Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
  • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
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  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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  • A61K31/33—Heterocyclic compounds
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  • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
• • A—HUMAN NECESSITIES
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • A—HUMAN NECESSITIES
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
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  • 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
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • A—HUMAN NECESSITIES
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  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1277—Preparation processes; Proliposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
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  • C12N2310/141—MicroRNAs, miRNAs
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

• the presently disclosed subject matter provides prodrugs (e.g., prodrugs of chemotherapeutic agents) comprising drug moieties bound to lipid moieties via disulfide-containing linkers.
• the presently disclosed subject matter also provides nanoparticles comprising the prodrugs.
• the nanoparticles can comprise, for example, a lipid coating layer containing the prodrug and a nanoscale coordination polymer (NCP) nanoparticle core, which can itself optionally comprise a chemotherapeutic agent analogue or prodrug or a combination of two chemotherapeutic agent analogues or prodrugs.
• NCP nanoscale coordination polymer
• the nanoparticles can be further combined with photosensitizers for photodynamic therapy (PDT), targeting agents, and/or immunotherapy agents, such as immunosuppression inhibitors.
• PDT photodynamic therapy
• immunotherapy agents such as immunosuppression inhibitors.
• the nanoparticle- based compositions of the presently disclosed subject matter can provide synergistic anti-cancer effects by combining multiple treatment modalities in a variety of cancers.
• DHA dihydroartemisinin
• DOPA dioleoyl-sn-glycero-3-phosphate
• DOPC 1 ,2-dioleoyl-sn-glycero-3-phosphate sodium salt
• DSPE-PEG 2k 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N- [amino(polyethylene glycol) 2 ooo]
• IC50 fifty percent inhibitory concentration
• ICP-MS inductively coupled plasma-mass spectrometry
• NCP nanoscale coordination polymer
• nm nanometer
• PBS phosphate buffered saline
• PD-L1 programmed death-ligand 1
• PEG polyethylene glycol
• PTX paclitaxel
• PVP polyvinylpyrrolidone
• RES reticuloendothelial system
• Photodynamic therapy where a systemic or locally administrated photosensitizer (PS) is excited by local light irradiation to produce reactive oxygen species (ROS), can selectively kill tumor cells while preserving adjacent normal tissue.
• PDT does not incur cross resistance with radiotherapy or chemotherapy, and therefore, can be useful in the treatment of cancer patients who have failed radiotherapy and chemotherapy.
• PDT provokes a strong acute inflammatory reaction observed as localized edema at the targeted site.
• the inflammation elicited by PDT is a tumor antigen nonspecific process orchestrated by the innate immune system.
• PDT is particularly effective in rapidly generating an abundance of alarm/danger signals, such as damage-associated molecular patterns (DAMPs), at the treated site that can be detected by the innate immunity alert elements.
• DAMPs damage-associated molecular patterns
• PDT can enhance antitumor immunity via stimulating dendritic cells by dead and dying tumor cells, leading to the recruitment and activation of CD8+ cytotoxic T cells (CTLs) followed by the formation of immune memory cells and resistance to subsequent tumor growth.
• CTLs cytotoxic T cells
• Some small molecule chemotherapeutics including oxaliplatin, paclitaxel, daunorubicin, docetaxel, doxorubicin, cyclophosphamide, dihydroartemisinin, and mitoxanthrone, can efficiently cause immunogenic cell death.
• Some chemotherapeutics are known to be immune-stimulatory. However, many small molecule chemotherapeutics are highly hydrophobic, making delivery of the anticancer agents to tumors difficult.
• prodrugs and/or other drug delivery platforms for chemotherapeutics to improve their delivery (e.g., their targeted delivery).
• prodrug and/or other drug delivery platforms that can combine chemotherapeutics, such as those chemotherapeutics with known immunogenic effects, with other treatment modalities, such as PDT modalities and/or immunotherapy agents (e.g., immunosuppression therapeutics), to provide enhanced anticancer therapy.
• chemotherapeutics such as those chemotherapeutics with known immunogenic effects
• other treatment modalities such as PDT modalities and/or immunotherapy agents (e.g., immunosuppression therapeutics)
• the presently disclosed subject matter provides a prodrug comprising: (a) a monovalent drug moiety; (b) a monovalent lipid moiety; and (c) a bivalent linker moiety comprising a biodegradable bond, wherein the monovalent drug moiety and the monovalent lipid moiety are linked through the linker.
• the monovalent drug moiety is a monovalent derivative of an anticancer drug compound.
• the monovalent drug moiety is a monovalent derivative of a drug compound selected from the group comprising Etoposide (ET), Paclitaxel (PTX), OTS964, NLG919, OTS167, OTSC41 , dihydroartemisin, Camptothecin (CPT), Doxorubicin, Docetaxel, Vincristine, Mitoxantrone, Artesunate, and Capecitabine.
• the biodegradable bond is a disulfide bond.
• the monovalent lipid moiety is a monovalent derivative of cholesterol, oleic acid, lyso-lipid or phosphocholine. In some embodiments, the monovalent lipid moiety is a cholesterol derivative and the monovalent lipid moiety and bivalent linker moiety together have the structu
• the monovalent lipid moiety is an oleic acid derivative and the monovalent lipid moiety and bivalent linker moiety together have the structure:
• the monovalent lipid moiety is a lyso-lipid derivative and the monovalent lipid moiety and bivalent linker moiety together have the structure:
• the monovalent lipid moiety is a phosphocholine derivative and the monovalent lipid moiety and bivalent linker together have the structure:
• the presently disclosed subject matter provides a method for treating a disease in a patient in need of treatment wherein the method comprises the steps of: administering to a patient a prodrug comprising a monovalent drug moiety, a monovalent lipid moiety, and a bivalent linker moiety comprising a biodegradable bond, optionally wherein administering the prodrug to the patient comprises administering a nanoparticle comprising the prodrug to the patient; and administering to the patient an immunotherapy agent.
• the prodrug is administered by administering a nanoparticle comprising the prodrug and wherein the nanoparticle comprises a nanoscale coordination polymer.
• the method further comprises administering to the patient a photosensitizer and/or a scintillator, and irradiating at least a portion of the patient with light and/or X-rays.
• the photosensitizer is a nanoparticle photosensitizer.
• the disease is cancer.
• the disease is selected from the group comprising a head tumor, a neck tumor, breast cancer, a gynecological tumor, a brain tumor, colorectal cancer, lung cancer, mesothelioma, a soft tissue sarcoma, skin cancer, connective tissue cancer, adipose cancer, lung cancer, stomach cancer, anogenital cancer, kidney cancer, bladder cancer, colon cancer, prostate cancer, central nervous system cancer, retinal cancer, blood cancer, neuroblastoma, multiple myeloma, lymphoid cancer, and pancreatic cancer.
• the method further comprises administering to the patient an additional treatment.
• the additional treatment is a cancer treatment, wherein said cancer treatment is selected from the group comprising surgery, radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, and gene therapy.
• the chemotherapy comprises administering a drug selected from the group comprising oxaliplatin, doxorubicin, daunorubicin, docetaxel, mitoxanthrone, paclitaxel, digitoxin, digoxin, and septacidin.
• the chemotherapy comprises administering 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, and an inorganic nanoparticle formulation.
• 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, and an inorganic nanoparticle formulation.
• the immunotherapy agent is selected from the group comprising an anti-CD52 antibody, an anti-CD20 antibody, anti-CD47 antibody, an anti-GD2 antibody, polysaccharide K, and a cytokine.
• the immunotherapy agent is selected from the group comprising Alemtuzumab, Ofatumumab, Rituximab, Zevalin, Adcetris, Kadcyla, and Ontak.
• the immunotherapy agent is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an IDO inhibitor, a CCR7 inhibitor, a OX40 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor.
• the cytokine is selected from the group comprising an interferon and an interleukin. In some embodiments, the cytokine is selected from the group comprising IFN-a, IFN- ⁇ , IL-2, IL-12 and TNF-a.
• the disease is a metastatic cancer.
• the presently disclosed subject matter provides a composition comprising zinc pyrophosphate and a prodrug comprising a monovalent drug moiety, a monovalent lipid moiety, and a bivalent linker moiety comprising a biodegradable bond.
• the zinc pyrophosphate is in the form of nanoparticles.
• the presently disclosed subject matter provides a composition comprising a prodrug comprising a monovalent drug moiety, a monovalent lipid moiety, and a bivalent linker moiety comprising a biodegradable bond; and a nanoscale coordination polymer comprising a cisplatin and/or oxaliplatin prodrug.
• the composition further comprises siRNA.
• the presently disclosed subject matter provides a nanoscale particle for delivery of therapeutic agents, said nanoscale particle comprising: a core comprising a metal-organic matrix material, optionally wherein the metal-organic matrix material comprises a coordination polymer; and a prodrug, wherein the prodrug comprises a monovalent drug moiety, a monovalent lipid moiety, and a bivalent linker moiety comprising a biodegradable bond.
• the nanoscale particle further comprises at least one nucleic acid chemotherapeutic agent.
• the nucleic acid chemotherapeutic agent is a siRNA, a miRNA, or an AS ODN.
• the at least one nucleic acid is attached to the metal- organic matrix material core via coordination bonds between phosphate groups on the nucleic acid and metal ions on an outer surface of the core.
• the at least one nucleic acid is selected from the group comprising survivin siRNA, ERCC-1 siRNA, P-glycoprotein siRNA (P-gp siRNA), Bcl-2 siRNA, or a mixture thereof.
• the nanoscale particle further comprises at least one photosensitizer.
• the nanoscale particle further comprises at least one non-nucleic acid chemotherapeutic agent incorporated in the metal-organic matrix material core, optionally wherein the at least one non- nucleic acid chemotherapeutic agent is incorporated in the metal-organic matrix material core via a covalent or coordination bond.
• the at least one non-nucleic acid chemotherapeutic agent incorporated in the metal-organic matrix material core is selected from the group comprising cisplatin or oxaliplatin prodrugs, gemcitabine, methotrexate, leucovorin, pemetrexed disodium, doxorubicin, vinblastine, vincristine, vindesine, cytarabine, azathioprine, melphalan, imatinib, anastrozole, letrozole, carboplatin, paclitaxel, docetaxel, etoposide, and vinorelbine.
• the nanoscale particle comprises at least two chemotherapeutic agents incorporated in the metal-organic matrix material core.
• the non-nucleic acid chemotherapeutic agent is cis, cis, trans-Pt(NH 3 )2Cl2(OEt)(02CCH2CH 2 COOH), optionally wherein the core comprises between about 10 weight % and about 50 weight % of the non-nucleic acid chemotherapeutic agent.
• the nanoscale particle has an average diameter of between about 20 nm and about 140 nm.
• the nanoscale particle further comprises one or more coating agents or layers covering at least a portion of the outer surface of the metal-organic matrix material core, wherein the one or more coating agents or layers are selected from a metal oxide, a polymer, a single lipid layer, a lipid bilayer, and combinations thereof.
• the metal-organic matrix material core is coated with a lipid bilayer comprising a cationic lipid and/or a functionalized lipid, wherein said functionalized lipid is a lipid functionalized with a group that can bond to a nucleic acid, and wherein at least one nucleic acid is covalently bonded to the functionalized lipid and/or attached to the cationic lipid via electrostatic interactions.
• the lipid bilayer comprises a mixture comprising one or more of a thiol- or dithiol- functionalized 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE), 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), and 1 ,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC).
• DSPE thiol- or dithiol- functionalized 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine
• DOTAP 1,2-dioleoyl-3-trimethylammonium propane
• DOPC 1 ,2-dioleoyl-sn- glycero-3-phosphocholine
• the one or more coating agents or layers further comprise a passivating agent, optionally a hydrophilic polymer; a targeting agent, optionally a RGD peptide; and/or an imaging agent, optionally a fluorescent moiety.
• the lipid bilayer further comprises one or more of 1 ,2-dioleoyl-sn-glycero-3- phosphate sodium salt (DOPA), cholesterol, and pegylated-DSPE.
• DOPA 1 ,2-dioleoyl-sn-glycero-3- phosphate sodium salt
• the metal-organic matrix material core comprises a metal bisphosphonate coordination polymer comprising a multivalent metal ion and a bisphosphonate.
• the multivalent metal ion is selected from the group comprising Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , and combinations thereof.
• the bisphosphonate is a chemotherapeutic prodrug, optionally a cisplatin or oxaliplatin prodrug.
• the prodrug comprises a monovalent derivative of DHA.
• the bisphosphonate is a bisphosphonate ester of cis, cis- trans- [Pt(NH 3 ) 2 Cl2(OI-l)2] (a cisplatin prodrug) or cis, trans- [Pt(dach)Cl2(OH) 2 ].
• the multivalent metal ion is Zn 2+ .
• the metal-organic matrix material core comprises between about 40 and about 50 weight % of bisphosphonate.
• the nanoscale particle further comprises a lipid single layer or lipid bilayer coating, optionally wherein one or more of survivin siRNA, P-gp siRNA, and Bcl-2 siRNA are attached to the coating.
• the nanoscale particle has a diameter between about 20 nm and about 180 nm. In some embodiments, the nanoscale particle has a diameter between about 90 nm and about 180 nm.
• the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising zinc pyrophosphate and a lipid-conjugate prodrug; a nanoscale coordination polymer comprising a cisplatin and/or oxaliplatin prodrug and a lipid- conjugate prodrug, or a composition comprising a nanoscale particle comprising a metal-organic matrix material core and a lipid-conjugate prodrug.
• the cancer is selected from lung cancer, pancreatic cancer, ovarian cancer, breast cancer and colon cancer.
• the cancer is ovarian cancer, optionally a cisplatin resistant ovarian cancer.
• the method further comprises administering to the subject an immunotherapy agent.
• the immunotherapy agent is selected from the group comprising an anti-CD52 antibody, an anti-CD20 antibody, anti-CD47 antibody an anti-GD2 antibody, a cytokine, and polysaccharide K.
• the immunotherapy agent is selected from the group comprising Alemtuzumab, Ofatumumab, Rituximab, Zevalin, Adcetris, Kadcyla, and Ontak.
• the immunotherapy agent is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an IDO inhibitor, a CCR7 inhibitor, an OX40 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor.
• the method further comprises administering to the subject a photosensitizer.
