the ROS for on-demand release of a therapeutic agent, thereby resulting in effective combination therapies.
  • the presently disclosed MOFs provide X-ray triggered release of chemotherapy and/or immunotherapy agents, providing for selective cytotoxic or immune activation in tumors. This can greatly reduce the general toxicity associated with chemotherapy and/or immunotherapy agents when provided in more conventional form, improving their therapeutic indices.
  • MOFs disclosed herein include nMOFs comprising an X-ray triggerable 7- ethyl-10-hydroxycamptothecin (SN38) prodrug and nMOFs with X-ray triggerable innate immune modulator prodrugs (e.g., resiquimod (R848) prodrugs) for synergistic radiotherapy (RT) and chemotherapy or immunotherapy.
  • nMOFs comprising an X-ray triggerable 7- ethyl-10-hydroxycamptothecin (SN38) prodrug and nMOFs with X-ray triggerable innate immune modulator prodrugs (e.g., resiquimod (R848) prodrugs) for synergistic radiotherapy (RT) and chemotherapy or immunotherapy.
  • SN38 X-ray triggerable 7- ethyl-10-hydroxycamptothecin
  • R848 X-ray triggerable innate immune modulator prodrugs
  • electron- dense Hfn oxo cluster or other X-ray absorbing, metal-containing secondary building units can serve as radiosensitizers to enhance hydroxyl radical generation for the triggered release of the therapeutic agent (e.g., the SN38 or innate immune modulator) of the covalently attached prodrug via hydroxylation of a 3,5-dimethoxylbenzyl carbonate followed by 1,4-elimination.
  • the therapeutic agent e.g., the SN38 or innate immune modulator
  • Hf-TP-SN An exemplary nMOF comprising a SN38 prodrug attached to a terphenyl-based bridging ligand, referred to herein as Hf-TP-SN, provided 5-fold higher release of SN38 from Hf-TP-SN than a comparable amount of a homogeneous counterpart, i.e., MeO-SN.
  • Hf-TP-SN plus X-ray irradiation can induce significant cytotoxicity to cancer cells in vitro and efficiently inhibits tumor growth in vivo with a high tumor growth inhibition index of 0.965 in a murine colon carcinoma model.
  • the term “about”, when referring to a value or to an amount of size (i.e., diameter), weight, concentration or percentage is meant to encompass variations of in one example ⁇ 20% or ⁇ 10%, in another example ⁇ 5%, in another example ⁇ 1%, and in still another example ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods.
  • the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
  • the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim.
  • the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
  • alkyl can refer to C1-20 inclusive, linear (z.e., "straightchain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (z.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • Lower alkyl refers to an alkyl group having 1 to about 8 carbon atoms (z.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.
  • Higher alkyl refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • alkyl refers, in particular, to C1-8 straight-chain alkyls.
  • alkyl refers, in particular, to C1-8 branched-chain alkyls.
  • Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxy carbonyl, oxo, and cycloalkyl.
  • alkyl chain there can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.
  • substituted alkyl includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • aryl is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety.
  • the common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine.
  • aryl specifically encompasses heterocyclic aromatic compounds.
  • the aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others.
  • the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.
  • the aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", wherein R' and R" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.
  • substituted aryl includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.
  • aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.
  • Heteroaryl refers to an aryl group that contains one or more noncarbon atoms (e.g., O, N, S, Se, etc) in the backbone of a ring structure.
  • Nitrogen-containing heteroaryl moieties include, but are not limited to, pyridine, imidazole, benzimidazole, pyrazole, pyrazine, triazine, pyrimidine, and the like.
  • Alkyl refers to an -alkyl-aryl group, optionally wherein the alkyl and/or aryl moiety is substituted.
  • Alkoxyl or “oxyalkyl” refer to an alkyl-O- group wherein alkyl is as previously described.
  • alkoxyl as used herein can refer to C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, /-butoxyl, and pentoxyl.
  • Alkylene refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.
  • the alkylene group can be straight, branched or cyclic.
  • the alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described.
  • An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.
  • arylene refers to a bivalent aromatic group, e.g., a bivalent phenyl or napthyl group.
  • the arylene group can optionally be substituted with one or more aryl group substituents and/or include one or more heteroatoms.
  • amino refers to the group -N(R)2 wherein each R is independently H, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, or substituted aralkyl.
  • aminoalkyl and alkylamino can refer to the group -N(R)2 wherein each R is H, alkyl or substituted alkyl, and wherein at least one R is alkyl or substituted alkyl.
  • Arylamine and “aminoaryl” refer to the group -N(R)2 wherein each R is H, aryl, or substituted aryl, and wherein at least one R is aryl or substituted aryl, e.g., aniline (i.e., -NHCeHs).
  • Dialkylamino refers to an -NRR' group wherein each of R and R' is independently an alkyl group and/or a substituted alkyl group as previously described.
  • exemplary alkylamino groups include ethylmethylamino, dimethylamino, and diethylamino.
  • halo refers to fluoro, chloro, bromo, and iodo groups.
  • hydroxyl and “hydroxy” refer to the -OH group.
  • mercapto or “thiol” refer to the -SH group.
  • carbboxylate when the term “carboxylate” is used in reference to an anion of a SBU, the term “carboxylate” can be used to refer to the anion HCCh' and, thus, can be synonymous with the term “formate”.
  • each R can be independently H, alkyl, aralkyl, aryl, or a negative charge (i.e., wherein effectively there is no R group present to bond to the oxygen atom, resulting in the presence of an unshared pair of electrons on the oxygen atom).
  • each R can be present or absent, and when present is selected from H, alkyl, aralkyl, or aryl.
  • bonding or “bonded” and variations thereof can refer to either covalent or non-covalent bonding. In some cases, the term “bonding” refers to bonding via a coordinate bond.
  • conjugation can refer to a bonding process, as well, such as the formation of a covalent linkage or a coordinate bond.
  • prodrug refers to a chemical entity that, upon administration to a recipient, is capable of providing (directly or indirectly) a pharmaceutically active compound (e.g. a known pharmaceutically active compound) or an active metabolite or residue thereof, either based on conditions already present in at least a portion of the recipient and/or based on conditions that can be introduced deliberately to at least a portion of the recipient.
  • a pharmaceutically active compound e.g. a known pharmaceutically active compound
  • an active metabolite or residue thereof either based on conditions already present in at least a portion of the recipient and/or based on conditions that can be introduced deliberately to at least a portion of the recipient.
  • the pharmaceutically active compound can be referred to as the “parent drug” or “parent compound” of the prodrug.
  • a prodrug can be a derivative of a pharmaceutically active compound that comprises one or more groups or bonds that can be cleaved under particular conditions, such as at a certain pH, in the presence of a particular type of enzyme, or in the presence of one or more other chemical entities).
  • groups or bonds can include, but are not limited to, an ester, carbamate, carbonate, phosphate ester, azo group or amide, depending upon the particular conditions that can cleave the groups or bonds.
  • the prodrug has less pharmaceutical activity than the parent compound (i.e., the pharmaceutically active compound upon which the structure of the prodrug is based and to which the prodrug can be transformed in vivo).
  • the prodrug compound has no measurable inhibitory activity prior to transformation to the parent compound.
  • metal-organic framework or “MOF” refers to a solid two- or three-dimensional network comprising both metal and organic components, wherein the organic components include at least one, and typically more than one carbon atom.
  • the material is crystalline. In some embodiments, the material is amorphous. In some embodiments, the material is porous.
  • the metal-organic matrix material is a coordination polymer, which comprises repeating units of coordination complexes comprising a metal-based secondary building unit (SBU), such as a metal ion or metal complex, and a bridging polydentate (e.g., bidentate or tridentate) organic ligand.
  • SBU metal-based secondary building unit
  • bridging polydentate e.g., bidentate or tridentate
  • the material contains more than one type of SBU or metal ion.
  • the material can contain more than one type of organic bridging ligand.
  • nanoscale metal-organic framework can refer to a nanoscale particle comprising an MOF.
  • nanoscale particle refers to a structure having at least one region with a dimension (e.g., length, width, diameter, etc.) of less than about 1,000 nm.
  • the dimension is smaller (e.g., less than about 500 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, or even less than about 30 nm).
  • the dimension is between about 30 nm and about 250 nm (e.g., about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nm).
  • the nanoparticle is approximately spherical.
  • the characteristic dimension can correspond to the diameter of the sphere.
  • the nanomaterial can be disc-shaped, plate-shaped (e.g., hexagonally plate-like), oblong, polyhedral, rod-shaped, cubic, or irregularly-shaped.
  • a “coordination complex” is a compound in which there is a coordinate bond between a metal ion and an electron pair donor, ligand or chelating group.
  • ligands or chelating groups are generally electron pair donors, molecules or molecular ions having unshared electron pairs available for donation to a metal ion.
  • coordinate bond refers to an interaction between an electron pair donor and a coordination site on a metal ion resulting in an attractive force between the electron pair donor and the metal ion.
  • coordinate bond refers to an interaction between an electron pair donor and a coordination site on a metal ion resulting in an attractive force between the electron pair donor and the metal ion.
  • the use of this term is not intended to be limiting, in so much as certain coordinate bonds also can be classified as having more or less covalent character (if not entirely covalent character) depending on the characteristics of the metal ion and the electron pair donor.
  • ligand refers generally to a species, such as a molecule or ion, which interacts, e.g., binds, in some way with another species. More particularly, as used herein, a “ligand” can refer to a molecule or ion that binds a metal ion in solution to form a “coordination complex.” See Martell, A, E,, and Hancock, R, D,, Metal Complexes in Aqueous Solutions, Plenum: New York (1996), which is incorporated herein by reference in its entirety. The terms “ligand” and “chelating group” can be used interchangeably.
  • bridging ligand can refer to a group that bonds to more than one metal ion or complex, thus providing a “bridge” between the metal ions or complexes.
  • Organic bridging ligands can have two or more groups with unshared electron pairs separated by, for example, an alkylene or arylene group. Groups with unshared electron pairs, include, but are not limited to, -CO2H, -NO2, amino, hydroxyl, thio, thioalkyl, -B(0H)2, -SO3H, PO3H, phosphonate, and heteroatoms (e.g., nitrogen, oxygen, or sulfur) in heterocycles.
  • heteroatoms e.g., nitrogen, oxygen, or sulfur
  • coordination site when used herein with regard to a ligand, e.g., a bridging ligand, refers to a unshared electron pair, a negative charge, or atoms or functional groups cable of forming an unshared electron pair or negative charge (e.g., via deprotonation under at a particular pH).
  • small molecule refers to a non-polymeric compound typically having a molecular weight below about 1000 daltons (Da), below about 900 Da, or below about 800 Da.
  • photosensitizer refers to a chemical compound or moiety that can be excited by light of a particular wavelength, typically visible or near-infrared (NIR) light, and produce a reactive oxygen species (ROS).
  • NIR visible or near-infrared
  • ROS reactive oxygen species
  • the photosensitizer in its excited state, can undergo intersystem crossing and transfer energy to oxygen (O2) (e.g., in tissues being treated by PDT) to produce ROSs, such as singlet oxygen ( X O2).
  • O2 oxygen
  • X O2 singlet oxygen
  • a PS can be excited (without exposure to light) via transfer of energy from a group that is capable of absorption of X-rays (e.g., an X- ray absorbing metal in a metal-containing SBU).
  • the PS can generate ROS, such as singlet oxygen, as part of a process referred to herein as “radiodynamic therapy” (RDT).