• the photosensitizer is a pyrolipid, wherein said pyrolipid is a lipid covalently attached to a porphyrin or a derivative or analog thereof.
• the presently disclosed subject matter provides a pharmaceutical formulation comprising a composition comprising a lipid-conjugate prodrug, optionally wherein the composition comprises a nanoscale particle, and a pharmaceutically acceptable carrier.
• the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof wherein the method comprises administering to the subject a composition comprising a nanoscale particle comprising a lipid-conjugate prodrug and a metal-organic matrix material core and a photosensitizer, and irradiating the subject or a treatment area of the subject with radiation having a wavelength suitable to activate the photosensitizer.
• the cancer is a head and neck cancer, optionally wherein the head and neck cancer is a cisplatin resistant head and neck cancer.
• the method further comprises administering to the subject an immunotherapy agent.
• the immunotherapy agent is selected from the group comprising an anti-CD52 antibody, an anti-CD20 antibody, anti-CD47 antibody, an anti-GD2 antibody, polysaccharide K, and a cytokine.
• the immunotherapy agent is selected from the group comprising a radiolabeled antibody, an antibody-drug conjugate, and a neoantigen.
• the immunotherapy agent is selected from the group comprising Alemtuzumab, Ofatumumab, Rituximab, Zevalin, Adcetris, Kadcyla, and Ontak.
• the immunotherapy agent is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an IDO inhibitor, a CCR7 inhibitor, a OX40 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor.
• lipid moiety-containing prodrugs, nanoparticles, and nanoparticle formulations comprising the prodrugs, as well as methods of treating disease using the nanoparticles and formulations.
• Figure 1A is a graph showing the antitumor efficacy of a cisplatin- containing nanoscale coordination polymer (NCP) particle of the presently disclosed subject matter further comprising a lipid coating layer comprising a cholesterol-linked etoposide prodrug and lipid-modified small interfering ribonucleic acids (i.e., NCP-1/ET/siRNAs) in a xenograft tumor model of a cisplatin-resistant ovarian cancer (A2780/CDDP).
• NCP nanoscale coordination polymer
• Tumor volume in cubic millimeters (mm 3 )
• a control i.e., a phosphate- buffered saline (PBS)-treated model, squares.
• PBS phosphate- buffered saline
• Figure 1 B is a graph showing the antitumor efficacy of a cisplatin- containing NCP particle of the presently disclosed subject matter further comprising a lipid coating layer comprising a cholesterol-linked etoposide prodrug and lipid-modified small interfering ribonucleic acids (i.e., NCP- 1/ET/siRNAs) in a xenograft tumor model of a cisplatin-resistant ovarian cancer (SKOV-3).
• NCP- 1/ET/siRNAs small interfering ribonucleic acids
• Tumor volume in cubic millimeters (mm 3 )
• a control i.e., a phosphate-buffered saline (PBS)-treated model, squares.
• PBS phosphate-buffered saline
• Figure 1 C is a graph showing the antitumor efficacy of a cisplatin- containing NCP particle of the presently disclosed subject matter further comprising a lipid coating layer comprising a cholesterol-linked etoposide prodrug and lipid-modified small interfering ribonucleic acids (i.e., NCP- 1/ET/siRNAs) in a xenograft tumor model of a small cell lung cancer (H82).
• Tumor volume in cubic millimeters (mm 3 )
• a control i.e., a phosphate-buffered saline (PBS)-treated model, squares.
• FIG. 2 is a series of graphs showing calriticulin (CRT) exposure on the cell surface of CT26 murine colorectal adenocarcinoma cells induced by platinum (Pt) and/or paclitaxel (PTX) at a concentration of 5 micromolar ( ⁇ ) as determined by flow cytometry analysis.
• CTR calriticulin
• oxaliplatin top left
• PTX top middle
• a combination of PTX and oxaliplatin top right
• NCP NCP comprising zinc (Zn) and an oxaliplatin analogue
• NCP-2 bottom left
• a combination of Zn and PTX bottom middle
• NCP-2 coated with a lipid coating comprising a cholesterol-linked PTX NCP-2/PTX, bottom right.
• Data in gray from phosphate buffered saline (PBS)-treated cells is shown in each graph as a control.
• PBS phosphate buffered saline
• Figure 3 is a graph showing the platinum (Pt) data from pharmacokinetics and biodistribution of a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol- modified paclitaxel (NCP-2/PTX) after intravenous (i.v.) injection in CT26 murine colorectal adenocarcinoma tumor bearing mice at a dose of 1 milligram per kilogram (mg/kg) expressed as percentage initial dose (% ID) of Pt per gram (g) of tissue.
• Pt concentration was analyzed via inductively coupled plasma-mass spectrometry (ICP-MS) at 5 minutes, and at 1 , 3, 5, 8, 24, and 48 hours, and is expressed in micrograms g).
• ICP-MS inductively coupled plasma-mass spectrometry
• Figure 4 is a graph showing the pharmacokinetics of free oxaliplatin (observed data, open circles; predicted, dark grey line) and of a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified paclitaxel (NCP-2/PTX) (observed data, black squares; predicted, light grey line) after intravenous (i.v.) injection in Beagle dogs at a dose of 1 milligram per kilogram (mg/kg).
• the platinum (Pt) concentrations were analyzed via inductively coupled plasma-mass spectrometry (ICP-MS) for up to 336 hours after injection.
• ICP-MS inductively coupled plasma-mass spectrometry
• Figure 5 is a graph showing the in vivo anticancer activity of a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified paclitaxel (NCP-2/PTX) (black squares) and of a combination of NCP-2/PTX and an anti-programmed death-ligand 1 (PD-L1 ) antibody (Pd-L1 Ab) (open circles) injected intraperitoneally into CT26 murine colorectal adenocarcinoma tumor bearing mice at an oxaliplatin analogue dose of 1 milligram per kilogram (mg/kg) and a paclitaxel dose of 2.24 mg/kg on the days indicated by the arrows (i.e.
• Figure 6 is a graph showing the in vivo anticancer activity of a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified paclitaxel (NCP-2/PTX) (black squares) and of a combination of NCP-2/PTX and an anti-programmed death-ligand 1 (PD-L1 ) antibody (Pd-L1 Ab) (open circles) injected intraperitoneally into MC38 murine colorectal carcinoma tumor bearing mice at an oxaliplatin analogue does of 1 milligram per kilogram (mg/kg) and a paclitaxel dose of 2.24 mg/kg on the days indicated by the arrows (i.e., days 7, 1 1 , 15, 19, 23, 31 , and 39). Tumor size (in cubic millimeters (mm 3 )) at different days following first treatment is shown. For comparison, data from mice injected with phosphate buffered saline (PBS, x-
• Figure 7 is a graph showing the in vivo anticancer activity of a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified paclitaxel (NCP-2/PTX) in CT26 murine colorectal adenocarcinoma tumor bearing mice of different immunocompetencies: athymic nude mice (black squares); BALB/c Rag -/- mice (open circles); and BALB/c mice (x-marked triangles).
• NCP-2/PTX was intraperitoneally injected into the mice at an oxaliplatin analogue does of 1 milligram per kilogram (mg/kg) on the days indicated by the arrows (days 7, 10, and 15).
• Figure 8 is a graph showing the in vivo anticancer activity of NCP particles comprising a zinc and oxaliplatin analogue core and a lipid coating layer (NCP-2) in MC38 murine colorectal carcinoma tumor bearing mice. Mice were injected intraperitoneally every four days for a total of five doses at an oxaliplatin analogue dose of 2 milligrams per kilogram (mg/kg).
• mice were injected with NCP-2 (open circles), NCP-2 particles that included a cholesterol-modified paclitaxel (PTX) in a lipid coating layer with a molar ratio of oxaliplatin analogue to PTX of 2: 1 (NCP-2/PTX 2: 1 ; left-pointing triangles); NCP-2 particles that included a cholesterol-modified paclitaxel (PTX) in the lipid layer with a molar ratio of oxaliplatin analogue to PTX of 1 : 1 (NCP-2/PTX 1 : 1 ; right-pointing triangles); NCP-2/PTX 2: 1 and 75 micrograms g) of an anti-programmed death-ligand 1 (PD-L1 ) antibody (NCP-2/PTX 2: 1 + 75 ⁇ g PD-L1 Ab; upward-pointing triangles), or NCP- 2/PTX 1 : 1 and 75 ⁇ g PD-L1 antibody (NCP-2/PTX1 : 1 +
• Figure 9A is a graph showing the probability of tumor-free mice after re-challenge with live tumor cells in mice previously vaccinated with nanoparticle-treated cancer cells.
• BALB/c mice were inoculated subcutaneously with CT26 murine colorectal adenocarcinoma cells that had been treated with light and a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified paclitaxel (NCP-2/PTX, dotted line). Seven days after inoculation, the mice were re-challenged with live CT26 cells. Probability of tumor-free mice after re-challenge is also shown for mice originally inoculated with phosphate buffered saline (PBS)-treated cancer cells (solid line).
• PBS phosphate buffered saline
• Figure 9B is a graph showing tumor growth curves of rechallenged tumors in mice inoculated with phosphate buffered saline (PBS, black squares) as a control or inoculated with CT26 murine colorectal adenocarcinoma cells that had been treated with light and a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified paclitaxel (NCP-2/PTX, open circles).
• PBS phosphate buffered saline
• NCP particle comprising a zinc and oxaliplatin analogue core
• NCP-2/PTX lipid coating layer comprising cholesterol-modified paclitaxel
• Figure 10 is a graph of in vivo anticancer activity of a NCP comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified mitoxantrone (NCP-2/MTX) in CT26 murine colorectal adenocarcinoma tumor bearing mice.
• NCP-2/MTX cholesterol-modified mitoxantrone
• Tumor size data (in cubic millimeters (mm 3 )) is provided for mice treated with phosphate buffered saline (PBS, black squares), NCP-2/MTX at an oxaliplatin analogue dose of 1 milligram per kilogram (mg/kg) and a mitoxantrone (MTX) dose of 0.58 mg/kg (open circles) or a combination of MCP-2/MTX and 75 microgram fag) of an anti-programmed death-ligand 1 (PD-L1 ) antibody (MCP-2/MTX + PD- L1 Ab, x-marked triangles). Treatment was performed every four days for a total of six doses beginning on the seventh day after tumor inoculation.
• PBS phosphate buffered saline
• MTX mitoxantrone
• Figure 1 1 is a graph of in vivo anticancer activity of a NCP comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified mitoxantrone (NCP-2/MTX) in MC38 murine colorectal carcinoma tumor bearing mice.
• NCP-2/MTX cholesterol-modified mitoxantrone
• Tumor size data (in cubic millimeters (mm 3 )) is provided for mice treated with phosphate buffered saline (PBS, downward-facing triangles), NCP-2/MTX at an oxaliplatin analogue dose of 2 milligram per kilogram (mg/kg) and a mitoxantrone (MTX) dose of 1 .16 mg/kg (black squares) or a combination of MCP-2/MTX and 75 microgram g) of an anti-programmed death-ligand 1 (PD-L1 ) antibody (MCP-2/MTX + PD-L1 Ab, open circles). Treatment was performed every four days starting on the twelfth day after tumor cell inoculation for a total of five doses.
• PBS phosphate buffered saline
• MTX mitoxantrone
• Figure 12A is a graph showing calriticulin (CRT) exposure on the cell surface of CT26 murine colorectal adenocarcinoma cells induced by dihydroartemisinin (DHA) at a concentration of 1 micromolar ( ⁇ ) as determined by flow cytometry analysis. Data in gray from phosphate buffered saline (PBS)-treated cells is shown in each graph as a control.
• CTR calriticulin
• Figure 12B is a graph showing calriticulin (CRT) exposure on the cell surface of MC38 murine colorectal carcinoma cells induced by dihydroartemisinin (DHA) at a concentration of 1 micromolar ( ⁇ ) as determined by flow cytometry analysis. Data in gray from phosphate buffered saline (PBS)-treated cells is shown in each graph as a control.
• CTR calriticulin
• Figure 13 is a graph showing the in vivo anticancer activity of a NCP particle comprising a zinc and oxaliplatin analogue coordination polymer core and a lipid coating layer comprising cholesterol-modified dihydroartemisinin (NCP-2/DHA, triangles) in CT26 murine colorectal adenocarcinoma tumor bearing mice.
• NCP-2/DHA cholesterol-modified dihydroartemisininin
• Zn/DHA open circles
• Figure 14 is a graph showing the percentage (%) cell viability of
• OX oxaliplatin
• DHA dihydroartemisinin
• Cells were treated with one of the following: free OX (black squares); free DHA (open circles); a combination of free OX and free DHA (OX + DHA, upward-pointing triangles); a lipid-coated nanoscale polymer particles comprising a coordination polymer of zinc (Zn) and oxaliplatin prodrug (NCP-3, downward-pointing triangles); a combination of free Zn and free DHA (Zn/DHA, diamonds), or NCP-3 comprising a lipid-modified DHA in its lipid coating layer (NCP-3/DHA, left-pointing, x-marked triangles).
• Figure 15 is a graph showing the percentage (%) cell viability of MC38 murine colorectal carcinoma cells treated with free and nanoparticle drug formulations of oxaliplatin (OX) and dihydroartemisinin (DHA).
• OX oxaliplatin
• DHA dihydroartemisinin
• Cells were treated with one of the following: free OX (black squares); free DHA (open circles); a combination of free OX and free DHA (OX + DHA, upward- pointing triangles); lipid-coated nanoscale polymer particles comprising a coordination polymer of zinc (Zn) and oxaliplatin prodrug (NCP-3, downward- pointing triangles); a combination of free Zn and free DHA (Zn/DHA, diamonds), or NCP-3 comprising a lipid-modified DHA in its lipid coating layer (NCP-3/DHA, left-pointing, x-marked triangles).
• free OX black squares
• free DHA open circles
• Figure 16 is a series of plots showing the percentage (%) of apoptotic and necrotic cells after different treatments quantified by cell cytometry.
• Figure 17 is a graph of high mobility group box-1 (HMBG-1 ) protein release/expression (in nanograms per milliliter (ng/mL)) in the medium of CT26 murine colorectal adenocarcinoma cells receiving oxaliplatin and/or dihydroartemisinin (DHA) treatment.