  • RDT radiodynamic therapy
  • the photosensitizer is a porphyrin, a chlorophyll, a dye, or a derivative or analog thereof, such as a porphyrin, chlorophyll or dye comprising one or more additional aryl or alkyl group substituents, having one or more carbon-carbon double bonds replaced by a carbon-carbon single bond, and/or comprising a substituent (e.g., a substituted alkylene group) that can covalently substituted with a bond to an organic bridging ligand).
  • porphyrins, chlorins, bacteriochlorins, or porphycenes can be used.
  • the photosensitizer can have one or more functional groups, such as carboxylic acid, amine, or isothiocyanate, e.g., for using in attaching the photosensitizer to another molecule or moiety, such as an organic bridging ligand or a SBU, and/or for providing an additional site or sites to enhance coordination or to coordinate an additional metal or metals.
  • the photosensitizer is a porphyrin or a derivative or analog thereof. Exemplary porphyrins include, but are not limited to, hematoporphyrin, protoporphyrin and tetraphenylporphyrin (TPP).
  • Exemplary porphyrin derivatives include, but are not limited to, pyropheophorbides, bacteriochlorophylls, chlorophyll a, benzoporphyrin derivatives, tetrahydroxyphenyl chlorins, purpurins, benzochlorins, naphthochlorins, verdins, rhodins, oxochlorins, azachlorins, bacteriochlorins, tolyporphyrins and benzobacteriochlorins.
  • Porphyrin analogs include, but are not limited to, expanded porphyrin family members (such as texaphyrins, sapphyrins and hexaphyrins), porphyrin isomers (such as porphycenes, inverted porphyrins, phthalocyanines, and naphthalocyanines), and TPP substituted with one or more functional groups.
  • the porphyrin is bis(p-benzoato)porphyrin (DBP).
  • the PS is a metal coordination complex comprising a metal (e.g., Ru or Ir) and one or more nitrogen donor ligands, e.g., one or more nitrogen-containing aromatic groups.
  • the one or more nitrogen donor ligands are selected from the group including, but not limited to, a bipyridine (bpy), a phenanthroline, a terpyridine, or a phenyl-pyridine (ppy), each of which can optionally be substituted with one or more aryl group substituents (e.g., on a carbon atom of the aromatic group).
  • cancer refers to diseases caused by uncontrolled cell division and/or the ability of cells to metastasize, or to establish new growth in additional sites.
  • malignant refers to cancerous cells or groups of cancerous cells.
  • cancers include, but are not limited to, skin cancers (e.g., melanoma), connective tissue cancers (e.g., sarcomas), adipose cancers, breast cancers, head and neck cancers, lung cancers (e.g., mesothelioma), stomach cancers, pancreatic cancers, ovarian cancers, cervical cancers, uterine cancers, anogenital cancers (e.g., testicular cancer), kidney cancers, bladder cancers, colorectal cancers (i.e., colon cancers or rectal cancers), prostate cancers, central nervous system (CNS) cancers, retinal cancer, blood, neuroblastomas, multiple myeloma, and lymphoid cancers (e.g., Hodgkin’s and nonHodgkin’s lymphomas).
  • skin cancers e.g., melanoma
  • connective tissue cancers e.g., sarcomas
  • metalstatic cancer refers to cancer that has spread from its initial site (i.e., the primary site) in a patient’s body.
  • anticancer drug refers to drugs (i.e., chemical compounds) or prodrugs known to, or suspected of being able to treat a cancer (i.e., to kill cancer cells, prohibit proliferation of cancer cells, or treat a symptom related to cancer).
  • chemotherapeutic refers to a non-PS molecule that is used to treat cancer and/or that has cytotoxic ability.
  • Such more traditional or conventional chemotherapeutic agents can be described by mechanism of action or by chemical compound class, and can include, but are not limited to, alkylating agents (e.g., melphalan), anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g., paclitaxel), epothilones, histone deacetylase inhibitors (e.g., vorinostat), inhibitors of topoisomerase I or II (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or precursors thereof (e.g., methotrexate), peptide antibiotics (e.g., bleomycin), platinum based agents (e.g., cisplatin or oxaliplatin), retinoids (e.g., tretinoin), and vinka alkaloids (e.
  • Metal-organic frameworks are solid two- or three-dimensional networks comprising both metal and organic components.
  • MOFs can include repeating units of coordination complexes comprising a metal-based secondary building unit (SBU), such as a metal ion or metal complex, and a bridging polydentate (e.g., bidentate or tridentate) organic ligand.
  • SBU metal-based secondary building unit
  • a bridging polydentate e.g., bidentate or tridentate
  • MOFs metal-organic frameworks
  • nMOFs nanoscale MOFs
  • the covalent linkage between the bridging ligand and the therapeutic agent can include a bond that is sensitive to (e.g., reactive or unstable to) particular conditions.
  • This bond can be viewed as an actionable trigger that can be used to release the therapeutic agent at a time and/or location of interest, e.g., after deliberately administering a treatment that can introduce the particular conditions to which the covalent linkage is sensitive.
  • 18 ' 27 X-ray stands out as an external stimulus that could be useful as a trigger. X-rays are capable of deep tissue penetration, 28 image-guided precise dosing, 29 ' 30 and radiotherapeutic effects through direct DNA damage and/or indirect cytotoxic effects (e.g., via generating reactive oxygen species (ROS)).
  • ROS reactive oxygen species
  • Heavy metal -based nMOFs can act as radioenhancers by enhancing energy deposition and ROS generation. 39 ' 41
  • heavy metal -nMOFs with covalently conjugated drugs that can be efficiently triggered by X-rays to release the drugs via enhanced ROS generation and radiation-induced cleavage of the drug molecules for synergistic radiotherapy and chemotherapy while reducing the systemic exposure of chemotherapeutics.
  • An example of such a nMOF disclosed herein is a Hf-TP-SN nMOF with an X-ray triggerable 7-ethyl-10- hydroxycamptothecin (SN38) prodrug for synergistic radiotherapy and chemotherapy.
  • SN38 is the active metabolite of irinotecan, a compound that has been used in the treatment of colorectal and pancreatic cancer. 42 ' 43
  • Hf-TP-SN was synthesized via a combination of pre-functionalization of terphenyl ligands with 3,5-dimethylbenzyl alcohol and post-synthetic modification with SN38 via a carbonate bond.
  • Hf-TP-SN Upon X-ray irradiation, electron-dense Hfn-SBUs served as radiosensitizers to enhance OH generation, leading to 5- fold higher release of SN38 from Hf-TP-SN than a homogeneous counterpart. Hf-TP-SN not only enhanced the radiotherapeutic efficacy but also achieved chemotherapeutic effect through on-demand release of SN38. Such a chemoradiotherapy strategy can effectively reduce the radiation dose required for tumor regression and minimizes the side effects of chemotherapy via burst release of SN38 inside the cancer cells.
  • ROS-responsive nanotherapeutics have emerged as a preferred antitumor strategy 60 ' 62 as the ROS concentration in the tumor tissue can be higher than that in normal organs.
  • Various ROS- responsive drug delivery systems with photodynamic effect or chemodynamic effect have been developed to enable ROS-triggered drug release.
  • Radiotherapy has provided important curative and palliative treatments to more than half of all cancer patients with tissue-penetrating ionizing radiations to kill cancer cells by directly inducing DNA damage or indirectly generating ROS to decompose biomolecules and disrupt the redox balance.
  • nMOFs Nanoscale metal-organic frameworks
  • 73,74,2 have shown exciting potential in RT and drug delivery because of their unique advantages, including highly porous structures, high surface areas, good biocompatibility, and molecular tunability.
  • Hf-DBP can provide a nanoplatform for designing nanotherapeutics by incorporating a high loading of ROS-responsive prodrugs into the framework.
  • QP-SN quaterphenyl dicarboxylate ligand conjugated with 7-ethyl-10- hydroxycamptothecin (SN-38, an active metabolite of irinotecan) 79 via a OH responsive 3,5- dimethoxylbenzyl carbonate linkage.
  • DBP ligand 194200 vs 19.2 A
  • QP-SN was introduced to Hf-DBP nMOF with photosensitizing DBP ligands to form a novel multi-functional mix-ligand Hf-DBP-QP-SN nMOF.
  • Hf-DBP-QP-SN achieved tumor-selective chemotherapy via a cascade of OH generation from electron-dense Hfn SBUs 80 , hydroxylation of the 3,5-dimethoxylbenzyl carbonate at ortho positions, and subsequent 1,4- elimination to release SN38. 38 Under the irradiation of X-rays, Hf-DBP-QP-SN effectively suppressed tumor growth due to synergistic tumor-targeted RT-RDT and chemotherapy. See Figures 14A and 14B.
  • nMOF comprising a quaterphenyl dicarboxylate ligand conjugated to pomalidomide, a thalidomide analog antineoplastic agent, through a picolinium-containing X-ray sensitive linkage
  • MOFs comprising X-ray-triggerable innate immune modulator prodrugs, which can be used for combination radiotherapy and immunotherapy or combination radiotherapy-radiodynamic therapy and immunotherapy.
  • a MOF comprising a X-ray-triggerable innate immune modulate prodrug
  • a MOF i.e., HF-DBP-QP-R848
  • R848 a prodrug of R848, as described hereinbelow. See Scheme 7, below.
  • the presently disclosed subject matter provides a MOF (e.g., a nMOF) comprising an irradiation-sensitive prodrug (e.g., a prodrug comprising a bond sensitive to X-rays or other ionizing radiation, or a bond sensitive to a product produced by absorption of X-rays or other ionizing radiation by another component of the MOF).
  • a prodrug e.g., a prodrug comprising a bond sensitive to X-rays or other ionizing radiation, or a bond sensitive to a product produced by absorption of X-rays or other ionizing radiation by another component of the MOF.
  • the prodrug can include a bond that is sensitive to radicals (e.g., hydroxyl radicals) produced by X-ray adsorption of a metal ion.
  • the presently disclosed subject matter provides a MOF (e.g., a porous nMOF) comprising an X-ray sensitive prodrug, wherein the MOF comprises metal-containing secondary building units (SBUs) linked together via organic bridging ligands, wherein at least one of the SBUs comprises a metal cation capable of absorbing X-rays, and wherein at least one of the organic bridging ligands is substituted by a group having the formula: -L-D, wherein L is a bivalent linker group comprising one or more bonds capable of radical-promoted bond cleavage and wherein D is a monovalent moiety of a therapeutic agent.
  • SBUs secondary building units
  • organic bridging ligands at least one of the SBUs comprises a metal cation capable of absorbing X-rays
  • at least one of the organic bridging ligands is substituted by a group having the formula: -L-D, wherein L is a
  • the presently disclosed nMOF comprises periodic repeats of metal-based SBUs and organic bridging ligands, wherein each SBU is bonded to at least one other SBU via coordinative bonding to the same organic bridging ligand and wherein one or more of the SBUs contain cations of a high Z-metal that can absorb ionizing irradiation energy, such as X-ray, y-ray, P-irradiation, or proton irradiation.
  • one or more of the SBUs are metal-oxo clusters with a structure that strongly absorbs ionizing irradiation energy.
  • the metal cation capable of absorbing X-rays is a cation of an element selected from the group comprising Hf, the lanthanide metals (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu), Ba, Ta, W, Re, Os, Ir, Pt, Au, Pb, and Bi.
  • the one or more SBUs comprise combinations of metals (either in the same SBU or in different SBUs).
  • the SBUs are metal oxo clusters comprising one or more of Hf, a lanthanide metal, Ba, Ta, W, Re, Os, Ir, Pt, Au, and Bi.
  • the oxo clusters comprise anions selected from oxide (O 2 ), hydroxide (OH ), S 2 ', SH', and formate (HCO2 ).
  • the oxo clusters are capped with anions derived from a strongly coordinating modulator, such as a monocarboxylic acid.