• HMBG-1 high mobility group box-1
• Treatments included free oxaliplatin, free DHA, cholesterol-modified DHA (chol-DHA), lipid-coated nanoscale polymer particles comprising a coordination polymer of zinc (Zn) and oxaliplatin prodrug (NCP-3), a combination of free Zn and DHA (Zn/DHA), a combination of free oxaliplatin and free DHA (oxaliplatin+DHA), NCP-3 including cholesterol-modified DHA in its lipid coating layer (NCP-3/DHA), and NCP-3 including oleic acid-modified DHA in its lipid coating layer (NCP- 3/OA-DHA).
• PBS phosphate buffered saline
• Figure 18A is a graph showing the pharmacokinetics of platinum measured by inductively coupled plasma-mass spectrometry (ICP-MS) in the plasma of SD/CD rats after intravenous injection of a NCP particle comprising zinc (Zn) and an oxaliplatin prodrug and including dihydroarteminisin incorporated in its lipid coating layer (NCP-3/DHA).
• ICP-MS inductively coupled plasma-mass spectrometry
• Figure 18B is a graph showing the pharmacokinetics of cholesterol- modified dihydroarteminisin (Chol-DHA) measured by liquid chromatography mass spectrometry (LC-MS) in the plasma of SD/CD rats after intravenous injection of a NCP particle comprising zinc (Zn) and an oxaliplatin prodrug and including dihydroarteminisin incorporated in its lipid coating layer (NCP- 3/DHA).
• Chol-DHA cholesterol- modified dihydroarteminisin measured by liquid chromatography mass spectrometry
• Figure 19 is a graph of the biodistribution of NCP particles comprising zinc (Zn) and an oxaliplatin prodrug and including dihydroarteminisin incorporated in its lipid coating layer (NCP-3/DHA) after intraperitoneal injection into CT26 murine colorectal adenocarcinoma tumor bearing mice.
• Platinum (Pt) from the oxaliplatin prodrug was analyzed by inductively coupled plasma-mass spectrometry (ICP-MS).
• Figure 20A is a graph showing the in vivo anticancer activity of NCP particles comprising zinc (Zn) and an oxaliplatin (OX) prodrug and including dihydroarteminisin (DHA) incorporated in its lipid coating layer (NCP-3/DHA) with or without checkpoint blockade immunotherapy against CT26 murine colorectal adenocarcinoma tumor bearing mice.
• NCP particles comprising zinc (Zn) and an oxaliplatin (OX) prodrug and including dihydroarteminisin (DHA) incorporated in its lipid coating layer (NCP-3/DHA) with or without checkpoint blockade immunotherapy against CT26 murine colorectal adenocarcinoma tumor bearing mice.
• DHA dihydroarteminisin
• mice treated with NCP-3/DHA left-pointing, x-marked triangles); the same particles without DHA in the lipid coating layer (NCP-3, downward-pointing triangles); a combination of free OX, free DHA, and an anti-programmed death-ligand 1 (PD-L1 ) antibody (Free OX + DHA + PD-L1 , half-filled hexagons); a combination of NCP-3 and PD-L1 antibody (NCP-3 + PD-L1 , diamonds); a combination of NCP-3/DHA and PD-L1 antibody (NCP-3/DHA + PD-L1 , stars), and a higher dosage (32 milligrams per kilogram (mg/kg)) of NCP-3/DHA (half-filled pentagons).
• PBS phosphate buffered saline
• Figure 20B is a graph showing the in vivo anticancer activity of NCP particles comprising zinc (Zn) and an oxaliplatin (OX) prodrug and including dihydroarteminisin (DHA) incorporated in its lipid coating layer (NCP-3/DHA) with or without checkpoint blockade immunotherapy against MC38 murine colorectal carcinoma tumor bearing mice.
• NCP particles comprising zinc (Zn) and an oxaliplatin (OX) prodrug and including dihydroarteminisin (DHA) incorporated in its lipid coating layer (NCP-3/DHA) with or without checkpoint blockade immunotherapy against MC38 murine colorectal carcinoma tumor bearing mice.
• DHA dihydroarteminisin
• mice treated with NCP-3/DHA left-pointing, x-marked triangles); the same particles without DHA in the lipid coating layer (NCP-3, downward-pointing triangles); a combination of free OX, free DHA, and an anti-programmed death-ligand 1 (PD-L1 ) antibody (Free OX + DHA + PD-L1 , half-filled pentagons); a combination of NCP-3 and PD-L1 antibody (NCP-3 + PD-L1 , diamonds); a combination of NCP-3/DHA and PD-L1 antibody (NCP-3/DHA + PD-L1 , hexagons), and a higher dosage (16 milligrams per kilogram (mg/kg)) of NCP-3/DHA and the PD-L1 antibody (circles).
• PBS phosphate buffered saline
• Figure 21 is a graph of gamma-interferon (IFN- ⁇ ) producing T cells detected in spenocytes harvested after treatment with an oxaliplatin (OX) and/or dihydroarteminisin (DHA)-containing or non-containing composition and stimulated with a MC38 tumor-specific lysine-serine-proline repeats (KSP) peptide.
• IFN- ⁇ gamma-interferon
• PBS phosphate buffered saline
• PD-L1 an anti-programmed death-ligand 1 antibody
• Free OX+DHA+PD- L1 Free OX+DHA+PD- L1
• Zn/DHA zinc
• Figure 22A is a graph showing the in vivo anticancer activity of a lipid-coated NCP particle comprising a core comprising a coordination polymer of zinc and an oxaliplatin prodrug and including a lipid-modified dihydroarteminisin (DHA) in the lipid-coating layer (NCP-3/DHA) against 4T1 triple negative breast cancer tumor bearing mice.
• Mice were treated with PD-L1 antibody (upward-pointing triangles), NCP-3/DHA (circles), or a combination thereof (downward-pointing triangles).
• PBS phosphate buffered saline
• Mice were injected intraperitoneal ⁇ with an oxaliplatin-equivalent dose of 8 milligrams per kilogram once every 3 days starting on day 10.
• Figure 22B is a graph showing the in vivo anticancer activity of a lipid-coated NCP particle comprising a core comprising a coordination polymer of zinc and an oxaliplatin prodrug and including a lipid-modified dihydroarteminisin (DHA) in the lipid-coating layer (NCP-3/DHA) against LL/2 non-small cell lung cancer tumor bearing mice.
• Mice were treated with PD- L1 antibody (circles), or a combination of antibody and NCP-3/DHA (triangles).
• PBS phosphate buffered saline
• Mice were injected intraperitoneal ⁇ with an oxaliplatin dose of 8 milligrams per kilogram once every 3 days starting on day 12.
• Figure 23A is a graph showing cytotoxicity in non-small cell lung cancer (NSCLC) A549 cells incubated for 72 hours with compositions including: cisplatin (squares), etoposide (ET, circles), paclitaxel (PTX, upward-pointing triangles), camptothecin (CPT, downward-pointing triangles), cholesterol modified ET (Chol-ET, diamonds), cholesterol- modified PTX (Chol-PTX, leftward-pointing triangles), or oleic acid-modified CPT (OA-CPT, rightward-pointing triangles).
• NSCLC non-small cell lung cancer
• Figure 23B is a graph showing cytotoxicity in non-small cell lung cancer (NSCLC) A549 cells incubated for 72 hours with compositions including: a NCP particle comprising a cisplatin analogue (NCP-1 , squares), NCP-1 with a lipid coating layer comprising oleic acid-modified comptothecin (OA-CPT) and a cholesterol-modified etoposide (Chol-ET) (NCP-1 /OA- CPT/Chol-Et, circles), NCP-1 with a lipid coating layer comprising OA-CPT and a cholesterol-modified paclitaxel (Chol-PTX) (NCP-1 /OA-CPT/Chol-PTX, upward-pointing triangles), or NCP-1 with a lipid coating layer comprising OA-CPT, Chol-ET and Chol-PTX (NCP-1 /OA-CPT/Chol-ET/Chol-PTX, downward-pointing triangles).
• NCP-1 N
• Figure 24A is a graph showing cellular uptake into CT26 murine colorectal adenocarcinoma cells of, from left to right, oxaliplatin, a NCP particle comprising zinc and an oxaliplatin prodrug (NCP-2), and NCP-2 comprising pyrolipid in a lipid coating layer (NCP-2@pyrolipid).
• Platinum (Pt) concentration in picomoles (pmol) per 10 5 cells) was determined by inductively-coupled plasma-mass spectrometry (ICP-MS) after 1 , 2, 4, and 24 hours.
• Figure 24B is a graph showing cellular uptake into CT26 murine colorectal adenocarcinoma cells of, from left to right, a NCP particle compfrising a zinc and oxaliplatin prodrug core and pyrolipid in a lipid coating layer (NCP-2@pyrolipid), and of porphysome. Pyrolipid concentration (in picomoles (pmol) per 10 5 cells) was determined by ultraviolet-visible spectrometry (UV-Vis) after 1 , 2, 4, and 24 hours.
• UV-Vis ultraviolet-visible spectrometry
• Figure 25A is a graph showing the percentage (%) of platinum remaining from the initial dose (ID) versus time from the pharmacokinetics and biodistribution of NCP particles comprising a zinc and oxaliplatin prodrug core and pyrolipid in a lipid coating layer (NCP-2@pyrolipid) after intravenous injection into CT26 murine colorectal adenocarcinoma tumor bearing mice.
• Platinum (Pt) was analyzed by inductively-coupled plasma mass spectrometry (ICP-MS).
• Figure 25B is a graph showing the percentage (%) of pyrolipid remaining from the initial dose (ID) versus time from the study described for Figure 25A. Pyrolipid was analyzed via ultraviolet-visible spectrometry (UV- Vis).
• Figure 26 is a graph showing the in vivo anticancer activity of a lipid- coated NCP particle comprising a core that comprises a polymer of zinc and an oxaliplatin analogue and a lipid-coating layer comprising pyrolipid (NCP- 2@pyrolipid) in CT26 murine colorectal adenocarcinoma tumor bearing mice.
• mice were intravenously injected with NCP-2@pyrolipid (downward- pointing triangles) or the same NCP particle without pyrolipid (NCP-2, circles) at an oxaliplatin analogue dose of 2 milligrams per kilogram (mg/kg) followed by irradiation (+) at 670 nanometers (nm), 100 milliWatts per square centimeter (mW/cm 2 ) for 30 minutes 24 hours post injection every four days for a total of two treatments.
• PBS phosphate buffered saline
• NCP-2@pyrolipid without irradiation upward-pointing triangles
• Figure 27 is a graph showing the in vivo anticancer activity of a lipid- coated NCP particle comprising a core that comprises a polymer of zinc and an oxaliplatin analogue and a lipid-coating layer comprising pyrolipid (NCP- 2@pyrolipid) in HT29 human colorectal tumor bearing mice.
• mice were intravenously injected with NCP-2@pyrolipid (downward-pointing triangles) or the same NCP particle without pyrolipid (NCP-2) (circles) at an oxaliplatin analogue dose of 2 milligrams per kilogram (mg/kg) followed by irradiation (+) at 670 nanometers (nm), 100 milliwatts per square centimeter (mW/cm 2 ) for 30 minutes 24 hours post injection every four days for a total of four treatments.
• PBS phosphate buffered saline
• NCP-2@pyrolipid without irradiation upward-pointing triangles.
• Figure 28A is a graph of serum gamma interferon (IFN- ⁇ ) concentration (in nanograms per liter (ng/L)) in CT26 murine colorectal adenocarcinoma tumor bearing mice on days 7, 8, 9, and 10 post tumor inoculation (days 0, 1 , 2, and 3 post first treatment) treated as described in Figure 26.
• IFN- ⁇ serum gamma interferon
• Figure 28B is a graph of serum interleukin 6 (IL-6) concentration (in nanograms per liter (ng/L)) in CT26 murine colorectal adenocarcinoma tumor bearing mice on days 7, 8, 9, and 10 post tumor inoculation (days 0, 1 , 2, and 3 post first treatment) treated as described in Figure 26.
• IL-6 serum interleukin 6
• Figure 28C is a graph of serum tumor necrosis factor alpha (TNF-a) concentration (in nanograms per liter (ng/L)) in CT26 murine colorectal adenocarcinoma tumor bearing mice on days 7, 8, 9, and 10 post tumor inoculation (days 0, 1 , 2, and 3 post first treatment) treated as described in Figure 26.
• TNF-a serum tumor necrosis factor alpha
• Figure 29A is a graph showing the probability of tumor-free mice after re-challenge with live tumor cells in mice previously vaccinated with nanoparticle-treated cancer cells.
• BALB/c mice were inoculated subcutaneously with CT26 murine colorectal adenocarcinoma cells that had been treated with light and a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising pyrolipid (NCP- 2@pyrolipid, dotted line). Seven days after inoculation, the mice were re- challenged with live CT26 cells. For comparison, the probability of tumor- free mice after re-challenge in mice originally vaccinated with phosphate buffered saline (PBS)-treated cancer cells is also shown (solid line).
• PBS phosphate buffered saline
• Figure 29B is a graph showing tumor growth curves of rechallenged tumors in mice inoculated with phosphate buffered saline (PBS, squares) as a control or inoculated with CT26 murine colorectal adenocarcinoma cells that had been treated with light and a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising pyrolipid (NCP- 2@pyrolipid, circles).
• PBS phosphate buffered saline
• Figure 29C is a graph showing the serum concentrations (in nanograms per liter (ng/L)) of, from left to right, interferon gamma (IFN- ⁇ ), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-a) (reported in nanograms per liter (ng/L) in the mice described for Figure 30A one day after the first tumor inoculation.
• IFN- ⁇ interferon gamma
• IL-6 interleukin 6
• TNF-a tumor necrosis factor alpha
• Figure 30A is a graph showing the abscopal effect of a NCP particle comprising an oxaliplatin analogue and coated with a lipid coating layer comprising pyrolipid (NCP-2@pyrolipid).
• the graph shows the tumor growth curves of tumors in the left flank of mice inoculated in both the left and right flanks with CT26 murine colorectal adenocarcinoma cells and then treated in the right flank with NCP-2@pyrolipid and light (NCP-2@pyrolipid (+), circles), NCP-2@pyrolipid without light (NCP-2@pyrolipid (-), downward-pointing triangles), a cisplatin bisphosphonate-containing NCP particle with a pyrolipid-containing lipid coating layer and light (NCP-1 ⁇ pyrolipid (+), upward-pointing triangles), or phosphate buffered saline and light (PBS (+), squares).
• Figure 30B is a graph showing the antitumor effect of a NCP particle comprising an oxaliplatin analogue and coated with a lipid coating layer comprising pyrolipid (NCP-2@pyrolipid).
• the graph shows the tumor growth curves of tumors in the right flank of mice inoculated in both the left and right flanks with CT26 murine colorectal adenocarcinoma cells and then treated in the right flank with NCP-2@pyrolipid and light (NCP-2@pyrolipid (+), circles), NCP-2@pyrolipid without light (NCP-2@pyrolipid (-), downward-pointing triangles), a cisplatin bisphosphonate-containing NCP particle with a pyrolipid-containing lipid coating layer and light (NCP-1 @pyrolipid (+), upward-pointing triangles), or phosphate buffered saline and light (PBS (+), squares).
• Figure 31 A is a graph of the tumor growth curves of tumors in the right flanks of mice inoculated in both the left and right flanks with MC38 murine colorectal carcinoma cells and then treated in the right flank with a NCP particle comprising an oxaliplatin analogue and coated with a lipid coating layer including pyrolipid (NCP-2@pyrolipid) and light (NCP- 2@pyrolipid (+), upward-pointing triangles), NCP-2@pyrolipid without light (NCP-2@pyrolipid (-), circles), NCP-2@pyrolipid, light, and an anti- programmed death-ligand 1 (PD-L1 ) antibody (NCP-2@pyrolipid (+) + anti- PD-L1 , diamonds), NCP-2@pyrolipid and anti-PD-L1 antibody without light (NCP-2@pyrolipid (-) + anti-PD-L1 , downward-pointing triangles) or phosphate buffered saline and light (PBS (+), squares).
• Figure 31 B is a graph of the tumor growth curves of tumors in the left flanks of mice inoculated in both the left and right flanks with MC38 murine colorectal carcinoma cells and then treated in the right flank with a NCP particle comprising an oxaliplatin analogue and coated with a lipid coating layer including pyrolipid (NCP-2@pyrolipid) and light (NCP-2@pyrolipid (+), upward-pointing triangles), NCP-2@pyrolipid without light (NCP-2@pyrolipid (-), circles), NCP-2@pyrolipid, light, and an anti-programmed death-ligand 1 (PD-L1 ) antibody (NCP-2@pyrolipid (+) + anti-PD-L1 , diamonds), NCP- 2@pyrolipid and anti-PD-L1 antibody without light (NCP-2@pyrolipid (-) + anti-PD-L1 , downward-pointing triangles) or phosphate buffered saline and light (PBS (+), squares).
• Figure 32A is a graph of the tumor growth curves of tumors in the right flanks of mice inoculated in both the left and right flanks with CT26 murine colorectal adenocarcinoma cells and then treated in the right flank with a NCP particle comprising an oxaliplatin analogue and coated with a lipid coating layer including pyrolipid (NCP-2@pyrolipid) and light and an anti-programmed death-ligand 1 (PD-L1 ) antibody (NCP-2@pyrolipid (+) + anti-PD-L1 , circles) or phosphate buffered saline and light (PBS (+), squares).
• NCP-2@pyrolipid pyrolipid
• PD-L1 anti-programmed death-ligand 1
• PBS (+) phosphate buffered saline and light
• Figure 32B is a graph of the tumor growth curves of tumors in the left flanks of mice inoculated in both the left and right flanks with CT26 murine colorectal adenocarcinoma cells and then treated in the right flank with a NCP particle comprising an oxaliplatin analogue and coated with a lipid coating layer including pyrolipid (NCP-2@pyrolipid) and light and an anti- programmed death-ligand 1 (PD-L1 ) antibody (NCP-2@pyrolipid (+) + anti- PD-L1 , circles) or phosphate buffered saline and light (PBS (+), squares).
• NCP-2@pyrolipid pyrolipid
• PD-L1 anti- programmed death-ligand 1
• PBS (+) phosphate buffered saline and light
• Figure 33A is a graph of tumor growth curves of 4T1 triple negative breast cancer xenografts treated with a NCP particle comprising a zinc- pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti- programmed death-ligand 1 (PD-L1 ) antibody (downward-pointing triangles), Zn@pyrolipid and an anti-PD-L1 antibody (upward-pointing triangles) or phosphate buffered saline (PBS) and light (squares).
• PD-L1 anti- programmed death-ligand 1
• Figure 33B is a graph of the tumor weights (in grams (g)) at the end of the treatments described in Figure 33A.
• Figure 34A is a graph of the tumor growth curves of tumors in the right flanks of mice inoculated in both the left and right flanks with 4T1 triple negative breast cancer cells and then treated in the right flank with a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD-L1 ) antibody (downward- pointing triangles), Zn@pyrolipid and an anti-PD-L1 antibody (upward- pointing triangles), or phosphate buffered saline (PBS) and light (PBS (+), squares).
• a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD
• Figure 34B is a graph of the tumor growth curves of tumors in the left flanks of mice inoculated in both the left and right flanks with 4T1 triple negative breast cancer cells and then treated in the right flank with a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD-L1 ) antibody (downward- pointing triangles), Zn@pyrolipid and an anti-PD-L1 antibody (upward- pointing triangles), or phosphate buffered saline (PBS) and light (PBS (+), squares).
• a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD
• Figure 34C is a graph showing the tumor weights (in grams (g)) of the tumors in left (left) and right (right) flanks of the mice treated as described for Figures 34A and 34B at the end of the treatment period.
• Figure 35A is a graph of the tumor growth curves of tumors in the right flanks of mice inoculated in both the left and right flanks with TUBO breast cancer cells and then treated in the right flank with a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD-L1 ) antibody (downward-pointing triangles), Zn@pyrolipid and an anti-PD-L1 antibody (upward-pointing triangles), or phosphate buffered saline (PBS) and light (squares).
• a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD-L1 ) antibody (down
• Figure 35B is a graph of the tumor growth curves of tumors in the left flanks of mice inoculated in both the left and right flanks with TUBO breast cancer cells and then treated in the right flank with a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD-L1 ) antibody (downward-pointing triangles), Zn@pyrolipid and an anti-PD-L1 antibody (upward-pointing triangles), or phosphate buffered saline (PBS) and light (squares).
• a NCP particle comprising a zinc-pyrophosphate core and a lipid coating layer comprising pyrolipid (Zn@pyrolipid) and light (+) (circles), with Zn@pyrolipid, light and an anti-programmed death-ligand 1 (PD-L1 ) antibody (down
• Figure 35C is a graph showing the tumor weights (in grams (g)) of the tumors in left (left) and right (right) flanks of the mice treated as described for Figures 35A and 35B at the end of the treatment period.
• Figure 36A is a graph of serum tumor necrosis factor alpha (TNF-a) concentration (in picograms per milliliter (pg/ml_)) in TUBO breast tumor bearing mice on days 10, 1 1 , 12, and 13 post tumor inoculation (days 0, 1 , 2, and 3 post first treatment) treated as described in Figures 35A and 35B.
• TNF-a serum tumor necrosis factor alpha
• Figure 36B is a graph of serum interferon gamma (IFN- ⁇ ) concentration (in picograms per milliliter (pg/ml_)) in TUBO breast tumor bearing mice on days 10, 1 1 , 12, and 13 post tumor inoculation (days 0, 1 , 2, and 3 post first treatment) treated as described in Figures 35A and 35B.
• Figure 36C is a graph of serum interleukin 6 (IL-6) concentration (in picograms per milliliter (pg/ml_)) in TUBO breast tumor bearing mice on days 10, 1 1 , 12, and 13 post tumor inoculation (days 0, 1 , 2, and 3 post first treatment) treated as described in Figures 35A and 35B.
• IFN- ⁇ serum interferon gamma
• Figure 37A is a graph showing the proportion of CD45+ lymphocytes in right and left flank TUBO breast tumors models of mice treated in the right flank with phosphate buffered saline (PBS, open circles), photodynamic therapy (PDT, squares) via injection of a zinc-pyrophosphate NCP particle with a pyrolipid-containing lipid coating layer and irradiation, injection of an anti-programmed death-ligand 1 (PD-L1 ) antibody (a-PD-L1 , triangles), or PDT and antibody (black circles). Data is collected on the twelfth day after the first treatment.
• PBS phosphate buffered saline
• PDT photodynamic therapy
• Figure 37B is a graph showing the proportion of CD8+ T cells in right and left flank TUBO breast tumors models of mice treated in the right flank with phosphate buffered saline (PBS, open circles), photodynamic therapy (PDT, squares) via injection of a zinc-pyrophosphate NCP particle with a pyrolipid-containing lipid coating layer and irradiation, injection of an anti- programmed death-ligand 1 (PD-L1 ) antibody (a-PD-L1 , triangles), or PDT and antibody (black circles). Data is collected on the twelfth day after the first treatment.
• PBS phosphate buffered saline
• PDT photodynamic therapy
• Figure 37C is a graph showing the proportion of CD4+ T cells in right and left flank TUBO breast tumors models of mice treated in the right flank with phosphate buffered saline (PBS, open circles), photodynamic therapy (PDT, squares) via injection of a zinc-pyrophosphate NCP particle with a pyrolipid-containing lipid coating layer and irradiation, injection of an anti- programmed death-ligand 1 (PD-L1 ) antibody (a-PD-L1 , triangles), or PDT and antibody (black circles). Data is collected on the twelfth day after the first treatment.
• PBS phosphate buffered saline
• PDT photodynamic therapy
• Figure 37D is a graph showing the proportion of B cells in right and left flank TUBO breast tumors models of mice treated in the right flank with phosphate buffered saline (PBS, open circles), photodynamic therapy (PDT, squares) via injection of a zinc-pyrophosphate NCP particle with a pyrolipid- containing lipid coating layer and irradiation, injection of an anti-programmed death-ligand 1 (PD-L1 ) antibody (a-PD-L1 , triangles), or PDT and antibody (black circles). Data is collected on the twelfth day after the first treatment.
• PBS phosphate buffered saline
• PDT photodynamic therapy
• Figure 38A is a graph showing the proportion of CD8+ T cells in tumor-draining lymph nodes of right and left flank TUBO breast tumors models of mice treated in the right flank with phosphate buffered saline (PBS, open circles), photodynamic therapy (PDT, squares) via injection of a zinc-pyrophosphate NCP particle with a pyrolipid-containing lipid coating layer and irradiation, injection of an anti-programmed death-ligand 1 (PD-L1 ) antibody (a-PD-L1 , triangles), or PDT and antibody (black circles). Data is collected on the twelfth day after the first treatment.
• PBS phosphate buffered saline
• PDT photodynamic therapy
• Figure 38B is a graph showing the proportion of CD4+ T cells in tumor-draining lymph nodes of right and left flank TUBO breast tumors models of mice treated in the right flank with phosphate buffered saline (PBS, open circles), photodynamic therapy (PDT, squares) via injection of a zinc-pyrophosphate NCP particle with a pyrolipid-containing lipid coating layer and irradiation, injection of an anti-programmed death-ligand 1 (PD-L1 ) antibody (a-PD-L1 , triangles), or PDT and antibody (black circles). Data is collected on the twelfth day after the first treatment. DETAILED DESCRIPTION
• the presently disclosed subject matter provides prodrugs comprising drug-lipid conjugates that include biodegradable linkages, such as disulfide bonds.
• the prodrugs can be prepared from small molecule chemotherapeutics, including, but not limited to, those that are known to have immunological effect, such as the ability to cause immune cell death.
• the lipid moieties of the prodrugs can enhance incorporation of the prodrugs in lipid coating layers of inorganic and/or metal-organic matrix nanoparticles or other nanoscale drug delivery platforms.
• the presently disclosed subject matter provides nanoparticles that contain chemotherapeutics, such as those that are known to cause immunogenic cell death or to be immune- stimulatory.
• the nanoparticle can comprise multiple chemotherapeutics to treat multiple cancer types.
• the nanoparticles can be combined with PDT modalities or used alone to cause immune-stimulation, leading to effective cancer therapy.
• nanoparticles can be further combined with immunotherapy agents, e.g., immunosuppression inhibitors that, for example, target the CTLA-4, PD-1/PD-L1 , IDO, LAG-3, CCR-7, or other pathways, or multiple immunosuppression inhibitors targeting a combination of these pathways, to elicit systemic antitumor immunity.
• immunotherapy agents e.g., immunosuppression inhibitors that, for example, target the CTLA-4, PD-1/PD-L1 , IDO, LAG-3, CCR-7, or other pathways, or multiple immunosuppression inhibitors targeting a combination of these pathways, to elicit systemic antitumor immunity.
• 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.
• Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes, but is not limited to, 1 , 1 .5, 2, 2.75, 3, 3.90, 4, and 5).
• 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 Ci -2 o inclusive, linear (i.e. , "straight-chain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.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 (i.e. , a C-i-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 C-i-8 straight-chain alkyls.
• alkyl refers, in particular, to Ci -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, alkoxycarbonyl, 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.
• 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 non-carbon 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.
• 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, 1 1 , 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 "alkylaminoalkyi”), 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., -NHC 6 H 5 ).
• thioalkyl can refer to the group -SR, wherein R is selected from H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl.
• thioaralkyl and thioaryl refer to -SR groups wherein R is aralkyl and aryl, respectively.
• diisulfide can refer to the -S-S- group.
• 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.
• 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.
• a “monovalent” as used herein refers to a chemical moiety that has one site available for chemical bonding to another chemical moiety.
• a “monovalent moiety” can be a part of whole molecule that is attached to the remainder of the whole molecule via an attachment at one site on the monovalent moiety.
• bivalent refers to a chemical moiety that has two sites available for chemical bonding to another chemical moiety or moieties.
• conjugate and “conjugated” as used herein can refer to the attachment (e.g., the covalent attachment) of two or more components (e.g., chemical compounds, polymers, biomolecule, particles, etc.) to one another.
• a conjugate can comprise monovalent moieties derived from two different chemical compounds covalently linked via a bivalent linker moiety (e.g., an optionally substituted alkylene or arylene).
• the linker can contain one or more biodegradable bond, such that one or more bonds in the linker can be broken when the prodrug is exposed to a particular physiological environment or enzyme.