  • the oxo clusters are capped with an anion selected from the group including, but not limited to, acetate, formate, benzoate, and trifluoroacetate.
  • the metal cation capable of absorbing X-rays is an Hf cation.
  • one or more of the SBUs comprise a Hf oxo cluster.
  • one or more of the SBUs can be selected from the group including, but not limited to an Hfe oxo cluster (e.g., HfeO4(OH)4(HCO2)n), a Hfn oxo cluster (e.g., HfnO8(OH)i4(HCO2)i8), an Hfi8 oxo cluster (e.g., Hfi8Oi2(OH)24(HCO2)24) and an Hf24 oxo cluster (e.g., Hf240i6(OH)34(HC02)3o).
  • Hfe oxo cluster e.g., HfeO4(OH)4(HCO2)n
  • HfnO8(OH)i4(HCO2)i8 e.g., HfnO8(OH
  • one or more of the SBUs comprise a Hfe oxo cluster (e.g., [HfeO4(OH)4(HCO2)e]) or an Hfn oxo cluster. In some embodiments, one or more of the SBUs comprise a Hfe oxo cluster. In some embodiments, one or more of the SBUs comprise a Hfn oxo cluster.
  • L has the structure: wherein: X is selected from the group comprising -O-, -NR’-, -O-Xi-O-, and -NH-Xi-NH-, wherein R’ is selected from H and alkyl, and Xi is alkylene. In some embodiments, X is selected from -O- and -NR’-. In some embodiments, X is -NH-.
  • the parent therapeutic agent is a therapeutic agent that comprises at least one functional group for covalently bonding the therapeutic agent to L.
  • the functional group can be used to form a portion of the group L (i.e., to provide one or more atoms to the structure of L).
  • D is the monovalent moiety that would be provided if the bond attaching the functional group to the therapeutic agent were cleaved.
  • the functional group is a carboxylate
  • D is the monovalent moiety that has the same structure as the parent therapeutic agent but with a free bonding site where the carboxylate group is in the parent therapeutic agent.
  • the functional group is a hydroxy or phenol group
  • D can have the same structure as the parent therapeutic agent but with a free bonding site where the hydroxy or phenol OH group is in the parent therapeutic agent.
  • the therapeutic agent is a chemotherapeutic agent or an immune modulator (i.e., a small molecule chemotherapeutic agent or small molecule immune modulator).
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • the chemotherapeutic agent is camptothecin or an analog and/or metabolite thereof, such as SN38.
  • the chemotherapeutic agent is pomalidomide, lenalidomide, or another thalidomide analog chemotherapeutic agent (e.g., another amino-substituted thalidomide analog chemotherapeutic agent).
  • the immune modulator is a TLR agonist or a STING agonist.
  • the therapeutic agent is selected from SN38, R848 (resiquimod), imiquimod, VTX-378 (motolimod), DSR-6434, ioxoribine, TLR7/8 agonist 1, Cu-712-9, neoseptin-3, and diABZI. See Figures 35A-35C, which shows MOF prodrugs of exemplary immune modulators.
  • the therapeutic agent is R848.
  • the at least one organic bridging ligand substituted by a group having the formula -L-D is an organic bridging ligand comprising an arylene group (e.g., a terphenyl (TP) moiety or a quaterphenyl (QP) moiety) and at least two groups, which can be the same or different, capable of coordinating with an SBU.
  • the at least two groups capable of coordinating with an SBU are selected from the group including, but not limited to, a carboxylate group, an aromatic or non-aromatic nitrogen-containing group (e.g., pyridine), a phenol, an acetylacetonate, a phosphonate, and a phosphate.
  • the at least one organic bridging ligand has a structure selected from:
  • the MOF further comprises at least one photosensitizer (PS).
  • PS photosensitizer
  • the MOF comprises at least one organic bridging ligand that is covalently attached to a PS or that is, itself, a PS.
  • the MOF comprises at least one organic bridging ligand comprising a porphyrin, a chlorin, a chlorophyll, a phthalocyanine, a ruthenium-bipyridine complex, iridium-phenylpyridine complex, or an iridium-bipyridine complex.
  • the PS or PS containing bridging ligands can generate reactive oxygen species (ROS) such as singlet oxygen superoxide ( O2 ), hydrogen peroxide (H2O2), and hydroxyl radicals ( OH), upon absorption of ionizing irradiation by the MOF (e.g., by the metal cation of a SBU).
  • ROS reactive oxygen species
  • the MOF comprises at least one organic bridging ligand comprising a porphyrin, optionally bis(p- benzoato)porphyrin (DBP).
  • the MOF is a mixed ligand MOF comprising at least two different organic bridging ligands, one that comprises a covalently attached radical sensitive prodrug and one that comprises a PS that can generate ROS when the MOF absorbs ionizing radiation.
  • the presently disclosed subject matter provides a method of treating a disease a disease in a subject in need thereof, the method comprising: (a) administering to the subject a MOF (e.g., a nMOF) of the presently disclosed subject matter, or a pharmaceutical composition thereof; and (b) irradiating at least a portion of the subject with X-rays.
  • a MOF e.g., a nMOF
  • the disease is cancer or another proliferative disease or pathogenic infection.
  • the disease is cancer.
  • the cancer is selected from the group including, but not limited to, a head tumor, a neck tumor, a head and neck tumor, a breast tumor, a gynecological tumor, a brain tumor, a colorectal cancer, a lung cancer, mesothelioma, a soft tissue sarcoma, and a pancreatic cancer.
  • the subject can be exposed to the ionizing irradiation energy in any suitable manner and/or using any suitable equipment, such as that currently being used for delivering X-rays in a medical or veterinary setting.
  • the X-ray source and/or output can be refined to enhance disease treatment.
  • the X-rays can be generated using a peak voltage, current and/or, optionally, a filter chosen to minimize DNA damage in the patient due to X-ray irradiation and maximize X-ray absorption by the scintillator.
  • the subjects are irradiated with a linear accelerator (LINAC), using conventional techniques, Intensity-Modulated Radiation Therapy (IMRT), Image Guided Radiation Therapy (IGRT), or Stereotactic Body Radio Therapy (SBRT), a 60 Co radiation source, an implanted radioactive seed such as the ones used in brachytherapy, an orthovoltage or supervoltage X-ray irradiator, a high energy electron beam generated from LINAC, or a proton source.
  • the irradiating can comprise generating X-rays using a tungsten or another metal target, Cobalt-60 sources (cobalt unit), linear accelerators (linacs), Ir-192 sources, and Cesium-137 sources.
  • the irradiating comprises passing the X-rays (e.g., the X-rays generated using a tungsten target) or other ionizing radiation through a filter prior to irradiation the subject.
  • the filter can comprise an element with an atomic number of at least 20.
  • the filter comprises copper (Cu).
  • the filter can have a thickness that is less than about 5 millimeters (mm).
  • the filter can have a thickness of less than about 4 mm (e.g., less than about 3 mm, less than out 1 mm, less than about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm, less than about 0.2 mm, or less than about 0.1 mm).
  • the X-rays can be generated using a peak voltage, current and/or, optionally, a filter chosen to minimize DNA damage in the patient due to X-ray irradiation and maximize X-ray absorption by the scintillator.
  • the X-rays are generated using a peak voltage that is less than about 230 kVp.
  • the peak voltage is less than about 225 kVp, less than about 200 kVp, less than about 180 kVp, less than about 160 kVp, less than about 140 kVp, less than about 120 kVp, less than about 100 kVp, or less than about 80 kVp.
  • the X-rays are generated using a peak voltage that is about 120 kVp.
  • step (b) comprises irradiating at least a portion of the subject with X-rays one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times).
  • X-rays are generated by placing radioactive sources inside the subject on a temporary or permanent basis.
  • a MOF of the presently disclosed subject matter is injected along with the implantation of a radioactive source.
  • the methods can further comprise administering to the subject an additional treatment (e.g., an additional cancer and/or immunotherapy treatment).
  • an additional cancer treatment can be selected on the basis of the cancer being treated and/or on other factors, such as the patient’s treatment history, overall health, etc., in accordance with the best judgement of the treating physician.
  • the additional cancer treatment can be selected from the group including, but not limited to, surgery, radiotherapy, conventional chemotherapy, toxin therapy, immunotherapy, cryotherapy and gene therapy.
  • the additional cancer treatment can comprise administering to the patient a conventional chemotherapeutic, such as, but not limited to, a platinum-containing agent (e.g., cisplatin or oxaliplatin or a prodrug thereof), doxorubicin, daunorubicin, docetaxel, mitoxanthrone, paclitaxel, digitoxin, digoxin, and septacidin or another conventional chemotherapeutic known in the art.
  • the additional chemotherapeutic agent can be present in the MOF (e.g., encapsulated or coordinatively or covalently bonded to the MOF).
  • the additional chemotherapeutic agent can be present in the same pharmaceutical composition or formulation as the MOF or in a separate pharmaceutical composition or formulation, administered prior to, simultaneously with, or after administration of the pharmaceutical composition or formulation comprising the MOF and/or the irradiation.
  • the additional cancer treatment can involve administering to the patient a drug formulation selected from the group comprising a polymeric micelle formulation, a liposomal formulation, a dendrimer formulation, a polymer-based nanoparticle formulation, a silica-based nanoparticle formulation, a nanoscale coordination polymer formulation, a nanoscale metal-organic framework formulation, and an inorganic nanoparticle (gold, iron oxide nanoparticles, etc.) formulation.
  • the drug formulation can be a formulation including a conventional chemotherapeutic.
  • D is a monovalent moiety of a chemotherapeutic agent, thereby providing to the subject, upon performing the irradiating of (b), a combination of chemotherapy and either radiotherapy (RT) or radiotherapy-radiodynamic therapy (RT- RDT).
  • the MOF comprises a PS (e.g., a DBP bridging ligand) and the method provides combination of chemotherapy and RT-RDT.
  • D is a monovalent moiety of SN38.
  • a higher percentage of the SN38 (or other chemotherapeutic agent) is released from the MOF upon performing the irradiating of (b) compared to the release of SN38 (or other chemotherapeutic agent) from a homogenous prodrug comprising the SN38 monovalent moiety (or other chemotherapeutic agent-derived monovalent moiety).
  • the term “homogenous prodrug” as used herein refers to a prodrug that is free of coordination bonding and/or that is not a MOF.
  • the homogenous SN38 prodrug is MeO-SN.
  • D is a monovalent moiety of an immune modulator, thereby providing to the subject, upon performing the irradiating of (b), a combination of immunotherapy and either RT or RT-RDT.
  • the MOF comprises a PS (e.g., a DBP bridging ligand) and the method provides, upon performing the irradiating of (b), a combination of immunotherapy and RT-RDT.
  • the presently disclosed methods can provide more selective treatment than use of a non-MOF (e.g., homogenous) prodrug or free therapeutic agent.
  • a non-MOF e.g., homogenous
  • release of the therapeutic agent can be controlled by directing the X-ray radiation treatment to regions at or near a disease site (e.g., a tumor).
  • a disease site e.g., a tumor.
  • the method provides selective cytotoxicity and/or immune activation in a tumor (i.e., higher cytotoxicity and/or immune activation in a tumor than in surrounding non-diseased tissue).
  • the presently disclosed methods can provide an improved therapeutic index for the therapeutic agent.
  • the presently disclosed method can provide effective results using a lower dose of X-ray radiation than conventional radiotherapy.
  • the presently disclosed MOF or a pharmaceutical composition thereof can be used in treating a disease or in the manufacture of a composition for treating a disease in a subject in need thereof.
  • the disease is cancer or another proliferative disease or pathogenic infection.