• prodrug can refer to a compound that, upon administration to a subject or sample, is capable of providing (directly or indirectly) another compound (i.e., a "parent compound") having a desired biological activity (e.g., anticancer activity).
• a desired biological activity e.g., anticancer activity
• the prodrug compound has less of the desired biological activity than the parent compound.
• the prodrug compound has no measurable biological activity prior to transformation to the parent compound.
• the prodrug itself has the desired activity.
• Transformation of the prodrug to the parent compound can take place in the presence of particular enzymes (e.g., esterases) or under certain biological conditions (e.g., at a physiologically relevant pH or in the presence of reducing agents present in a physiological environment).
• the prodrug is initially transformed into another prodrug, which is then transformed (sometimes much more slowly) into the parent compound.
• Prodrugs can provide increased bioavailability and/or enhanced delivery to a biological compartment (e.g., a lysosome, the brain or lymphatic system, etc.) relative to a parent compound.
• the prodrug can be more compatible with a particular delivery platform or formulation than the parent compound.
• bonding or “bonded” and variations thereof can refer to covalent, coordinative, or non-covalent bonding. In some cases, the term “bonding” refers to bonding via a coordinate bond. The term “conjugation” can refer to a bonding process, as well, such as the formation of a covalent linkage or a coordinate bond.
• 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. P., 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(OH) 2 ,
• heterocycles 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).
• the term "metal-organic matrix material” refers to a solid material comprising both metal and organic components, wherein the organic components include at least one, and typically more than one carbon atom.
• the material can be crystalline or amorphous.
• the matrix material is porous.
• the metal-organic matrix material is a coordination polymer, (e.g., a nanoscale coordination polymer (NCP)) which comprises repeating units of coordination complexes comprising metal ions and bridging polydentate (e.g., bidentate) ligands (e.g., organic ligands).
• NCP nanoscale coordination polymer
• the matrix material contains more than one type of metal ion.
• the matrix material can contain metal clusters.
• the matrix material is a metal-organic framework (MOF) comprising a coordination complex network that comprises bridging organic ligands.
• MOF metal-organic framework
• metal-organic framework can refer 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.
• the material is amorphous.
• 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. In some embodiments, the material can contain more than one type of organic bridging ligand.
• an MOF typically refers to a metal- organic matrix material wherein the metal component is a metal cluster or metal oxo cluster
• NCP can refer to a metal-organic matrix material where the metal component is a metal ion.
• NCP as used herein typically refers to a metal organic matrix material
• an NCP can refer to a coordination polymer that does not include a carbon-containing ligand.
• the NCP can refer to zinc pyrophosphate.
• 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, less than about 30 nm or even less than about 20 nm).
• the dimension is between about 20 nm and about 250 nm (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 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.
• the nanoparticle can comprise a core region (i.e., the space between the outer dimensions of the particle) and an outer surface (i.e., the surface that defines the outer dimensions of the particle).
• the nanoparticle can have one or more coating layers surrounding or partially surrounding the nanoparticle core.
• a spherical nanoparticle can have one or more concentric coating layers, each successive layer being dispersed over the outer surface of a smaller layer closer to the center of the particle.
• Embedded can refer to a agent that is bound, for example covalently bound or bound via a coordinative bond, inside or on the surface of the core of the particle (e.g., to a coordination site of a bridging ligand or to a metal ion).
• agents can be "sequestered”, “entrapped”, or “trapped” (i.e., non-covalently encapsulated) inside pores, cavities or channels in the metal-organic matrix core of a NCP or MOF particle or interact with a NCP or MOF material via hydrogen bonding, London dispersion forces, or any other non-covalent interaction.
• polymer and “polymeric” refer to chemical structures that have repeating units (i.e., multiple copies of a given chemical substructure).
• Polymers can be formed from polymerizable monomers.
• a polymerizable monomer is a molecule that comprises one or more moieties that can react to form bonds (e.g., covalent or coordination bonds) with moieties on other molecules of polymerizable monomer.
• bonds e.g., covalent or coordination bonds
• each polymerizable monomer molecule can bond to two or more other molecules/moieties.
• a polymerizable monomer will bond to only one other molecule, forming a terminus of the polymeric material.
• Polymers can be organic, or inorganic, or a combination thereof.
• inorganic refers to a compound or composition that contains at least some atoms other than carbon, hydrogen, nitrogen, oxygen, sulfur, phosphorous, or one of the halides.
• an inorganic compound or composition can contain one or more silicon atoms and/or one or more metal atoms.
• organic polymers are those that do not include silica or metal atoms in their repeating units.
• exemplary organic polymers include polyvinylpyrrolidone (PVO), polyesters, polyamides, polyethers, polydienes, and the like.
• PVO polyvinylpyrrolidone
• Some organic polymers contain biodegradable linkages, such as esters or amides, such that they can degrade overtime under biological conditions.
• hydrophilic polymer as used herein generally refers to hydrophilic organic polymers, such as but not limited to, polyvinylpyrrolidone
• PVP polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxy-propyloxazoline, polyhydroxypropylmethacrylamide, polymethy- acrylamide, polydimethylacrylamide, polyhydroxylpropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene-imine (PEI), polyethyleneglycol (i.e., PEG) or another hydrophilic poly(alkyleneoxide), polyglycerine, and polyaspartamide.
• PEI polyethylene-imine
• PEG polyethyleneglycol
• hydrophilic poly(alkyleneoxide) polyglycerine, and polyaspartamide.
• hydrophilic refers to the ability of a molecule or chemical species to interact with water. Thus, hydrophilic polymers are typically polar or have groups that can hydrogen bond to water.
• an imaging agent refers to a chemical moiety that aids in the visualization of a sample.
• an imaging agent can be a "contrast agent”, and can refer to a moiety (a specific part of or an entire molecule, macromolecule, coordination complex, or nanoparticle) that increases the contrast of a biological tissue or structure being examined.
• the contrast agent can increase the contrast of a structure being examined using, for example, magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET) imaging, single photon emission computed tomography (SPECT) imaging, or a combination thereof (i.e., the contrast agent can be multimodal).
• MRI magnetic resonance imaging
• PET positron emission tomography
• SPECT single photon emission computed tomography
• MRI contrast agent refers to a moiety that effects a change in induced relaxation rates of water protons in a sample.
• optical imaging agent or “optical contrast agent” refer to a group that can be detected based upon an ability to absorb, reflect or emit light (e.g., ultraviolet, visible, or infrared light). Optical imaging agents can be detected based on a change in amount of absorbance, reflectance, or fluorescence, or a change in the number of absorbance peaks or their wavelength maxima. Thus, optical imaging agents include those which can be detected based on fluorescence or luminescence, including organic and inorganic dyes.
• fluorophore and “fluorescent moiety” refer to species that can be excited by visible light or non-visible light (e.g., UV light).
• fluorophores include, but are not limited to: quantum dots and doped quantum dots (e.g., a semiconducting CdSe quantum dot or a Mn-doped CdSe quantum dot), fluorescein, fluorescein derivatives and analogues, indocyanine green, rhodamine, triphenylmethines, polymethines, cyanines, phalocyanines, naphthocyanines, merocyanines, lanthanide complexes or cryptates, fullerenes, oxatellurazoles, LaJolla blue, porphyrins and porphyrin analogues and natural chromophores/fluorophores such as chlorophyll, carotenoids, flavonoids, bilins, phytochrome, phycobilin
• 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 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 ( 1 0 2 ).
• O2 oxygen
• the photosensitizer is a porphyrin, a chlorophyll, a dye, or a derivative or analog thereof.
• phophyrins, 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 use in attaching the photosensitizer to another molecule or moiety, such as an organic bridging ligand, 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.
• expanded porphyrin family members such as texaphyrins, sapphyrins and hexaphyrins
• porphyrin isomers such as porphycenes, inverted porphyrins, phthalocyanines, and naphthalocyanines
• pyrolipid refers to a conjugate of a lipid and a porphyrin, porphyrin derivative, or porphyrin analog.
• the pyrolipid can comprise a lipid conjugate wherein a porphyrin or a derivative or analog thereof is covalently attached to a lipid side chain.
• Pyrolipids and pyrolipid synthesis are described, for example, in U.S. Patent Application Publication No. 2014/0127763, which is incorporated herein by reference in its entirety.
• lyso-lipid refers to a lipid in which one or more acyl group has been removed.
• 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, colon cancers, prostate cancers, central nervous system (CNS) cancers, retinal cancer, blood, neuroblastomas, multiple myeloma, and lymphoid cancers (e.g., Hodgkin's and non-Hodgkin's lymphomas).
• skin cancers e.g., melanoma
• connective tissue cancers e.g., sarcomas
• adipose cancers e.g., breast cancers,
• 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.
• scintillator refers to a moiety or compound that exhibits luminescence (emits light, e.g., light in the visible or NIR range) when excited by ionizing radiation, such as x-rays.
• Nanoscale coordination polymers are an emerging class of self-assembled, hybrid nanomaterials whose properties can be tuned by varying the molecular building blocks. NCPs can provide desirable drug delivery platforms for treating disease. See, for example, PCT International Patent Application Publications Nos. WO 2013/009701 and WO 2015/069926, the disclosures of each of which are incorporated herein by reference in their entireties. For instance, NCPs can be used to enhance the delivery of small molecule drugs and biologies to tumor sites via the enhanced permeability and retention (EPR) effect by taking advantage of the leaky blood vasculatures and reduced lymphatic drainage in tumors.
• EPR enhanced permeability and retention
• NCPs can provide drug delivery by incorporating drug compounds or analogues as part of the particle metal-organic matrix core material, e.g., by covalent linkage or coordination to a matrix component, or, additionally or alternatively, by embedding the drug or drug analogue in pores in the matrix material core.
• the particles can comprise coating layers surrounding all or a part of the particle core, wherein therapeutic agents (e.g., therapeutic small molecules or biological agents, e.g., or nucleic acids, proteins or antibodies), photosensitizers, targeting agents, passivating agents, and/or detection/imaging agents can be incorporated into the coating layers.
• the presently disclosed subject matter is based on an approach for providing a prodrug suitable for incorporation into NCP particle lipid coating layers, e.g., to provide enhanced delivery of the corresponding parent drugs and for providing NCP combination therapies including the parent drug.
• the prodrugs can also be useful for incorporation into other nanoparticle drug delivery platforms, such as, but not limited to, polymeric micelles, liposomes, dendrimers, polymer-based nanoparticles, silica-based nanoparticles, nanoscale metal-organic frameworks (MOFs), and inorganic nanoparticles (e.g., gold nanoparticles, iron oxide nanoparticles, etc.).
• a suitable prodrug can be provided by conjugating a drug compound to a lipid moiety via a linkage that will degrade in vivo.
• the linkage is a disulfide bond.
• the prodrug is a drug-lipid conjugate, wherein the drug-lipid conjugate comprises a linker moiety comprising a disulfide.
• the drug compound is a chemotherapeutic agent or a small molecule inhibitor for immunotherapy.
• the drug compound comprises a hydroxyl or phenol group and the drug compound can be covalently attached to a linker via a bond involving the oxygen atom of the hydroxyl or phenol.
• the drug compound can comprise a thiol, primary or secondary amine, or carboxylic acid group that can be used as an attachment site to a linker group as part of forming a prodrug.
• the prodrug comprises a drug compound selected from the group including, but not limited to, Etoposide (ET), Camptothecin (CPT), dihydroartemisinin (DHA), Paclitaxel (PTX), OTSC41 , OTS964, OTS167 (see Matsuo et al.. Science Translational Medicine, 22 Oct 2014: 259ra145), Doxorubicin, Docetaxel, Vincristine, Mitoxantrone (MTX), artesunate (ART), Capecitabine, and NLG919 (see U.S. Patent Application Publication No.
• Etoposide Etoposide
• CPT Camptothecin
• DHA dihydroartemisinin
• PTX Paclitaxel
• OTSC41 OTS964, OTS167
• Doxorubicin Docetaxel
• Vincristine Vincristine
• ART artesunate
• Capecitabine and NLG919
• lipid such as, but not limited to, cholesterol (Choi), oleic acid (OA), a lyso-lipid, or phosphocholine
• linker e.g., a disulfide-containing linker.
• the structure of OTSC41 is shown at the top left of Scheme 1 , below, while the structures of OTS964 and NLG919 are shown in the top right and bottom of Scheme 1 , respectively.
• the prodrugs can be readily loaded onto NCP particles during the lipid coating process driven by hydrophobic interactions between the prodrug lipid component (e.g., the Choi or OA) and phospholipids in the lipid coating.
• the prodrugs retain the antitumor activity of their corresponding parent drugs due to intracellular release of active parent drugs under the reducing environment inside cells.
• the presently disclosed subject matter provides a prodrug comprising a lipid-conjugated therapeutic agent parent drug.
• the prodrug comprises: (a) a monovalent drug moiety, (b) a monovalent lipid moiety, and (c) a bivalent linker moiety comprising a linkage that will degrade in vivo, such as a disulfide bond, wherein the monovalent drug moiety and the monovalent lipid moiety are linked (e.g., covalently linked) through the linker.
• the monovalent drug moiety and the monovalent lipid moieties can be monovalent derivatives of a drug compound and a lipid, respectively.
• the monovalent derivative can be a deprotonated derivative of a drug compound or lipid that comprises a hydroxyl, thiol, amino, or carboxylic acid group.
• the monovalent drug moiety is a monovalent derivative of a chemotherapeutic agent and/or of a small molecule inhibitor for immunotherapy.
• the drug is a chemotherapeutic compound known to cause immunogenic cell death or to be immune- stimulatory.
• chemotherapeutic compounds include, but are not limited to ET, PTX, OTS964, NLG919, OTS167, OTSC41 , DHA, CPT, Doxorubicin, Docetaxel, Vincristine, MTX, ART, and Capecitabine.
• the monovalent lipid moiety is a monovalent derivative of cholesterol, oleic acid, a lyso-lipid or phosphocholine.
• the bivalent linker moiety can be a derivative of a dihydroxy-substituted disulfide, such as, but not limited to, bis(2-hydroxyethyl) disulfide, dihydroxydiphenyl disulfide, bis(2-hydroxypropyl) disulfide, and the like.
• linkers can be activated for attachment to a drug or lipid by reaction with phosgene or triphosgene, for example, to provide chloroformate groups.
• the monovalent lipid moiety is a cholesterol derivative and the monovalent lipid moiety and bivalent linker moiety together have the structure:
• monovalent lipid moiety is an oleic acid derivative and the monovalent lipid moiety and bivalent linker moiety together have the structure:
• the monovalent lipid moiety is a lyso-lipid derivative and the monovalent lipid moiety and bivalent linker moiety together have the structure:
• R is a saturated or unsaturated acyl group.
• R is a C 8 -C 2 6 (e.g., a C 8 , Ci 0 , Ci 2 , Ci 4 , Ci 6 , Ci 8 , C 2 o, C22, C 24 or C 2 e) saturated or unsaturated acyl group.
• R is selected from oleyl, stearyl or palmitoleyl.
• the monovalent lipid moiety is a phosphocholine derivative and the monovalent lipid moiety and bivalent linker together have the structure:
• the monovalent drug moiety and the monovalent lipid moiety are each bonded to a hydroxyl group of bis-(2- hydroxyethyl) disulfide linker (or another linker comprising two hydroxyl groups) via carbonate bonds.
• one or both of the drug moiety and the lipid moiety can be bonded to the linker via carbamate, thioester, ester, amide, ether, or amine linkages.
• any other suitable linker comprising a biodegradable bond can be used in the prodrugs of the presently disclosed subject matter.
• Scheme 2, above shows additional exemplary biodegradable linkers suitable for the presently disclosed prodrugs. These linkers can be based on linkers that comprise a phenol group or groups that can form a phenol ester (or carbonate or carbamate) with the lipid and/or drug moiety. Suitable linkers also include linkers that can form a benzyl ester (or carbonate or carbamate) with the lipid or drug moiety. Thus, in some embodiments, suitable linkers can be prepared from phenols or benzyl alcohols.
• biodegradable linkers include, but are not limited to, linkers based on hydrazones, thiazolidines, thio-substituted succinimides (e.g., prepared from Michael addition of a thiol to a maleimide), other disulfides (i.e., disulfides other than dihydroxy-substituted disulfides), dicarboxylic acids, 3- (hydroxyphenyl)propionic acids, and the like.
• linkers based on hydrazones thiazolidines
• thio-substituted succinimides e.g., prepared from Michael addition of a thiol to a maleimide
• other disulfides i.e., disulfides other than dihydroxy-substituted disulfides
• dicarboxylic acids i.e., 3- (hydroxyphenyl)propionic acids, and the like.
• the presently disclosed subject matter provides a composition comprising a lipid conjugate prodrug as described herein for use in treating a disease in a subject in need of treatment thereof.
• the composition for use in treating disease comprises a nanoparticle comprising the prodrug (e.g., present as a component of a lipid coating layer present as one or more coating layers coating at least a portion of the surface of the nanoparticle core).
• the composition or use in treating a disease further comprises one or more additional therapeutic agents, e.g., one or more chemotherapeutic agents (or analogues or prodrugs thereof), one or more immunotherapy agents, one or more targeting agents, one or more imaging agent, one or more scintillator, one or more photosensitizer, or any mixture thereof.
• additional therapeutic agents e.g., one or more chemotherapeutic agents (or analogues or prodrugs thereof), one or more immunotherapy agents, one or more targeting agents, one or more imaging agent, one or more scintillator, one or more photosensitizer, or any mixture thereof.
• Photodynamic therapy is a phototherapy that combines three non-toxic components— a photosensitizer (PS), a light source, and tissue oxygen— to cause toxicity to malignant and other diseased cells.
• PS photosensitizer
• tissue oxygen tissue oxygen
• ROS reactive oxygen species
• PDT can lead to localized destruction of diseased tissues via selective uptake of the PS and/or local exposure to light, providing a minimally invasive cancer therapy.
• An alternative approach is to use nanocarriers to selectively deliver therapeutic or PDT agents to tumors via the enhanced permeation and retention effect (EPR) and sometimes, via active tumor targeting with small molecule or biologic ligands that bind to overexpressed receptors in cancers.
• EPR enhanced permeation and retention effect
• Nanoscale particles such as those constructed from NCPs and comprising lipid or other coating layers, can be used as a nanocarrier platform for therapeutic and PDT agents.
• NCPs can combine many beneficial features into a single delivery platform, including tunable chemical compositions and crystalline structures; high porosity; and bio-degradability.
• PDT can selectively kill tumor cells while preserving adjacent normal tissue. PDT does not incur cross-resistance with radiotherapy or chemotherapy, and therefore, is useful in the treatment of cancer patients who have not responded significantly to traditional radiotherapy and/or chemotherapy. PDT can provoke a strong acute inflammatory reaction observed as localized edema at the targeted site. The inflammation elicited by PDT is a tumor antigen nonspecific process orchestrated by the innate immune system. PDT is particularly effective in rapidly generating an abundance of alarm/danger signals, such as damage-associated molecular patterns (DAMPs), at the treated site that can be detected by the innate immunity alert elements.
• DAMPs damage-associated molecular patterns
• PDT-mediated enhancement of antitumor immunity is believed due to the stimulation of dendritic cells by dead and dying tumor cells and can be accompanied by the recruitment and activation of CD8+ cytotoxic T cells (CTLs) followed by the formation of immune memory cells and resistance to subsequent tumor growth.
• CTLs cytotoxic T cells
• the presently disclosed subject matter provides a method that combines PDT (or X-ray PDT) with immunotherapy.
• the presently disclosed subject matter provides a method of treating a disease in a patient in need of treatment thereof, the method comprising the steps of: administering to a patient a nanoparticle photosensitizer or a scintillator; irradiating at least a portion of the patient with light and/or X-rays; and administering to the patient an immunotherapy agent.
• the nanoparticle photosensitizer comprises a NCP.
• the nanoparticle comprises a lipid-conjugate prodrug of the presently disclosed subject matter.
• the disease is cancer.
• the cancer can be selected from a cancer such as, but not limited to, a head tumor, a neck tumor, breast cancer, a gynecological tumor, a brain tumor, colorectal cancer, lung cancer, mesothelioma, a soft tissue sarcoma, skin cancer, connective tissue cancer, adipose cancer, lung cancer, stomach cancer, anogenital cancer, kidney cancer, bladder cancer, colon cancer, prostate cancer, central nervous system cancer, retinal cancer, blood cancer, neuroblastoma, multiple myeloma, lymphoid cancer and pancreatic cancer.
• the cancer is a metastatic cancer
• the immunotherapy agent can be a known small molecule inhibitor of programmed cell death protein 1 (PD-1 ), programmed death-ligand 1 (PD- L1 , also known as CD274), cytotoxic T lymphocyte associated protein 4 (CTLA-4), indolamine 2,3-deoxygenase (I) (IDO), or C-C chemokine receptor type 7 (CCR7).
• PD-1 programmed cell death protein 1
• PD- L1 also known as CD274
• CTL-4 cytotoxic T lymphocyte associated protein 4
• IDO indolamine 2,3-deoxygenase
• CCR7 C-C chemokine receptor type 7
• Small molecule IDO inhibitors include, for instance, INCB24360 and NLG919, among others.
• the small molecule inhibitor can be provided as a lipid conjugate prodrug.
• the immunotherapy agent can be an antibody or antibody fragment.
• Immunotherapeutic antibodies for oncology include, but are not limited to, anti-CD51 antibodies (e.g., Alemtuzumab), anti-CD20 antibodies (e.g., Ofatumumab and Rituximab), anti-CD47 antibodies, and anti-GD2 antibodies.
• anti-CD51 antibodies e.g., Alemtuzumab
• anti-CD20 antibodies e.g., Ofatumumab and Rituximab
• anti-CD47 antibodies e.g., Ofatumumab and Rituximab
• anti-GD2 antibodies e.g., anti-GD2 antibodies.
• Immunotherapy agents can also include conjugated monoclonal antibodies, including, but not limited to, radiolabeled antibodies (e.g., Ibritumomab tiuxetan (Zevalin), etc.) and chemo-labeled antibodies (also known as antibody-drug conjugates (ADCs)), e.g., Brentuximab vedotin (Adcetris), Ado-trastuzumab emtansine (Kadcyla), denileukin diftitox (Ontak), etc.
• radiolabeled antibodies e.g., Ibritumomab tiuxetan (Zevalin), etc.
• chemo-labeled antibodies also known as antibody-drug conjugates (ADCs)
• ADCs antibody-drug conjugates
• immunotherapy agents include cytokines, such as, but not limited to, interferons (i.e., IFN-a, INF- ⁇ ), interleukins (i.e. IL-2, IL-12), and tumor necrosis factor alpha (TNF-a) and the like.
• cytokines such as, but not limited to, interferons (i.e., IFN-a, INF- ⁇ ), interleukins (i.e. IL-2, IL-12), and tumor necrosis factor alpha (TNF-a) and the like.
• immunotherapeutic agents include, for example, polysaccharide-K, neoantigens, etc.
• the immunotherapy agent is selected from the group comprising an anti-CD52 antibody, an anti-CD20 antibody, anti-CD47 antibody, an anti-GD2 antibody, polysaccharide K and a cytokine.
• the immunotherapy agent is selected from the group comprising Alemtuzumab, Ofatumumab, Rituximab, Zevalin, Adcetris, Kadcyla and Ontak.
• the immunotherapy agent is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an IDO inhibitor, a CCR7 inhibitor, a OX40 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor.
• the immunotherapy agent is a prodrug (e.g., a lipid conjugate prodrug) of a small molecule inhibitor immunotherapy agent.
• the cytokine is selected from the group comprising an interferon and an interleukin. In some embodiments, the cytokine is selected from the group comprising IFN-a, IFN- ⁇ , IL-2, IL-12 and TNF-a.
• the presently disclosed subject matter proves a combination of PDT based on a nanoparticle PS and immunotherapy for the treatment of multiple cancer types.
• the method comprises administering to the patient an additional treatment (i.e., in addition to the photosensitizer or scintillator and the immunotherapy agent).
• the additional treatment is a cancer treatment or a treatment for a side effect of the cancer (e.g., a pain medication).
• the additional treatment or additional cancer treatment is selected from the group including, but not limited to surgery, radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy and gene therapy.
• the chemotherapy can comprise administering a drug selected from the group comprising oxaliplatin, doxorubicin, daunorubicin, docetaxel, mitoxanthrone, paclitaxel, digitoxin, digoxin, dihydroartemisinin, and septacidin.
• a drug formulation such as a polymeric micelle formulation, a liposomal formulation, a dendrimer formulation, a polymer-based nanoparticle formulation, a silica-based nanoparticle formulation, a NCP formulation, and an inorganic nanoparticle formulation.
• irradiating with light can comprise irradiating with visible or near infrared light.
• the light can have a wavelength between about 630 nm and about 1400 nm (e.g., about 670 nm).
• the light can have a wavelength of from about 630 nm to about 740 nm (e.g. 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, or about 740 nm).
• PDT can be combined with inhibitor-based immunotherapy and/or other immunotherapy to cause systemic rejection of established tumors using adaptive immune response, e.g., cytotoxic T cells.
• adaptive immune response e.g., cytotoxic T cells.
• immunotherapeutic agents not only the effective eradication of a primary tumor, but also suppression/eradication of a distant metastatic tumor or tumors can be accomplished using nanoparticle (e.g., NCP particle)- based PDT effects.
• the antitumor efficacy can be enhanced by adding chemotherapeutics that are known to cause immunogenic cell death.
• inorganic materials are known to strongly absorb X-rays and convert the absorbed X-ray energy to visible and near-infrared light.
• the emitted near-infrared light from these X-ray scintillating nanomaterials can then be absorbed by the nearby photosensitizers to enable X-ray induced PDT effects.
• Other types of materials can also achieve X-ray induced PDT. When this X-ray induced PDT is combined with immune checkpoint inhibitors, excellent radioimmunotherapy can be obtained.
• the subject can be irradiated with X-rays 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 presently disclosed subject matter provides a method that combines treating a subject with one or more immunotherapy agents, one or more chemotherapeutic agents, or with a combination of an one or more immunotherapy agents and one or more chemotherapeutic agents wherein the method comprises administering to a subject at least one of the agents in the form of a lipid-conjugate prodrug.
• the method comprises administering a lipid-conjugate prodrug to a subject and administering an immunotherapy agent to the subject.
• the immunotherapy agent can by any suitable immunotherapy agent, such as one of the immunotherapy agents described above with regard to the combination therapies involving PDT.
• the lipid-conjugate prodrug can be administered in the form of a nanoparticle, e.g., wherein the lipid-conjugate prodrug is present in a lipid coating layer of a nanoparticle, such as, but not limited to a NCP particle.
• the method comprises administering the prodrug (e.g., as part of a nanoparticle), an immunotherapy agent, and at least one additional treatment, such as, but not limited to a cancer treatment (e.g. surgery, radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy and gene therapy.
• the chemotherapy can comprise administering a drug selected from the group comprising oxaliplatin, doxorubicin, daunorubicin, docetaxel, mitoxanthrone, paclitaxel, digitoxin, digoxin, dihydroartemisinin, and septacidin.
• the chemotherapy can comprise administering a drug formulation such as one of the chemotherapy drug formulations described herein above with regard to combination therapies involving PDT ,
• the lipid-based prodrugs described herein can be combined with any suitable additional drug delivery platform.
• the presently disclosed subject matter provides a composition comprising zinc pyrophosphate (i.e., an inorganic compound comprising Zn 2+ cations and pyrophosphate anions) and a lipid conjugate prodrug (e.g., a prodrug comprising a drug moiety linked to a lipid moiety via a linker comprising a disulfide bond).
• the zinc pyrophosphate can be provided in the form of a nanoparticle.
• the zinc pyrophosphate nanoparticle can be coated with a lipid coating layer, wherein the lipid coating layer can comprise the lipid conjugate prodrug.
• the presently disclosed subject matter provides a composition comprising a NCP comprising a cisplatin and/or oxaliplatin analogue or prodrug (e.g., as a bridging ligand in the NCP) and a lipid conjugate prodrug.
• the NCP can be provided in the form of a nanoparticle.
• the NCP particle can be coated with a lipid coating layer comprising the prodrug.