  • the disease is cancer.
  • the cancer is selected from a head tumor, a neck tumor, a head and neck tumor, a breast tumor, a gynecological tumor, a brain tumor, a colorectal cancer, a lung cancer, mesothelioma, a soft tissue sarcoma, and a pancreatic cancer.
  • the use is a use performed in combination with exposing at least a portion of the subject to ionizing radiation (e.g., X-rays).
  • ionizing radiation e.g., X-rays.
  • the use provides selective cytotoxicity and/or immune activation in a tumor (i.e., higher cytotoxicity and/or immune activation in a tumor than in surrounding non-diseased tissue).
  • the presently disclosed uses can provide an improved therapeutic index for the therapeutic agent.
  • the present method selectively releases chemotherapeutic agents and immune modulators in tumors during radiotherapy without causing significant systemic exposure of these agents, thereby reducing the toxicity from conventional systemic administration of chemotherapy and immunotherapy.
  • the presently disclosed subject matter provides a pharmaceutical composition or formulation comprising a MOF (e.g., a nMOF) as described herein and a pharmaceutically acceptable carrier, e.g., a pharmaceutically acceptable carrier that is pharmaceutically acceptable in humans.
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier that is pharmaceutically acceptable in humans.
  • the composition can also include other components, such as, but not limited to anti-oxidants, buffers, bacteriostatics, bactericidal antibiotics, suspending agents, thickening agents, and solutes that render the composition isotonic with the bodily fluids of a subject to whom the composition is to be administered.
  • compositions of the presently disclosed subject matter comprise, in some embodiments, a composition that includes a pharmaceutically acceptable carrier. Any suitable pharmaceutical formulation can be used to prepare the compositions for administration to a subject. In some embodiments, the composition and/or carriers can be pharmaceutically acceptable in humans.
  • suitable formulations can include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostatics, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the subject; and aqueous and non-aqueous sterile suspensions that can include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze- dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use.
  • Some exemplary ingredients are sodium dodecyl sulfate (SDS), in one example in the range of 0.1 to 10 mg/ml, in another example about 2.0 mg/ml; and/or mannitol or another sugar, for example in the range of 10 to 100 mg/ml, in another example about 30 mg/ml; and/or phosphate-buffered saline (PBS).
  • SDS sodium dodecyl sulfate
  • PBS phosphate-buffered saline
  • formulations of this presently disclosed subject matter can include other agents conventional in the art having regard to the type of formulation in question.
  • sterile pyrogen-free aqueous and non-aqueous solutions can be used.
  • compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e. living organism, such as a patient).
  • a subject i.e. living organism, such as a patient.
  • the subject or patient is a human subject, although it is to be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective with respect to all vertebrate species, including mammals, which are intended to be included in the terms “subject” and “patient”.
  • a mammal is understood to include any mammalian species for which employing the compositions and methods disclosed herein is desirable, particularly agricultural and domestic mammalian species.
  • the methods of the presently disclosed subject matter are particularly useful in warm-blooded vertebrates.
  • the presently disclosed subject matter concerns mammals and birds. More particularly provided are methods and compositions for mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans), and/or of social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), rodents (such as rats, mice, hamsters, guinea pigs, etc.), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses.
  • carnivores other than humans such as cats and dogs
  • swine pigs, hogs, and wild boars
  • rodents such as rats, mice
  • poultry such as turkeys, chickens, ducks, geese, guinea fowl, and the like
  • livestock including, but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.
  • Suitable methods for administration of a composition of the presently disclosed subject matter include, but are not limited to intravenous and intratumoral injection, oral administration, subcutaneous administration, intraperitoneal injection, intracranial injection, and rectal administration.
  • a composition can be deposited at a site in need of treatment in any other manner, for example by spraying a composition within the pulmonary pathways.
  • the particular mode of administering a composition of the presently disclosed subject matter depends on various factors, including the distribution and abundance of cells to be treated and mechanisms for metabolism or removal of the composition from its site of administration. For example, relatively superficial tumors can be injected intratum orally. By contrast, internal tumors can be treated following intravenous injection.
  • the method of administration encompasses features for regionalized delivery or accumulation at the site to be treated.
  • a composition is delivered intratum orally.
  • selective delivery of a composition to a target is accomplished by intravenous injection of the composition followed by photodynamic treatment (light irradiation) of the target.
  • compositions of the presently disclosed subject matter can be formulated as an aerosol or coarse spray. Methods for preparation and administration of aerosol or spray formulations can be found, for example, in U.S. Patent Nos. 5,858,784; 6,013,638; 6,022,737; and 6,136,295.
  • An effective dose of a composition of the presently disclosed subject matter is administered to a subject.
  • An “effective amount” is an amount of the composition sufficient to produce detectable treatment.
  • Actual dosage levels of constituents of the compositions of the presently disclosed subject matter can be varied so as to administer an amount of the composition that is effective to achieve the desired effect for a particular subject and/or target. The selected dosage level can depend upon the activity of the composition and the route of administration.
  • 2,5-Dibromobenzyl amine (6) 2,5-Dibromobenzyl amine (6) was prepared based on reported procedures with modification in the second step. 51 To a solution of 7M ammonia (NH3) in MeOH (100 mL, 0.70 mol) was added methyl 2,5-dibromobenzoate (4, 10.0 g, 0.034 mol) at RT; the resulting dissolved solution was evenly separated into five 20-mL vials, sealed, and stirred for 3 days.
  • 7M ammonia NH3
  • MeOH 100 mL, 0.70 mol
  • the white precipitate formed was separated and collected via filtration, dispersed in hexanes (100 mL), recollected via filtration, and dried under vacuum to produce 2,5-dibromobenzamide (5, 8.58 g, 0.031 mol, 90%).
  • IM borane-tetrahydrofuran complex (BH3 in THF) was added in excess (35 mL, 0.035 mol) to 5 (4.11 g, 0.0144 mol); the resulting mixture was stirred at reflux for 24 hours and then quenched with concentrated HC1 (3.5 mL). The solution was then refluxed for 2 more hours and cooled to RT. The suspension was filtered to obtain the white solid, which was then dissolved in water and basified to pH ⁇ 10 by addition of sat. sodium carbonate (Na2COs) while the solution was stirring. The reaction mixture was extracted with EA (30 mL) 3 times.
  • the resulting mixture was stirred at 40 °C for 1 day; it was then allowed to cool to RT before being acidified (while being stirred) to pH ⁇ l-2 by addition of concentrated HC1; the acidic solution was left to stir for 2 hours to ensure the completion of the acid-base reaction.
  • the resulting solution was filtered, washed with H2O and dried under vacuum to produce H2TP-OH (9, 0.493 g, 0.91 mmol, 90%).
  • Hf-TP-OH Synthesis of Hf-TP-OH.
  • HfCL and H2TP-OH were separately dissolved in dimethylformamide (DMF) at a concentration of 2 mg/mL.
  • 500 pL HfCL solution and 500 pL H2TP-OH were then combined in a 1-dram vial with the addition of 1 pL trifluoracetic acid and 5 pL water as modulators.
  • the mixture was heated in an oven at 80 °C for 1 day, after which the white precipitate was collected by centrifugation and sequentially washed with DMF, 1% tri ethylamine (TEA) in ethanol (EtOH) (v/v), and EtOH to afford Hf-TP-OH in 88% yield based on H2TP-OH.
  • DMF dimethylformamide
  • Hf-TP-SN via post-synthetic modification.
  • 10 mL Hf-TP-OH was washed with dry acetonitrile (ACN) twice and dispersed in ACN with a ligand concentration of 3.0 mM.
  • 4-nitrophenyl chloroformate (15.1 mg, 75 pmol), and TEA (12.5 pL, 90 pmol) were then added, and the solution was stirred for 2 days to afford Hf-TP-NCh nMOF.
  • Hf-MOFs Digestion of Hf-MOFs for UV-Vis spectroscopic measurements. 50 pL Hf-TP-OH or Hf-TP-SN solution, 900 pL DMSO and 50 pL H3PO4 were mixed and sonicated for 1 hour and let stand overnight, after which the mixture was diluted to a proper concentration for UV- Vis measurement.
  • the absorbance of H2TP-OH at 304 nm was used to calculate the concentration of H2TP-OH in Hf-TP-OH or Hf-TP-SN after comparison with the standard curve of H2TP-OH in DMSO, while the absorbance of SN38 at 390 nm was used to calculate the concentration of SN38 in Hf-TP-SN after comparison with the standard curve of SN38 in DMSO.
  • Hf-TP-SN Digestion of Hf-TP-SN for quantification of SN38 by LC-MS.
  • 100 pL IM NaHCOs solution was added to 100 pL Hf-TP-SN dispersion in water (total SN concentration: 100 pM).
  • the mixture was sealed and sonicated for 20 minutes, after which 100 pL PBS (200 mM, pH 4.0) was added to adjust the pH to 7.
  • 100 pL saturated NaCl solution and 150 pL ethyl acetate (EA) were then added before vortexing the mixture for 1 minute.
  • the EA layer after centrifugation was analyzed with LC-MS.
  • Hf-TP-SN Stability of Hf-TP-SN in PBS.
  • Hf-TP-SN was dispersed in 1 mL PBS (1 mM) with a Hf concentration of 5.2 mM. 200 uL suspension was taken after incubation for 1, 2, 4, 8, and 24 hours and centrifuged for PXRD measurement.
  • the PBS solution with the same DCFH concentration served as a blank control.
  • the fluorescence signal (em. 520/20 nm) was collected with a Synergy HTX microplate reader (ex. 485/20 nm; Agilent Technologies, Santa Clara, California, United States of America). Hydroxyl radical generation in test tubes. Hydroxyl radical ( OH) generation under irradiation was detected by the APF assay.
  • APF assay was added to the PBS suspension of Hf-TP-OH or Hf-TP-SN at the same Hf concentration.
  • the concentration of APF was 5 pM while that of Hf was 40 pM.
  • the PBS solution with the same APF concentration served as a blank control.
  • the fluorescence signal (em. 520/20 nm) was collected with a Synergy HTX microplate reader (ex. 485/20 nm; Agilent Technologies, Santa Clara, California, United States of America).
  • Hf-TP-SN or MeO-SN Hydroxyl radical triggered SN38 release in test tubes.
  • Hf-TP-SN or MeO-SN was dispersed in H2O at the same concentration of total SN38 (100 pM).
  • FeCh, (+)-Sodium L- ascorbate, Na2(EDTA) 2H2O (ethylenediaminetetraacetic acid, disodium salt dihydrate) was firstly dissolved in water to reach a concentration of 10 mM separately (10.5 mM for Na2(EDTA) 2H2O). Then, 100 pL of each solution and 50 pL H2O2 (200 mM in H2O) was added to Hf-TP-SN or MeO-SN solution to generate hydroxyl radical in situ and to trigger the release of SN38.
  • Hf-TP-SN For control group, only H2O2 was added. After 8h incubation at RT, additional 100 pL IM NaHCCh was added to Hf-TP-SN to digest the nMOF, after which the mixture was sealed and sonicated for 20 minutes before the addition of 100 pL PBS (200 mM, pH 4) to adjust the pH to 7. 100 pL saturated NaCl solution and 150 pL ethyl acetate (EA) were added before vortexing the Hf-TP-SN or MeO-SN mixture for 1 minute. The EA layer after centrifugation was analyzed by LC-MS.
  • EA ethyl acetate
  • 100 pL Hf-TP-SN or MeO-SN suspension was irradiated with 10 Gy X-ray.
  • 100 pL IM NaHCOs was added to Hf- TP-SN to digest the nMOF, after which the mixture was sealed, sonicated for 20 minutes, and sat 15h before the addition of 100 pL PBS (200 mM, pH 4.0) to adjust the pH to 7.