• the composition can further include a nucleic acid therapeutic agent, such as a siRNA, a miRNA or an AS ODN.
• the presently disclosed subject matter provides a nanoscale particle for delivery of therapeutic agents, wherein said nanoscale particle comprises: a core comprising a metal-organic matrix material and a prodrug comprising a drug-lipid conjugate.
• the prodrug can be present in a lipid coating layer surrounding all or a portion of the surface of the particle core.
• the drug-lipid conjugate comprises a monovalent drug moiety, a monovalent lipid moiety and a bivalent linker moiety comprising a biodegradable bond, e.g., a disulfide bond.
• the monovalent drug moiety can be, for example, a monovalent derivative of an anticancer drug and/or a small molecule immunotherapy agent.
• the monovalent drug moiety can be a monovalent derivative of a drug such as, but not limited to, ET, PTX, OTS964, NLG919, OTS167, OTSC41 , DHA, CPT, Doxorubicin, Docetaxel, Vincristine, MTX, ART, and Capecitabine.
• the lipid moiety is a monovalent derivative of CHOL, OA, a lyso-lipid, or phosphocholine.
• the bivalent linker is a bivalent derivative of bis(2-hydroxyethyl) disulfide or a similar compound.
• the nanoparticle can comprise any suitable metal-organic matrix.
• the metal-organic matrix comprises a coordination polymer (e.g., a NCP).
• Neoplastic cells grow within the context of the host environment, and can respond to numerous physical, chemical and cellular challenges. Therefore, those cells develop multiple strategies to control the tumor-host interaction. In order for a neoplasm to grow and spread, it needs to obtain sufficient oxygen and nutrients to break down the extracellular matrix (ECM) in order to invade surrounding tissues and metastasize, and to evade the host immune response.
• ECM extracellular matrix
• RNA interference (RNAi) technology can be used to target the molecules involved in angiogenesis, invasion/metastasis, and immune evasion for cancer therapy.
• target genes include: (1 ) growth factors (e.g., VEGF, EGF, FGF, PDGF, IL-8, and IGF-1 ); (2) proteases and protease inhibitors (e.g., Cathepsin, MMP2, Stromelysin, and uPA); (3) oncogenes (e.g., c-myc, ras, c-src, v-raf, c-jun, and VEGFR); (4) signal transduction (e.g., thymidine and phosporylase); (5) enzymes (e.g., RAS- farnesyl, transferase, Geranyl, and Transferase); (6) cytokines (e.g., IL-1 , IL- 6, and IL-8); and (7) endogenous stimulator (e.g., Ang-1 , Angiostatin II,
• growth factors e.g., VEGF, EGF, FGF, PDGF,
• RNAi targeting multidrug resistance (MDR) genes e.g., ABCB1 , ABCB4, and ABCB5
• DNA repair mechanisms are important for the maintenance of genomic stability and thus are potential therapeutic targets for cancer.
• cancer cells can overexpress proteins related to DNA repair in order to restore therapy-induced DNA damage.
• target genes include excision repair cross-complementing 1 (ERCC1 ), X-ray repair cross-complementing protein 1 (XRCC1 ), ribonucleotide reductase, double-strand break signaling/repair proteins ATM, and DNA-dependent protein kinase catalytic subunit.
• MicroRNAs are a class of small, non-coding RNAs that post-transcriptionally control the translation and stability of mRNAs. miRNAs are responsible for maintaining a proper balance of various biological processes, including proliferation, differentiation, and cell death. In cancer, the loss of tumor-suppressive miRNAs enhances the expression of target oncogenes, whereas increased expression of oncogenic miRNAs can repress target tumor suppressor genes. Cancer-related miRNAs have been classified as oncogenic (such as miR-155, miR-21 , and miR-17 ⁇ 29), tumor- suppressive (such as miR-15, miR-16, LIN28, DICER), and context- dependent (such as miR-146 and miR-29) genes. Delivering tumor- suppressive miRNAs and silencing oncogenic miRNAs have been successful in various mouse models.
• oncogenic such as miR-155, miR-21 , and miR-17 ⁇ 29
• tumor- suppressive such as miR-15, miR-16, LIN28, DICER
• miRNAs Owing to the ability of miRNAs to target signaling pathways that are often perturbed in cancer, miRNAs also have the potential to sensitize resistant cells. MDR usually involves the increased excretion of a drug through ATP-binding cassette (ABC) transporters. Two of these ABC transporters, ABCC3 and ABCC6, are induced directly by SOX2. miR-9 is identified as a negative regulator of SOX2. Forced expression of miR-9 in a chemotherapy-resistant glioma stem cell lines suppresses SOX2 expression, leading to reduced ABC transporter expression and hence drug retention.
• ABC ATP-binding cassette
• Oligonucleotides are unmodified or chemically modified single- stranded DNA molecules. In general, they are relatively short (13-25 nucleotides) and hybridize to a unique sequence in the total pool of targets present in cells.
• Antisense oligonucleotides are single-stranded DNA fragments found to be able to inhibit mRNA translation.
• Antitumor AS ODNs are targeted to the genes that are involved in cell division, angiogenesis, metastasis, and cell survival in the presence of apoptotic signals including Bcl-2, Survivin, MDM2, Bcl-XL, RelA, RAS, RAF, BCR- ABL, JNK1 ,2, TERT, c-myc, and c-myb. Since the majority of cancer cells differ in gene expression profile from normal cells, AS ODNs can be used to specifically suppress the tumor growth with minimal consequences for normal cells. For example, Genta Inc. (Berkeley Heights, New Jersey, United States of America) has developed an 18-mer phosphothioate AS ODN that is complementary to Bcl-2, known as GenasenseTM. In addition, AS ODNs targeting MDM2 have been shown to potentiate the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents.
• the nanoparticle can further comprise at least one nucleic acid therapeutic agent (e.g., a nucleic acid chemotherapeutic agent).
• the nucleic acid chemotherapeutic agent can be a small interfering ribonucleic acid (siRNA), a miRNA, or an AS ODN.
• the nucleic acid chemotherapeutic agent can be covalent linked to a lipid molecule and/or attached to a lipid molecule via electrostatic interactions, wherein said lipid molecule can form part of a lipid coating layer surrounding all or a portion of the surface of the nanoparticle core.
• nucleic acid can be attached to the metal-organic matrix material core via coordination bonds between phosphate groups on the nucleic acid and metal ions on an outer surface of the core.
• nucleic acids such as siRNAs, miRNAs, and AS ODNs, can be directly loaded to the surfaces of NCPs via coordination bonds between metal ions on the NCP outer surface and phosphate groups on nucleic acids.
• the nanoparticles can comprise, for example, a single siRNA or pooled siRNAs (including several siRNAs targeting different anticancer pathways).
• siRNAs can include, but are not limited to, the following: EGFR/ErbB1 siRNA, ErbB2/HER2/Neu siRNA, IGF-1 R siRNA, K-ras siRNA, R-ras siRNA, BRAF siRNA, ABL siRNA, c-Src siRNA, Met siRNA, c-Myc siRNA, N-Myc siRNA, Cyclin-D1 siRNA, PI3K siRNA, AKT siRNA, NF- ⁇ siRNA, EWS/FLI-1 siRNA, HIF siRNA, HPV E7 siRNA, E2F4 siRNA, HPV E6 siRNA, Hdmx siRNA, Notch-1 siRNA, Delta-like-1 siRNA, FLIP siRNA, BCL-2 siRNA, BCL-XL siRNA, Survivin siRNA, XIAP siRNA, Telomerase siRNA, ID1 siRNA, Cks-1 siRNA, Skp-2 siRNA, cathepsin L siRNA, VEGF siRNA, EGF siRNA
• the miRNAs can include, but are not limited to, the following: miR-9, miR-15, miR-16, miR-34, miR-181 , miR-200, miR 200c, miR-342, miR-630, let-7, LIN28, and DICER.
• the particles can also include one or more antisense oligonucleotides (AS ODNs).
• AS ODNs antisense oligonucleotides
• Gene targets of the AS ODNs used can include, but are not limited to, the following: Bcl-2, Survivin, MDM2, Bcl-XL, RelA, RAS, RAF, BCR-ABL, JNK1 ,2, TERT, c-myc, and c-myb.
• the nucleic acid can be used to suppress an immunotherapy target.
• the nucleic acid can be, for example, a siRNA targeting PD- 1/PD-L1 , a siRNA targeting IDO or a siRNA targeting CCR7.
• one nucleic acid is used.
• a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different nucleic acids is used.
• the nanoparticle can comprise at least one nucleic acid agent selected from the group comprising, but not limited to, survivin siRNA, ERCC-1 siRNA, P-glycoprotein siRNA (P-gp siRNA), Bcl-2 siRNA, or a mixture thereof.
• the nanoparticle can further comprise at least one photosensitizer (PS).
• PS photosensitizer
• the PS is embedded or sequestered in the nanoparticle core (e.g., in pores or hollow interior cavities) or covalently attached to an organic moiety within the NCP matrix.
• the PS is covalently or non-covalently attached to a lipid coating layer or layers (i.e., a single lipid layer, lipid bilayer, or combination thereof) surrounding a portion of the outer surface of the nanoparticle core.
• Any suitable PS can be used, such as but not limited to, porphyrins, chlorophylls dyes, or derivatives or analogs thereof.
• the coating layer or layers includes a lipid single layer or lipid bilayer comprising a pyrolipid, i.e., a lipid covalently attached to a porphyrin or a derivative or analog thereof.
• the nanoparticle can further comprise at least one non-nucleic acid chemotherapeutic agent incorporated in the metal- organic matrix material core.
• the at least one non-nucleic acid chemotherapeutic agent can be incorporated in the metal-organic matrix material core via a covalent or a coordination bond.
• the agent can embedded or sequestered in pores or hollow cavities within the core. Any suitable non-nucleic acid chemotherapeutic agent can be used.
• the non-nucleic acid chemotherapeutic agent is selected from the group comprising, but not limited to cisplatin or oxaliplatin prodrugs, gemcitabine, methotrexate, leucovorin, pemetrexed disodium, doxorubicin, vinblastine, vincristine, vindesine, cytarabine, azathioprine, melphalan, imatinib, anastrozole, letrozole, carboplatin, paclitaxel, docetaxel, etoposide, and vinorelbine.
• the particle can comprise a single non-nucleic acid chemotherapeutic agent incorporated into the metal-organic matrix core or can comprise combinations of 2, 3, 4, 5, 6, or more non-nucleic acid chemotherapeutic agents incorporated in the metal-organic matrix material core.
• the presently disclosed subject matter can, in some embodiments, provide a combination of chemotherapies based on a nanoparticle for the treatment of multiple cancer types or a combination of nanoparticle-based chemotherapy and immunotherapy for the treatment of multiple cancer types.
• the non-nucleic acid chemotherapeutic agent is an analogue or prodrug of oxaliplatin or cisplatin.
• a suitable cisplatin analogue or prodrug can comprise a platinum coordination complex comprising two NH 3 platinum ligands and two chloro ligands (i.e., the four Pt ligands typically present in cisplatin), and at least one or two additional ligand or ligands (e.g., comprising five or six Pt ligands in total), wherein at least one additional Pt ligand or ligands comprises at least two groups that can coordinate to a metal ion.
• at least one metal ligand of the platinum coordination complex can coordinate both to the platinum ion of the platinum coordination complex and to a metal ion in a second coordination complex (e.g., another Pt coordination complex).
• Such a ligand can comprise two or more amino, hydroxyl, and/or carboxylate groups, or a combination of such groups.
• the non-nucleic acid chemotherapeutic agent is cis, cis, trans- Pt(NH 3 )2Cl2(OEt)(02CCH2CH 2 COOH).
• the nanoparticle core can comprise between about 10 weight % and about 50 weight % of the non-nucleic acid chemotherapeutic agent (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, or about 50 weight % of the non-nucleic acid chemotherapeutic agent).
• the non-nucleic acid chemotherapeutic agent e.g., about 10, 15, 20, 25, 30, 35, 40, 45, or about 50 weight % of the non-nucleic acid chemotherapeutic agent.
• the nanoscale particle has an average diameter of less than about 500 nm or less than about 250 nm. In some embodiments, the particle has an average diameter of between about 20 nm and about 200 nm. In some embodiments, the nanoscale particle has an average diameter of between about 20 nm and about 140 nm (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, and about 140 nm).
• the nanoscale particle further comprises one or more coating agents or layers covering at least a portion of the outer surface of the metal-organic matrix material core, wherein the one or more coating agents or layers are selected from a metal oxide, a polymer, a single lipid layer, a lipid bilayer, and combinations thereof.
• a spherical nanoparticle can have one or more concentric coating layers, each successive layer being dispersed over the outer surface of a smaller layer closer to the center of the particle.
• the metal-organic matrix material core is coated with a lipid bilayer comprising a cationic lipid and/or a functionalized lipid, wherein said functionalized lipid is a lipid functionalized with a group that can bond to a nucleic acid, and wherein at least one nucleic acid is covalently bonded to the functionalized lipid and/or attached to the cationic lipid via electrostatic interactions.
• the lipid bilayer comprises a mixture comprising one or more of a thiol- or dithiol-functionalized 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), and 1 ,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC).
• DSPE thiol- or dithiol-functionalized 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine
• DOTAP 1,2-dioleoyl-3-trimethylammonium propane
• DOPC 1 ,2-dioleoyl-sn- glycero-3-phosphocholine
• the lipid bilayer or lipid single layer can comprise or further comprise one or more of the group including but not limited to, cholesterol, 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1 ,2-Distearoyl- sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2- dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA), and pegylated-DSPE.
• DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
• DSPC 1 ,2-Distearoyl- sn-glycero-3-phosphocholine
• DOPC 1,2-dioleoyl-3-trimethylammonium propane
• DOPA di
• the lipid bilayer comprises or further comprises one or more of 1 ,2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA), cholesterol, and pegylated-DSPE.
• the one or more coating agents or layers further comprise a passivating agent, such as, but not limited to, a hydrophilic polymer (e.g., PEG); a targeting agent, such as, but not limited to a RGD peptide, an aptamer, an oligonucleotide, a polypeptide, an antibody or antibody fragment, or a polysaccharide; and/or an imaging agent, such as an optical imaging agent (e.g., a fluorescent moiety).