  • CT26 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% HyClone penicillin-streptomycin 100X solution, and cultured in a humidified atmosphere containing 5% CO2 at 37°C.
  • the cytotoxicity of H2TP-OH, Hf-TP- OH, and Hf-TP-SN on CT26 cells was detected by MTS assay.
  • CT26 cells were seeded in 96-well plates at a density of 2500 cells/well. Different concentrations of H2TP-OH, Hf-TP- OH and Hf-TP-SN were added and 24 hours later 10% (v/v) of MTS reagent was added to each well. 90 minutes later the absorbance of each well at 490 nm was read by a Synergy HTX plate reader (Agilent Technologies, Santa Clara, California, United States of America) to calculate cell viability.
  • the plates were rinsed once with PBS, fixed by 4% paraformaldehyde for 20 minutes, and washed with PBS twice at RT.
  • the 6-well plates were scanned with a cell analysis system sold under the tradename INCUCYTE® S3 (Sartorius Bioanalytical Instruments, Inc., Brooklyn Center, Minnesota, United States of America) in the whole well mode with a 4* objective.
  • the colonies were identified with software available under the tradename INCUCYTE® 2021 A (Sartorius Bioanalytical Instruments, Inc., Brooklyn Center, Minnesota, United States of America) in a cellular resolution and the confluence was used as a parameter to calculate the plating efficiency (PE) and surviving fraction (SF):
  • PE Confluence (0 Gy, PBS)ICe ⁇ # (0 Gy, PBS)
  • D was the radiation dose
  • Cell # (D, MOF) was the number of cells seeded for a certain radiation dose D and a certain treatment group.
  • the dose modifying ratio at a 10% was used as a parameter to assess radiosensitization effect and defined as the ratio of doses under reference conditions to produce a 10% 52 :
  • DMRio% DMRio% (PBS) / DMRio% (MOF)
  • the cells were washed with PBS for three times and further incubated with Hoechst 33342 (10 pg mL' 1 ) in PBS for 10 minutes in the cell incubator. Finally, the cells were washed with PBS 3 times and observed on a Leica Stellaris 8 confocal microscope (Leica Microsystems, Wetzlar, Germany).
  • CT26 cells were seeded in 96-well plates at a density of 2500 cells/well. Different concentrations of SN38 and Me2TP-SN were added and 48 hours later, 10% (v/v) of MTS reagent was added to each well. 90 minutes later the absorbance of each well at 490 nm was read by a Synergy HTX plate reader (Agilent Technologies, Santa Clara, California, United States of America) to calculate cell viability.
  • Hf-TP-SN Hf: 50 pM
  • the cellular uptake of Hf-TP-SN was evaluated on CT26 cells.
  • the cells were seeded in 6-well plates at a density of 2 * 10 5 /well and cultured overnight.
  • the cells were incubated in a 37 °C incubator for 1, 2, and 6 hours. At each time point, the medium was aspirated, the cells were washed with PBS three times, collected by centrifugation, and counted with a hemocytometer.
  • the cell pellets were digested with nitric acid for 24 h and the concentration of Hf was detected by ICP-MS.
  • ROS generation To demonstrate the generation of ROS, CT26 cells were seeded in cell culture dishes at a density of 1.5 * I O 5 and cultured overnight. Hf-TP-SN or Hf-TP-OH was added at a Hf concentration of 50 pM and further incubated in a 37°C incubator for 4 hours. The cells were washed with PBS solution 3 times and 1 mL cell culture medium containing with 30 pM DCFH-DA was added and incubated at 37°C for another 30 minutes. Then the cells were irradiated with X-ray (3 Gy) and cultured in the cell incubator for another 24 h.
  • Hf-TP-SN or Hf-TP-OH was added at a Hf concentration of 50 pM and further incubated in a 37°C incubator for 4 hours. The cells were washed with PBS solution 3 times and 1 mL cell culture medium containing with 30 pM DCFH-DA was added and incubated at 37°C for another 30 minutes. Then the cells were
  • CT26 cells were seeded in cell culture dishes at a density of 1.5x 10 5 .
  • the cells were treated in the same way as in the clonogenic assay. 24 hours after radiation, the cells were washed with PBS and fixed with 4% paraformaldehyde at RT for 20 minutes. The cells were again rinsed with PBS, blocked and permeabilized with 5% FBS + 0.3% Triton-X in PBS at RT for 1 hour. After blocking, cells were incubated with the y-H2AX primary antibody (1 :500) in 1% BSA + 0.3% Triton-X in PBS at RT for 1 hour.
  • the cells were then washed with PBS and incubated with the Alexa Fluor 488 conjugated secondary antibody (1 :3000) in 1% BSA + 0.3% Triton-X in PBS at RT for 1 hour. Afterwards, the cells were washed with PBS and further incubated with Hoechst 33342 (10 pg ml 1 ) in PBS for 10 minutes at 37 °C to visualize cell nuclei, respectively. Finally, cells were washed with PBS 3 times and observed on a Leica Stellaris 8 confocal microscope (Leica Microsystems, Wetzlar, Germany).
  • BALB/c mice (6-8 weeks) were obtained from Charles River Laboratories, Inc (Wilmington, Massachusetts, United States of America) and bred in house at the animal facility at the University of Chicago (Chicago, Illinois, United States of America).
  • CT26 tumor models were established on BALB/c mice by inoculating 2 x 10 6 cells/mouse subcutaneously onto the right flanks at day 0, respectively. When CT26 tumors reached ⁇ 85 mm 3 , the mice were randomized for treatments.
  • PBS, irinotecan, Hf-TP, or Hf-TP-SN was intratumorally injected with an equivalent metal dose of 0.5 pmol in 20 pL PBS and SN38 dose of 0.047 pmol in 20 pL PBS.
  • the dose of irinotecan was 0.047 pmol in 20 pL PBS. 6-8 hours later, the mice were anaesthetized with 2.5% (v/v) isofl urane/Ch and mounted onto the X-Rad 225 irradiator.
  • the CT26 tumors were irradiated with 2 Gy X-ray/fraction for 3 consecutive days.
  • TGI tumor growth inhibition index
  • mice were established and treated in the same way as in antitumor efficacy experiments.
  • the mice were euthanized one day after the last irradiation.
  • the tumors were excised and fixed in 4% PFA for 48 h and 70% ethanol for 1 day.
  • the tissues were embedded in paraffin, sectioned and stained for H&E, y-H2AX, Ki67 and TUNEL by Human Tissue Resource Center at the University of Chicago. Briefly, the slides were deparaffinized and rehydrated using xylenes and serial dilutions of ethanol to distilled water.
  • the slides were treated with antigen retrieval buffer (AR9640; Leica Biosystems, Wetzlar, Germany) and heated in a steamer at 97°C for 20 minutes. After washing with tris-buffered saline (TBS), the slides were incubated with primary y-H2AX antibody (1 :400) or primary Ki67 antibody (Thermo Fisher Scientific, Clone# SP6, 1 :400; Thermo Fisher Scientific, Waltham, Massachusetts, United States of America) at RT for 1 hour in a wet chamber.
  • antigen retrieval buffer AR9640; Leica Biosystems, Wetzlar, Germany
  • TBS tris-buffered saline
  • primary Ki67 antibody Thermo Fisher Scientific, Clone# SP6, 1 :400; Thermo Fisher Scientific, Waltham, Massachusetts, United States of America
  • the slides were washed with TBS, then y-H2AX and Ki67 slides were incubated with anti-rabbit-polymer (Bond Polymer Refine Detection, Leica Biosystems, DS9800; Leica Biosystems, Wetzlar, Germany) for 30 minutes at RT.
  • the antigen-antibody binding was detected with the 3,3 '-Diaminobenzidine (DAB) (DAKO, K3468; Agilent Technologies, Santa Clara, California, United States of America) system.
  • DAB 3,3 '-Diaminobenzidine
  • Tissue sections were then immersed in hematoxylin for counterstaining and covered with cover glasses.
  • the slides were scanned on a CRi Pannoramic SCAN 40* whole slide scanner (3DHISTECH LTD, Budapest, Hungary). The images were analyzed with QuPath-0.2.3 software.
  • SN38 was conjugated to Hf-TP-OH nMOF via the 3,5-dimethoxylbenzyl carbonate linkage, which can be cleaved by hydroxyl radical ( OH).
  • 38 Hf-TP-SN was synthesized via a combination of pre-functionalization and post-synthetic modification. See Figure 1. The terphenyl dicarboxylate ligand was pre-functionalized with dimethoxybenzyl alcohol via an amide bond, incorporated into a Hf-nMOF, and then post-synthetically modified with SN38 via a carbonate bond.
  • Hf-TP-OH nMOF with Hfn SBUs was synthesized via a solvothermal reaction between HfCh, H2TP-OH, trifluoracetic acid, and water in N, N- dimethylformamide at 80 °C for 1 day.
  • Hf-TP-OH was treated with 4-nitrophenyl chloroformate followed by SN38 to afford Hf-TP-SN.
  • Hf-TP-OH and Hf-TP-SN both displayed a nanoplate morphology with a diameter of ⁇ 70 nm and a thickness of ⁇ 10 nm by transmission electron microscopy (TEM). See Figures 2 A and 2C.
  • HRTEM high-resolution TEM
  • FFT fast Fourier transform
  • Hf-TP-OH and Hf-TP-SN were 125 ⁇ 4 and 124 ⁇ 2 nm (see Figure 3 A), respectively, while their ( ⁇ -potentials were -11.2 ⁇ 1.2 and -10.3 ⁇ 0.4 mV (see Figure 3B), respectively, suggesting that Hf-TP-OH maintained the nanoscale size and surface charge after post-synthetic modification.
  • Powder X-ray diffraction (PXRD) studies confirmed that Hf-TP-SN and Hf-TP-OH adopted the same structure as Hfn-TP MOF consisting of Hfi2( «-O)8( «-OH)8( /2-OH)6 SBUs and TP ligand in a hep topology. 44 See Figure 3C.
  • Hf-TP-SN retained its crystallinity after incubation in PBS (1 mM, pH 7.4) for 24 hours (see Figure 3D), ensuring its stability for biological applications.
  • UV-Vis spectroscopic analysis of digested Hf-TP-SN indicated -16% of TP-OH ligands were conjugated with SN38 (see Figures 4A-4E) after comparing the absorbance of TP-OH ligand and SN38 with their standard curves.
  • Liquid chromatography-mass spectrometry (LC-MS) analysis indicated the trapping of 2.6% free SN38 (relative to total SN38) in the pores after digesting Hf-TP-SN using NaHCOi 45 and extraction with ethyl acetate.
  • ICP-MS inductively coupled plasma mass spectrometry
  • Me2TP-SN A molecular counterpart, Me2TP-SN, was synthesized from Me2TP-OTBS (5) to support the post-synthetic modification and to examine the cytotoxicity of the prodrug. See Scheme 2. Because of low aqueous solubility of Me2TP-SN, MeO-SN was synthesized from 1 and used as a homogeneous control. See Scheme 3.
  • ROS generation was evaluated by 2’,7’-dichlorodihydrofluorescein (DCFH) assay. 46
  • the total ROS signals in PBS, Hf-TP-OH, and Hf-TP-SN groups all increased linearly with X-ray doses.
  • the relative enhancements of Hf-TP-OH and Hf-TP-SN over PBS were 47% and 19%, respectively See Figure 5 A.
  • Aminophenyl fluorescein (APF) assay showed that Hf- TP-OH and Hf-TP-SN enhanced OH generation by 96% and 59%, respectively, over PBS.
  • the reduced ROS and hydroxyl radical signals from Hf-TP-SN are likely due to the consumption of OH by the 3,5-dimethoxylbenzyl carbonate linkage to release SN38.
  • H2TP-OH ligand and Hf-TP-OH nMOF did not show obvious cytotoxicity to CT26 colon carcinoma cells at a TP concentration of 100 pM (see Figures 6A and 6B), indicating their non-toxic nature without X-ray irradiation.
  • clonogenic assay showed that Hf-TP-OH possessed strong radiosensitizing property with a dose modifying ratio at 10% survival fraction (DMRio%) of 1.255 (see Figure 6C) due to enhanced OH generation via radiosensitization as probed by hydroxyphenyl fluorescein (HPF) assay. See Figure 6D.
  • cytotoxicity of SN38 and Me2TP-SN38 was also evaluated. While SN38 showed a half-maximal inhibitory concentration (IC50) of 0.584 pM (see Figure 7 A), 33 Me2TP-SN38 had much lower cytotoxicity with an IC50 of 13.5 pM (see Figure 7B), suggesting the successful construction of SN38 prodrug via the dimethoxybenzyl carbonate masking group.
  • IC50 half-maximal inhibitory concentration
  • Hf-TP-SN showed efficient and time-dependent cellular uptake as quantified by ICP-MS. See Figure 7C.
  • Hf-TP-SN showed a significantly increased DMRio% of 2.566 over Hf-TP-OH (1.255), likely due to the combined chemo-radiotherapeutic effects of the released SN38 and X-ray irradiation. See Figure 6C.
  • Hf-TP-OH(+) and Hf-TP-SN(+) exhibited 2.29- and 9.30-fold higher intracellular ROS signals than PBS(+), respectively, at an X-ray dose of 3 Gy. See Figures 8A-8D.
  • the stronger ROS signal in Hf-TP-SN(+) group likely resulted from the oxidative pressure of the released SN38 on the cells and the radiosensitizing effect of electron-dense Hfn SBUs.
  • DNA double strand breaks were investigated in CT26 cells via detecting the expression of y-H2AX, a phosphorylated protein biomarker for DSBs.
  • 49 PBS(+) induced a small amount of red y-H2AX fluorescence due to X-ray’s ability to cause DNA damage.
  • 50 More pronounced DSBs were observed in Hf-TP-OH(+) and Hf-TP-SN(+) groups, while no fluorescence was observed in Hf-TP-OH(-) group (see Figure 9), which supports potent radiosensitization by Hf-nMOFs.
  • Hf-TP-SN(-) also showed significantly enhanced y-H2AX signal over PBS control, likely due to the entrapped SN38 in the pores of Hf-TP-SN. Free SN38 inhibits the nuclear enzyme topoisomerase I during DNA replication, leading to DSBs. 42 ’ 43
  • a subcutaneous CT26 tumor model was established to assess the in vivo anticancer efficacy of Hf-TP-SN(+).
  • CT26 tumor-bearing mice were intratumorally injected with PBS, irinotecan (a prodrug of SN38, 0.046 pmol), 34 Hf-TP-OH (0.5 pmol Hf), or Hf-TP-SN (0.5 pmol Hf and 0.046 pmol SN38).
  • the tumors were exposed to X-ray at the dose of 2 Gy.
  • X-ray irradiation was repeated on two consecutive days (for a total of 6 Gy). Tumor volumes and mouse body weights were monitored daily until the PBS(-) end point. See Figures 11A-11D.
  • TGI tumor growth inhibition index
  • irinotecan(+) and Hf-TP-OH(+) enhanced tumor growth inhibition with TGIs of 0.695 and 0.869, respectively.
  • Hf-TP-SN(+) potently regressed tumors with a TGI value of 0.965 and completed tumor eradication in 40% mice.
  • Hf-TP-SN(-) treatment modestly inhibited tumor growth with a TGI of 0.362.
  • Hf-TP-SN(+) treatment increased the expression of y-H2AX (see Figure 12, top row) reduced cell proliferation with lower Ki67 signal (see Figure 12, row second from top), and increased cell apoptosis in TUNEL staining. See Figure 12, row second from bottom.
  • H&E staining showed distinctive cellular damage in the tumors treated with Hf-TP-OH(+) and Hf-TP-SN(+); minimal cellular damage was observed in PBS(+) and irinotecan(+) groups. See Figure 12, bottom row.
  • Hf-TP-SN nMOF was prepared comprising an X-ray triggerable SN38 prodrug for synergistic radiotherapy and chemotherapy. More particularly, Hf-TP-SN was synthesized via a combination of pre-functionalization of terphenyl ligands with 3,5- dimethylbenzyl alcohol and post-synthetic modification with SN38 via a carbonate bond. Upon X-ray irradiation, electron-dense Hfn-SBUs served as radiosensitizers to enhance OH generation, leading to 5-fold higher release of SN38 from Hf-TP-SN than a homogeneous counterpart.
  • Hf-TP-SN not only enhanced the radiotherapeutic efficacy but also achieved chemotherapeutic effect through on-demand release of SN38.
  • Such a chemoradiotherapy strategy effectively reduces the radiation dose required for tumor regression and minimizes the side effects of chemotherapy via burst release of SN38 inside cancer cells.
  • the postsynthetic modification step only requires a hydroxyl or amino group in a drug for nucleophilic substitution, a variety of therapeutic agents can be grafted onto the Hf-TP-OH to form novel Hf-TP-conjugated drugs for disease management. This highlights the use of nMOFs in multi-modality cancer treatment via on-demand, triggered release of therapeutic agents.
  • H2QP 2”-((4-(hydroxymethyl)-2,6-dimethoxybenzamido)methyl)-[l,l’:4’,l”:4”,l”’- quaterphenyl]-4,4”’-dicarboxylic acid
  • Hf-DBP-QP Synthesis of Hf-DBP-QP.
  • HfCh, H2DBP and H2QP-OH were separately dissolved in DMF at a concentration of 2, 3.5 and 3.5 mg/mL, respectively.
  • 500 pL HfCh solution, 100 pL H2DBP and 400 pL H2QP-OH were then combined in a 1-dram vial with the addition of 55 pL acetic acid and 5 pL water as modulators.
  • the mixture was heated in an oven at 80 °C for 2 days.
  • the purple precipitate was collected by centrifugation and sequentially washed with DMF, 1% triethylamine (TEA) in ethanol (EtOH) (v/v), and EtOH, and then dispersed in EtOH for storage.
  • TAA triethylamine
  • EtOH ethanol
  • Hf-DBP-QP-SN 10 mL Hf-DBP-QP was washed with dry acetonitrile (ACN) twice and dispersed in ACN with a QP concentration of 2.0 mM. 4-Nitrophenyl chloroformate (10.1 mg, 50 pmol), and TEA (8.3 pL, 60 pmol) were then added, and the solution was stirred for 2 days to afford Hf-DBP-QP -NO2 nMOF.
  • ACN dry acetonitrile
  • H2QP at 304 nm was used to calculate the concentration of H2QP-OH in Hf- DBP-QP after comparison with the standard curve of H2QP-OH in DMSO while the absorption of H2DBP at 408 nm was used to calculate the concentration of H2DBP in Hf- DBP-QP or Hf-DBP-QP-SN after comparison with the standard curve of H2DBP in DMSO.
  • Hf-DBP-QP-SN Digestion of Hf-DBP-QP-SN for Quantification of SN38 by LC-MS.
  • 100 pL IM NaHCCh solution was added to 100 pL Hf-DBP-QP-SN dispersion in PBS (SN concentration of approximately 100 pM).
  • the mixture was sealed and sonicated for 20 min.
  • 100 pL saturated NaCl solution and 150 pL ethyl acetate (EA) were added and the mixture was vortexed for 1 min.
  • the EA layer after centrifugation was analyzed by LC-MS.
  • Hf-DBP-QP-SN Stability Test of Hf-DBP-QP-SN in PBS.
  • Hf-DBP-QP-SN was dispersed in 1 mL PBS (1 mM) with a Hf concentration of 5 mM. 200 uL suspension was taken after incubation for 1, 2, 4, 8, and 24 h and centrifuged for PXRD measurement.
  • the concentration of DCFH was 5 pM while the concentration of Hf was 40 pM.
  • the PBS solution with the same DCFH concentration served as a blank control.
  • the fluorescence signal (em. 520/20 nm) was collected with a Synergy HTX microplate reader (ex. 485/20 nm; Agilent Technologies, Santa Clara, California, United States of America).
  • Hydroxyl radical generation in Test Tubes Hydroxyl radical (OH) generation under irradiation was detected by the APF assay.
  • APF assay was added to the PBS suspension of Hf-DBP, Hf-DBP-QP or Hf-DBP-QP-SN at the same Hf concentration. In the final mixture, the concentration of APF was 5 pM while that of Hf was 40 pM.
  • the fluorescence signal (em. 520/20 nm) was collected with a Synergy HTX microplate reader (ex. 485/20 nm; Agilent Technologies, Santa Clara, California, United States of America).
  • Hf-DBP-QP-SN or MeO-SN was dispersed in H2O at the same concentration of total SN38 (100 pM).
  • FeCh, (+)- Sodium L-ascorbate, Na2(EDTA) 2H2O (ethylenediaminetetraacetic acid, disodium salt dihydrate) was firstly dissolved in water to reach a concentration of 10 mM separately (10.5 mM for Na2(EDTA) 2H2O).
  • Hf-DBP-QP-SN or MeO-SN solution 100 pL of each solution and 50 pL H2O2 (200 mM in H2O) was added to Hf-DBP-QP-SN or MeO-SN solution to generate hydroxyl radical in situ and to trigger the release of SN38.
  • H2O2 100 pL was added to Hf-DBP-QP-SN to digest the nMOF, after which the mixture was sealed and sonicated for 20 minutes before the addition of 100 pL PBS (200 mM, pH 4) to adjust the pH to 7.
  • CT26 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (filtered, VWR International, Radnor, Pennsylvania, United States of America), 1% Penicillin-Streptomycin 100X solution sold under the tradename HYCLONE® HyClone Laboratories, Logan, Utah, United States of America), and cultured in a humidified atmosphere containing 5% CO2 at 37°C.
  • Mycoplasma was tested on a regular basis using a detection kit sold under the tradename MYCOALERT® (Lonza Walkersville, Inc., Walkersville, Maryland, United States of America)
  • the cytotoxicity of H2QP ligand, Hf-DBP-QP and Hf-DBP-QP-SN on CT26 cells were detected by MTS assay.
  • CT26 cells were seeded in 96-well plates at a density of 2500 cells/well. Different concentrations of Hf-DBP-QP-SN were added and 24 hours later 10% (v/v) of MTS reagent was added to each well. 90 minutes later the absorbance of each well at 490 nm was read by a Synergy HTX plate reader (Agilent Technologies, Santa Clara, California, United States of America) to calculate cell viability.
  • CT26 cells were seeded in 96-well plates at a density of 2500 cells/well. Different concentrations of SN38 or Me2QP-SN were added and 48 hours later 10% (v/v) of MTS reagent was added to each well. 90 minutes later the absorbance of each well at 490 nm was read by a Synergy HTX plate reader (Agilent Technologies, Santa Clara, California, United States of America) to calculate cell viability.
  • CT26 cells were seeded in 6-well plates at a density of 2 * 10 5 /well and cultured overnight.
  • Hf-DBP-QP Hf: 50 pM
  • Hf-DBP-QP-SN Hf: 50 pM
  • was added at an equivalent metal concentration into medium (n 3) and incubated for 1, 2, 3, 4 and 6 hours in a 37 °C incubator.
  • the medium was aspirated, the cells were washed with PBS for three times, collected by centrifugation, and counted with a hemocytometer.
  • Hf-DBP-QP and Hf-DBP-QP-SN were detected on a flow cytometer (sold under the tradename LSRFORTESSATM 4-15 HTS, BD Biosciences, Franklin Lakes, New Jersey, United States of America) and analyzed by FlowJo software (FlowJo LLC, Ashland, Oregon, United States of America).
  • Intracellular ROS Generation To demonstrate the generation of total ROS, CT26 cells were seeded in the cell culture dishes at a density of 1.5* 10 5 and cultured overnight. Hf- DBP-QP-SN or Hf-DBP-QP was added at an equivalent metal concentration of 50 pM and further incubated in a 37 °C incubator for 4 hours. The cells were washed with PBS solution for 3 times and 1 mL cell culture medium containing with 20 pM DCFH-DA was added and incubated at 37 °C for another 30 min. Then, the cells were irradiated with X-ray (3 Gy) and continued to be cultured in the cell incubator for another 12 h.
  • Hf- DBP-QP-SN or Hf-DBP-QP was added at an equivalent metal concentration of 50 pM and further incubated in a 37 °C incubator for 4 hours. The cells were washed with PBS solution for 3 times and 1 mL cell culture medium containing with 20 pM DCFH
  • CT26 cells were seeded in the cell culture dishes at a density of 1.5* 10 5 and cultured overnight.
  • Hf-DBP-QP-SN or Hf-DBP-QP was added at an equivalent metal concentration of 50 pM and further incubated in a 37 °C incubator for 4 hours.
  • the cells were washed with PBS solution for 3 times and 1 mL cell culture medium containing with 30 pM DCFH-DA was added and incubated at 37 °C for another 30 min. Then the cells were irradiated with X-ray (3 Gy) and continued to be cultured in the cell incubator for another 24 h.
  • OH in cancer cells was also detected according to the similar method as above.
  • CT26 cells were seeded in the cell culture dishes at a density of 1.5 * 10 5 and cultured overnight.
  • Hf-DBP-QP-SN or Hf-DBP-QP was added at an equivalent metal concentration of 50 pM and further incubated in a 37 °C incubator for 4 hours.
  • the cells were washed with PBS solution for 3 times and 1 mL cell culture medium containing with 10 pM HPF was added and incubated at 37°C for another 30 min. Then the cells were irradiated with X-ray (3 Gy) and continued to be cultured in the cell incubator for another 24 h.
  • Hf-DBP-QP or Hf-DBP-QP-SN was added at an equivalent metal concentration of 50 pM for 4 hours and irradiated with X- ray (3 Gy). 24 hours later, the cells were washed with PBS, trypsinized to afford single cell suspensions, The cells were stained with the dead cell apoptosis kit with annexin V Alexa Fluor 488 & PI and resuspended in the binding buffer for flow cytometric analysis (Annexin- V in FITC channel, PI in PE-dazzle 594 channel).
  • RT250 orthovoltage X-ray machine model (Philips, Andover, Massachusetts, United States of America) with fixed setting at 250 kVp, 15 mA and a built-in 1 mm Cu filter was used for X-ray irradiation in test tube and in vitro experiments.
  • the cells were washed with PBS twice and then trypsinized to afford single cell suspensions.
  • the cells were counted and diluted, then 200 cells were seeded in each well of 6-well plates and cultured in 2 mL medium for another 7 days.
  • the plates were rinsed once with PBS, fixed by 4% paraformaldehyde for 20 minutes at room temperature, and washed with PBS once.
  • the 6-well plates were then scanned and analyzed with a cell analysis system sold under the tradename INCUCYTE® S3 (Sartorius Bioanalytical Instruments, Inc., Brooklyn Center, Minnesota, United States of America) in the whole well mode with a 4 objective.
  • the colonies were identified with software sold under the tradename INCUCYTE® 2021A (Sartorius Bioanalytical Instruments, Inc., Brooklyn Center, Minnesota, United States of America) in a cellular resolution and the confluence was used as a parameter to calculate the plating efficiency (PE)and surviving fraction (SF)
  • PE plating efficiency
  • SF surviving fraction
  • the dose modifying ratio at a 10% was used as a parameter to assess radiosensitization effect and defined as the ratio of doses under reference conditions to produce a 10% SF:
  • DMR 10% DNA Damage.
  • CT26 cells were seeded in cell culture dishes at a density of 1.5x 10 5 .
  • the cells were treated in the same way as in the clonogenic assay. 24 hours after radiation, the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes. The cells were again rinsed with PBS, blocked and permeabilized with 5% FBS + 0.3% Triton-X in PBS at room temperature for 1 hour. After blocking, the cells were incubated with the y-H2AX primary antibody (1 :500) in 1% BSA + 0.3% Triton-X in PBS at 4 °C overnight.
  • the cells were then washed with PBS and incubated with the Alexa Fluor 488 conjugated secondary antibody (1 :3000) in 1% BSA + 0.3% Triton- X in PBS at room temperature for 1 hour. Afterwards, the cells were washed with PBS and further incubated with Hoechst 33342 (10 pg mL' 1 ) in PBS for 10 min at 37 °C to visualize cell nuclei, respectively. Finally, cells were washed by PBS for 3 times and observed on a Leica Stellaris 8 confocal microscope (Leica Microsystems, Wetzlar, Germany).
  • X-RAD 225 image-guided biological irradiator (Precision X- ray Inc., Madison, Connecticut, United States of America) was used with voltage at 225 kVp, current at 13 mA, a 0.3 mm Cu filter, and a 15 mm collimator for animal therapy.
  • the X-ray dose rate of X-RAD 225 was 0.04167 Gy/second.
  • BALB/c mice (6-8 weeks) were obtained from Charles River Laboratories, Inc (Wilmington, Massachusetts, United States of America) and bred in house.
  • CT26 tumor model was established on BALB/c mice by inoculating 2 * 10 6 cells/mouse subcutaneously onto the right flanks at day 0, respectively.
  • CT26 tumors reached to -105 mm 3 , the mice were randomized for irradiation treatment.
  • mice 6-8 hours later, the mice were anaesthetized with 2.5% (v/v) isoflurane/O2 and mounted onto the X-Rad 225 irradiator.
  • the CT26 tumors were irradiated with 2 Gy X-ray/fraction for 3 consecutive days.
  • the mice were euthanized, and the tumors and major organs were sectioned for hematoxylin- eosin (H&E) staining to evaluate general toxicity.
  • H&E hematoxylin- eosin
  • TGI tumor growth inhibition index
  • the slides were treated with antigen retrieval buffer (AR9640; Leica Biosystems, Wetzlar, Germany) and heated in a steamer over 97°C for 20 minutes). After washing with tris-buffered saline (TBS), the slides were incubated with primary y-H2AX antibody (1 :400) or primary Ki67 antibody (Clone# SP6, 1 :400; Thermo Fisher Scientific, Waltham, Massachusetts, United States of America) at room temperature for 1 hour in a wet chamber.
  • antigen retrieval buffer AR9640; Leica Biosystems, Wetzlar, Germany
  • TBS tris-buffered saline
  • the slides were washed with TBS, then y-H2AX and Ki67 slides were incubated with anti-rabbit-polymer (Bond Polymer Refine Detection, DS9800; Leica Biosystems, Wetzlar, Germany) for 30 minutes at room temperature.
  • the antigen-antibody binding was detected with the 3,3'- Diaminobenzidine (DAB) (DAKO, K3468; Agilent Technologies, Santa Clara, California, United States of America) system.
  • DAB 3,3'- Diaminobenzidine
  • Tissue sections were then immersed in hematoxylin for counterstaining and covered with cover glasses.
  • the slides were scanned on a CRi Pannoramic SCAN 40* whole slide scanner (3DHISTECH LTD, Budapest, Hungary) and analyzed with the QuPath-0.2.3 software.
  • the 5,15-di (p-benzoato)porphyrin ligand (H2DBP) was synthesized following reported procedures. 81
  • the quaterphenyl dicarboxylic acid (H2QP) with 3, 5 -dimethoxyl benzyl alcohol was synthesized from 2,7-dibromo-9J/-fluoren-9-one (12) in 6 steps via intramolecular ring-opening, amide formation with ammonia, borane reduction, amide coupling with 4-(((tert-butyldimethylsilyl)oxy)methyl)-2,6-dimethoxybenzoic acid, Suzuki coupling with (4-(methoxycarbonyl)phenyl)boronic acid, and base-catalyzed hydrolysis.
  • Hf-DBP-QP The mix-ligand nMOF, Hf-DBP-QP, was synthesized solvothermally by heating a mixture of HfCU, H2DBP, H2QP, acetic acid, and water in N, 7V-dimethylformamide at 80 °C for 1 day.
  • Hf-DBP-QP was postsynthetically modified by treatment with 4-nitrophenyl chloroformate followed by SN38 to afford Hf-DBP-QP-SN with SN38 conjugated to the QP ligand via a hydroxyl radical-responsive 3, 5 -dimethoxyl benzyl carbonate linkage.
  • Hf-DBP-QP displayed a similar nanoplate morphology as previously reported Hf- DBP with a diameter of ⁇ 120 nm and a thickness of ⁇ 20 nm by transmission electron microscopy (TEM). See Figure 15 A. After postsynthetic modification, Hf-DBP-QP-SN retained the nanoplate morphology without obvious variation of diameters. See Figure 15B.
  • Hf-DBP-QP and Hf-DBP-QP-SN were measured to be 81 ⁇ 3 nm and 96 ⁇ 3 nm (see Figure 15C), respectively, while their ( ⁇ -potentials were -18.4 ⁇ 0.8 mV and -21.7 ⁇ 0.5 mV (see Figure 15D), respectively, by dynamic light scattering (DLS).
  • Hf-DBP-QP and Hf-DBP-QP- SN displayed similar patterns as Hf-DBP, corresponding to the Hfn-nMOF structure with Hfi2( /3-O)8(//3-OH)8( /2-OH)6 SBUs and linear dicarboxylate ligands in a hep topology. 39 See Figure 16A. Moreover, Hf-DBP-QP-SN retained its crystallinity after incubation in PBS (ImM, pH 7.4) for 24 hours (see Figure 16B), suggesting the good stability of Hf-DBP-QP- SN in physiologically relevant environments.
  • PBS ImM, pH 7.4
  • UV-Vis spectroscopic analysis of digested Hf-DBP-QP determined the QP/DBP ratio to be 3.29 (see Figure 17) by comparing the absorbance of QP and DBP ligands with their standard curves (see Figures 18A-18D), which was consistent with the QP/DBP ratio (3.55) used in the synthesis.
  • the total loading of SN38 was quantified by high performance liquid chromatography (HPLC) after digesting Hf-DBP-QP-SN in 10% trifluoracetic acid in EA, indicating -25% of QP ligands were conjugated with SN38.
  • HPLC high performance liquid chromatography
  • Liquid chromatography-mass spectrometry (LC-MS) analysis indicated the trapping of 1.5% free SN38 (relative to total SN38) in the pores after digesting Hf-DBP-QP-SN using NaHCCh and extraction with ethyl acetate.
  • Hf-DBP-QP-SN was determined to be Hfi2( /3-O)8(//3-OH)8( /2-OH)6(QP- SN)0.88(QP)2.74(DBP)1.36(OH)8.04(H 2 O)8.04(SN38)0.01.
  • a molecular counterpart, Me 2 QP-SN was synthesized from compound 17 using the same synthetic route to support the post-synthetic modification and to examine the toxicity of the prodrug. See Scheme 5. Because of low aqueous solubility of Me 2 QP-SN, we synthesized MeO-SN from compound 1 and used it as a homogeneous control. See Scheme 3, above.
  • Hf-DBP-QP-SN In terms of total ROS, the relative enhancements of Hf-DBP and Hf-DBP-QP over PBS were 76% and 21%, respectively (see Figure 19C), while Hf-DBP-QP-SN was comparable with PBS.
  • the reduced ROS and hydroxyl radical signals from Hf-DBP-QP-SN are likely due to the consumption of OH by the 3,5-dimethoxylbenzyl carbonate linkage to release SN38, while the incorporation of DBP ligand significantly improved total ROS signal by generating singlet oxygen through the RDT process. 82,83
  • H2QP ligand and Hf-DBP-QP were assessed by 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay on CT26 cells. No obvious toxicity was observed at a Hf concentration of up to 200 pM or at a H2QP concentration of up to 60.33 pM. See Figure 21. This result suggests the biocompatibility of the mix-ligand Hf-DBP-QP nMOF.
  • Me2QP-SN and Hf-DBP-QP-SN showed no obvious toxicity (see Figures 22B and 22C) at an equivalent SN38 concentration of up to 25 pM or a Hf concentration of up to 300 pM (corresponding to 22.3 pM SN38 in Hf-DBP-QP-SN). This result shows that Me2QP-SN and Hf-DBP-QP-SN serve as prodrugs with low intrinsic toxicity.
  • CT26 cells were incubated with Hf-DBP-QP-SN for 1, 2, 3, 4 and 6 h, and cellular content of DBP was detected by flow cytometry. See Figure 23. CT26 cells showed efficient uptake of Hf-DBP- QP-SN in a time-dependent manner.
  • Hf-DBP-QP Hydroxyphenyl fluorescein
  • Hf-DBP-QP-SN(+) generated 26.33% ROS + cells, which was significantly higher than Hf-DBP-QP(+) (4.82%). See Figures 27A-27D. This difference could be caused by the oxidative pressure by released SN38 molecules. 43
  • Hf-DBP-QP and Hf-DBP-QP-SN exhibited longterm anti-proliferative effect with a radiation enhancement factor (REFio) of 1.147, and 2.52, respectively. See Figures 28 A and 28B.
  • the radioenhancement of Hf-DBP-QP likely resulted from RT-RDT process, while the higher radioenhancing effect of Hf-DBP-QP-SN was likely due to the synergistic effect between RT-RDT and chemotherapy resulted from the released SN38 triggered by OH.
  • Flow cytometry was used to assay the exposure of phosphatidylserine (PS) of CT26 cells by annexin V/propidium iodide (PI) apoptosis staining. See Figure 28C. Compared with PBS(-) and PBS(+) groups, Hf-DBP-QP(+) treatment gave an increased ratio of early and late apoptotic cells (29%), suggesting effective radioenhancement effect of Hf-DBP-QP.
  • PS phosphatidylserine
  • PI propidium iodide
  • Hf- DBP-QP-SN ⁇ Hf- DBP-QP-SN ⁇
  • Hf-DBP-QP(+) treatment showed an increase of necrosis, early and late apoptosis (44.81%), supporting the successful release of the SN38 to result in a higher proportion of apoptotic and necrotic cells than Hf-DBP-QP(+) treatment.
  • DNA damage was measured by y-H2AX assay. See Figure 29.
  • Hf-DBP-QP(+) treatment showed stronger red fluorescence than PBS(+) treated cells, suggesting the RT- RDT process could effectively lead to more DNA damage.
  • Hf-DBP-QP-SN(+) treated cancer cells showed stronger DNA damage than Hf-DBP-QP(+) treated cells, likely due to DNA damage by released SN38.
  • Hf-DBP-QP-SN(+) treatment effectively generated ROS through the RT-RDT process and caused oxidative stress by the released SN38.
  • the CT26 tumor model was established on BALB/c mice by inoculating 2 x 10 6 cells/mouse subcutaneously onto the right flanks.
  • Hf-DBP-QP-SN(-) exhibited no obvious tumor inhibition effect, supporting low toxicity of the nMOF nanotherapeutics. Irradiation of CT26 tumors with 2 Gy X-ray by 3 fractions moderately inhibited tumor growth with a tumor growth inhibition index (TGI) of 0.474, while Hf-DBP-QP(+) enhanced tumor growth inhibition with a TGI of 0.744. Irinotecan(+) exhibited a TGI of 0.641 due to the additive effect of radiotherapy and chemotherapy.
  • TGI tumor growth inhibition index
  • Hf-DBP-QP-SN(+) treatment gave an impressive TGI of 0.935 (see Figure 30A), demonstrating the synergistic effect between RT-RDT and chemotherapeutic effect of SN38 released from Hf-DBP-QP-SN in the tumors.
  • Hf-DBP-QP-SN(+) and control groups were also evaluated by analyzing tumor slices by hematoxylin and eosin (H&E) and TdT-mediated dUTP nick end labeling (TUNEL) staining. See Figures 31.
  • H&E hematoxylin and eosin
  • TUNEL TdT-mediated dUTP nick end labeling
  • Hf-DBP-QP- SN(+) treatment led to the most damaged and TUNEL positive cells, supporting the synergistic antitumor effects from RT-RDT and chemotherapy from released SN38.
  • y-H2AX and Ki67 staining further demonstrated that Hf-DBP-QP-SN(+) effectively induced the strongest DNA damage and inhibited tumor proliferation while greatly reducing systemic toxicity of traditional chemotherapeutic drugs. See Figure 31.
  • the mice in all treatment groups showed similar and steady body weight growth patterns (see Figure 30B), suggesting the lack of toxicity from these treatments. The treatments did not cause any histological abnormality in major organs (see Figure 32), further supporting the safety of Hf-DBP-QP-SN(+) treatment.
  • Hf-DBP-QP-SN(+) treatment significantly increased the median survival to 38 days from 29 days for Hf-DBP-QP(+) treatment. See Figure 33.
  • PBS(+) only modestly increased mouse median survival to 21 days from 18 days for PBS(-).
  • QP-SN new functional ligand
  • conjugated SN38 via a OH-cleavable linker
  • QP-SN and photosensitizing DBP ligands were used to prepare a novel mix-ligand nMOF, Hf-DBP-QP-SN, as a potent nMOF radiosensitizer and nanotherapeutic for RT-RDT and chemotherapy via X-ray triggered release of SN38 in tumors.
  • Test tube in vitro, and in vivo studies demonstrated that X-ray could precisely stimulate the release of SN38 from Hf-DBP-QP-SN in tumors through a cascade of OH generation under X-ray irradiation, hydroxylation of the 3,5- dimethoxylbenzyl carbonate at ortho positions, and subsequent 1,4-elimination to release SN38.
  • Hf-DBP-QP-SN plus X-ray treatment significantly inhibit the tumor growth with a TGI of 0.935 via synergistic RT-RDT and X-ray induced chemotherapy.
  • a MOF comprising a X-ray-triggerable innate immune modulate prodrug
  • a MOF was prepared comprising a prodrug of R848, as described hereinbelow. See Scheme 7, below.
  • a homogenous R848 prodrug was also prepared. See Scheme 6, below.
  • X-ray-triggerable prodrugs of other innate immune modulators see Figures 35A-35C can also be prepared.
  • Hf-DBP-QP-NCh nMOF (4.73 pmol based on QP) was redispersed in ACN before the addition of R848 (1.5 mg, 4.73 pmol) and TEA (1.2 pL, 10 pmol); the solution was then stirred for 3 days.
  • the as-synthesized Hf-DBP-QP-R848 was washed with ACN 10 times to remove remaining R848 to less than 1% than conjugated R848. The measurement was quantified by LC-MS.
  • Bone marrow-derived dendritic cells were obtained by the following procedure: Female C57BL/c mice aged 6 to 8 weeks were euthanized, and bone marrow cells were extracted from the femur and tibia using insulin syringes containing RPMI-1640.
  • Sterile ACK buffer (Corning, Glendale, Arizona, United States of America) was used to lyse the red blood cells, and the remaining cells were cultured in RPMI-1640 complete medium supplemented with 20 ng/mL recombinant mouse granulocyte-macrophage colonystimulating factor (GM-CSF, R&D Systems, Minneapolis, Minnesota, United States of America) and 10 ng/mL recombinant murine interleukin-4 (IL-4, PeproTech, Cranbury, New Jersey, United States of America). On day 4, the entire medium was discarded and replaced with a fresh, warm medium containing 20 ng/mL GM-CSF and 10 ng/mL IL-4.
  • GM-CSF mouse granulocyte-macrophage colonystimulating factor
  • IL-4 murine interleukin-4
  • BMDCs medium suspension containing these cells
  • the purity of the cells was assessed using flow cytometry with CD1 Ic-PE/Cy5.5 (N418) antibodies.
  • BMDCs were plated in 96-well plates at a density of 5* 10 4 cells per well.
  • Various concentrations of Hf-DBP-QP-R848 were irradiated with 0, 1, 5, 10, or 50 Gy and then added to the cells. The cells were incubated for 24 hours. The culture supernatants were collected and analyzed using enzyme-linked immunosorbent assay (ELISA) to measure IL-6 levels. See Figure 34.
  • ELISA enzyme-linked immunosorbent assay
  • N-alkyl-picolinium-based prodrugs The radiation-sensitive release of N-alkyl-picolinium-based prodrugs has recently been reported. 85 As described in Schemes 8 and 9, below, a pomalidomide prodrug nMOF was prepared containing a bridging ligand with a picolinium-based linkage that can be cleaved via a radiation-induced radical-promoted mechanism.
  • a pyridine-substituted carbamate of small molecule chemotherapeutic pomalidomide was prepared by reacting pomalidomide with phosgene and then with 4-(hydroxymethylpyridine).
  • Other chemotherapeutics such as lenalidomide, can be used in place of pomalidomide.
  • a mix-ligand nMOF, Hf-DBP-QP-BrPC was synthesized solvothermally by heating a mixture of HfCU, H2DBP, BrPC-QP, acetic acid, and water in N, A -di methyl form am ide at 80 °C for 1 day.
  • Hf-DBP-QP-PC was post-synthetically modified by treatment with the pyridine-substituted carbamate of pomalidomide afford Hf- DBP-QP-pomalidamide, i.e., a nMOF with pomalidomide covalently linked to a QP ligand by a X-ray sensitive picolinium linkage.
  • Radiotherapy activates picolinium prodrugs in tumors. Nature Chemistry 2024. doi.org/10/1038/s41557-024-01501-4.

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Abstract

L'invention concerne une structure organométallique (MOF) comprenant un promédicament sensible aux rayons X. Par exemple, la MOF peut comprendre des unités de construction secondaires (SBU) contenant du métal qui sont liées ensemble par l'intermédiaire de ligands de pontage organiques où la SBU comprend un cation métallique qui peut absorber les rayons X et où au moins un ligand de pontage organique est substitué par une fraction monovalente dérivée d'un agent thérapeutique, tel qu'un agent de chimiothérapie et/ou d'immunothérapie, par l'intermédiaire d'un groupe qui comprend une liaison capable de clivage de liaison favorisée par des radicaux. L'invention concerne également des méthodes d'utilisation du MOF pour traiter des maladies, telles que le cancer. Les méthodes peuvent comprendre la combinaison d'une radiothérapie ou d'une thérapie radiodynamique-radiothérapie avec une chimiothérapie ou une immunothérapie.
PCT/US2024/036893 2023-07-05 2024-07-05 Structures organométalliques à échelle nanométrique avec promédicaments déclenchables par rayons x pour radiothérapie, chimiothérapie et immunothérapie combinées Pending WO2025010407A2 (fr)

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