• a passivating agent such as, but not limited to, a hydrophilic polymer (e.g., PEG)
• a targeting agent such as, but not limited to a RGD peptide, an aptamer, an oligonucleotide, a polypeptide,
• the metal-organic matrix material core comprises a metal bisphosphonate coordination polymer comprising a multivalent metal ion and a bisphosphonate.
• the multivalent metal ion can be any suitable multivalent metal ion.
• the multivalent metal ion is a divalent metal ion, such as, but not limited to, Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , and combinations thereof.
• the multivalent metal ion is Zn 2+ .
• the bisphosphonate is itself a metal complex wherein two metal ligands are phosphonate-containing groups and wherein the phosphonate groups are available for coordinating to the multivalent metal ion.
• the bisphosphonate metal complex can comprise other ligands (i.e., those that do not contain a phosphonate group) that can include mono- and bidentate metal ligands, such as, but not limited to, halo (e.g., CI, Br, F, or I), NH 3 , alkylamino, hydroxyl, alkoxy, diols and diamines (e.g., diaminocyclohexane).
• the bisphosphonates can be prepared by providing a suitable metal complex, such as a metal complex with two hydroxyl ligands, and contacting the metal complex with, for example, diethoxyphosphinyl isocyanate, diethoxyphosphinyl isothiocyanate, diethoxyphosphinyl- containing carboxylic anhydride, or diethoxyphosphinyl-containing acyl chloride to form metal ligands that can provide phosphonate groups available for further coordinative bonding.
• a suitable metal complex such as a metal complex with two hydroxyl ligands
• the bisphosphonate is a platinum metal complex (e.g., cisplatin, oxaliplatin, or a similar complex) wherein two platinum ligands have been replaced by or conjugated to phosphonate-containing groups that are not involved in coordinating to the platinum.
• the metal bisphosphonate coordination polymer particle can comprise a lipid coating layer comprising a prodrug of DHA.
• the bisphosphonate is a chemotherapeutic prodrug, optionally a cisplatin or oxaliplatin prodrug, such as, but not limited to bisphosphonate ester of cis, cis-trans- [Pt(NH 3 )2CI 2 (OH)2] (a cisplatin prodrug) or cis, trans-[Pt(dach)CI 2 (OH) 2 ].
• metal-organic matrix material core comprises between about 40 and about 50 weight % of bisphosphonate (e.g., about 40,
• the particle can further comprise one or more coating layers.
• the one or more coating layers comprise a lipid single layer or lipid bilayer coating.
• one or more nucleic acid therapeutic agents such as one or more of survivin siRNA, P-gp siRNA, and Bcl-2 siRNA, are attached to the coating.
• the nanoscale particle has a diameter between about 20 nm and about 180 nm. In some embodiments, the nanoscale particle has a diameter between about 90 nm and about 180 nm (e.g., about 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or about 180 nm).
• compositions are provided for use in treating a disease (e.g., cancer) alone or in combination with one or more additional therapeutic agents, e.g., one or more chemotherapeutic agents (or analogues or prodrugs thereof), one or more immunotherapy agents, one or more targeting agents, one or more imaging agent, one or more scintillator, one or more photosensitizer, or any mixture thereof.
• additional therapeutic agents e.g., one or more chemotherapeutic agents (or analogues or prodrugs thereof)
• immunotherapy agents e.g., one or more chemotherapeutic agents (or analogues or prodrugs thereof)
• one or more immunotherapy agents e.g., one or more targeting agents, one or more imaging agent, one or more scintillator, one or more photosensitizer, or any mixture thereof.
• the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof, wherein the method comprises administering to the subject a composition comprising a nanoscale particle as described herein.
• the method of treating cancer comprises administering to the subject a composition comprising a nanoscale particle comprising: a core comprising a metal-organic matrix material, and a prodrug comprising a drug-lipid conjugate.
• the cancer can be any cancer in need of treatment, such as, but not limited to a skin cancer (e.g., a melanoma), a connective tissue cancer (e.g., a sarcoma), an adipose cancer, a breast cancer, a head and neck cancer, a lung cancer (e.g., mesothelioma), a stomach cancer, a pancreatic cancer, an ovarian cancer, a cervical cancer, an uterine cancer, an anogenital cancer
• a skin cancer e.g., a melanoma
• a connective tissue cancer e.g., a sarcoma
• an adipose cancer e.g., a breast cancer, a head and neck cancer
• a lung cancer e.g., mesothelioma
• a stomach cancer e.g., a pancreatic cancer
• an ovarian cancer e.g., a cervical cancer, an
• testicular cancer e.g., testicular cancer
• kidney cancer e.g., a kidney cancer, a bladder cancer, a colon cancer, a prostate cancer, a central nervous system (CNS) cancer, a retinal cancer, a blood cancer, a neuroblastoma, multiple myeloma, or a lymphoid cancer
• CNS central nervous system
• the cancer is lung cancer, pancreatic cancer, ovarian cancer, breast cancer or colon cancer.
• the cancer is a metastatic cancer and/or a chemo and/or radio-resistant cancer.
• the cancer is ovarian cancer.
• the ovarian cancer is a cisplatin resistant cancer.
• the method further comprises administering to the subject an immunotherapy agent.
• the immunotherapy agent is selected from an antibody, a small molecule inhibitor, a small molecule inhibitor prodrug, a cytokine, and polysaccharide K.
• the immunotherapy agent is selected from the group including, but not limited to, an anti-CD52 antibody, an anti-CD20 antibody, an anti-CD47 antibody an anti-GD2 antibody, a cytokine, and polysaccharide K.
• the immunotherapy agent is selected from the group including, but not limited to, Alemtuzumab, Ofatumumab, Rituximab, Zevalin, Adcetris, Kadcyla and Ontak.
• the immunotherapy agent is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an IDO inhibitor, a CCR7 inhibitor, an OX40 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor.
• more than one (e.g., 2, 3, 4, 5, 6, or more) immunotherapy agent can be administered
• the nanoscale particle further comprises a photosensitizer (PS).
• PS photosensitizer
• the nanoscale particle can comprise any suitable PS.
• the PS is a pyrolipid, wherein said pyrolipid is a lipid covalently attached to a porphyrin or a derivative or analog thereof.
• the method of treating cancer can further comprise irradiating the subject or a treatment area of the subject with radiation (e.g., visible or near infrared light) having a wavelength suitable to activate the photosensitizer.
• radiation e.g., visible or near infrared light
• the method can comprise irradiating the subject with light at a wavelength between about 630 nm and about 740 nm (e.g., 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, or about 740 nm).
• the PS-containing nanoparticle can be used to treat any cancer, such as one of the cancers described hereinabove.
• the cancer is a head and neck cancer, optionally a cisplatin resistant head and neck cancer.
• the chemotherapy and PDT can be further combined with immunotherapy and an immunotherapy agent can be administered to the subject in addition to or as part of a PS-containing nanoparticle.
• the immunotherapy agent is selected from the group including, but not limited to, an anti-CD52 antibody, an anti- CD20 antibody, an anti-CD20 antibody, anti-CD47 antibody, an anti-GD2 antibody, polysaccharide K and a cytokine.
• the immunotherapy agent is selected from a radiolabeled antibody, an antibody- drug conjugate, and a neoantigen.
• the immunotherapy agent is selected from the group comprising Alemtuzumab, Ofatumumab, Rituximab, Zevalin, Adcetris, Kadcyla and Ontak.
• the immunotherapy agent is a small molecule inhibitor, such as, but not limited to, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an IDO inhibitor, a CCR7 inhibitor, a OX40 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor.
• the presently disclosed subject matter provides a pharmaceutical formulation comprising a nanoscale particle as described herein (e.g., a nanoscale particle comprising a core comprising a metal-organic matrix material comprising a coordination polymer and a prodrug comprising a lipid-drug conjugate, optionally further comprising a PS and/or an immunotherapy agent) together with a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier can be pharmaceutically acceptable in humans.
• the presently disclosed subject matter provides nanoparticles that can comprise multiple chemotherapeutic agents, alone or in combination with one or more immunotherapy agents.
• the presently disclosed subject matter also provides nanoparticles that contain a PS for use in PDT, alone or in combination with one or more immunotherapy agents and/or one or more chemotherapy agents.
• These nanoparticle-based therapies can be used to treat multiple cancer types and to treat cancer more efficiently by targeting multiple pathways.
• compositions of the presently disclosed subject matter comprise, in some embodiments, a composition that includes a pharmaceutically acceptable carrier.
• a suitable pharmaceutical formulation can be used to prepare the compositions for administration to a subject.
• the composition and/or carriers can be pharmaceutically acceptable in humans.
• suitable formulations can include aqueous and nonaqueous 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 nonaqueous 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), 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
• ruminants such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels
• 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 intratumorally. 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 intratumorally.
• 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. VI. Doses
• 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 (e.g., cytotoxic or PDT activity or chemotherapeutic loading) of the composition and the route of administration.
• lipids Choi
• PTX Paclitaxel
• DHA dihydroartemisinin
• NLG919 Camptothecin
• OA oleic acid
• the synthesis of the prodrugs can involve the conversion of the hydroxyl group in either the lipid (Choi) or the anticancer drug (CPT) into an acyl chloride, followed by the direct conjugation with drugs (ET, PTX, NLG919) or lipids (OA) that also have hydroxyl group(s).
• the disulfide bond can be introduced into the prodrugs either via first functionalizing anticancer drugs with bis(2-hydroxyethyl) disulfide (OH-S-S-OH), followed by lipid conjugation (Chol-ET, Chol-PTX, and Chol-NLG919), or modifying the lipid with OH-S-S-OH first, followed by the anticancer drug conjugation (OA-
• DHA-S-S-OH To a mixture of DHA (200 mg, 0.7 mmol, 1 eq.) and 4-N,N- dimethylaminopyridine (DMAP, 200 mg, 1 .64 mmol, 2.3 eq.) in anhydrous tetrahydrofuran (THF, 4ml), a solution of triphosgene (70 mg, 0.24 mmol, 0.34 eq.) in anhydrous DCM (1 ml_) was added dropwise over an ice bath with stirring.
• DMAP 4-N,N- dimethylaminopyridine
• Chol-Boc-MTX (410 mg, 0.33 mmol) was dissolved in 20 ml trifluoroacetic acid (TFA) and stirred at room temperature for 30 min. The excess TFA was removed by nitrogen gas stream. The residue was recrystallized in tetrahydrofuran/hexane to yield 300 mg Chol-MTX TFA salt.
• the salt could be further converted to HCI salt by dissolving it in DCM, washing with saturated NaHC0 3 solution, 1 M HCI and removing solvent.
• Nanoscale Coordination Polymer Core-shell Nanoparticles Carrying Etoposide for the Treatment of Small Cell Lung Cancer Synthesis of Zn-pyrophosphate particles
• Microemulsions were first formed by the addition of 4 mL Na 4 P 2 O 7 - 10H 2 O (25 mg/mL in water) and 4 mL Zn(NO 3 ) 2 -6H 2 O (100 mg/mL in water) to two separate surfactant system mixtures (100 mL, 0.3 M TritonX- 100, 1 .5 M hexanol in cyclohexane).
• the separate microemulsions were stirred vigorously for 15 min at room temperate, 400 ⁇ of DOPA solution (200 mg/mL in CHCI 3 ) was added to Na 4 P 2 O 7 - 10H 2 O solution and the stirring was continued for 15 min until a clear solution formed.
• Zn/ET was obtained by adding a 80 ⁇ _ THF solution of DSPC, cholesterol, Chol-ET (molar ratio 1 : 1 :0.3, 1 : 1 :0.5 or 1 : 1 : 1 ), DSPE-PEG2k (20 mol%) and Zn-pyrophosphate bare particles to 500 ⁇ _ 30% (v/v) EtOH/H 2 O at 60 °C. THF and EtOH were evaporated and the solution was allowed to cool to room temperature before use. The particle size and distribution were determined by DLS. The loading of etoposide was determined by UV-Vis (see Table 1 ). Table 1. Characterization of Zn/ET particles.
• Nanoscale Coordination Polymer Core-shell Nanoparticles Carrying Paclitaxel for the Treatment of Non-Small Cell Lung Cancer
• Zn/PTX was obtained by adding a 80 ⁇ THF solution of DSPC, cholesterol, Chol-PTX (molar ratio 1 : 1 :0.3), DSPE-PEG2k (20 mol%) and Zn-pyrophosphate bare particles to 500 ⁇ 30% (v/v) EtOH/H 2 0 at 60 °C. THF and EtOH were evaporated and the solution was allowed to cool to room temperature before use.
• the particle size and distribution were determined by DLS, having a Z-average diameter of 94.15 ⁇ 0.61 nm, number-average diameter of 55.83 ⁇ 6.20 nm, PDI of 0.1 19 ⁇ 0.01 , zeta- potential of -0.67 ⁇ 1 .02 mV.
• the Zn/PTX particle has a drug loading of 7.14%.
• H460 and A549 cells seeded on 96-well plates at a density of 2000 cells/well were treated with Zn/PTX and free PTX at various etoposide concentrations for 72 h. The cell viability was then detected by MTS assay (Promega, USA) and the IC 50 values were calculated accordingly.
• PTX showed IC 50 of 10.79 ⁇ 0.44 ⁇ and 10.09 ⁇ 0.06 ⁇ against H460 and A549 cells after incubation for 72 h, respectively.
• Zn/PTX showed a similar IC 50 of 12.03 ⁇ 0.43 ⁇ and 1 1 .07 ⁇ 0.13 ⁇ , respectively, compared to free PTX.
• DHA Dihydroartemisinin
• Microemulsions were first formed by the addition of 0.2 ml_ Na 4 P 2 O 7 - 10H 2 O (25 mg/mL in water) and 0.2 ml_ Zn(NO 3 ) 2 6H 2 O (100 mg/ml_ in water) to two separate surfactant system mixtures (5 ml_, 0.3 M TritonX-100, 1 .5 M hexanol in cyclohexane). The separate microemulsions were stirred vigorously for 15 min at room temperate, 20 uL of DOPA solution (200 mg/mL in CHCI 3 ) was added to Na 4 P 2 O 7 - 10H 2 O solution and the stirring was continued for 15 min until a clear solution formed.
• DOPA solution 200 mg/mL in CHCI 3
• Zn/DHA was obtained by adding a 80 ⁇ _ THF solution of DOPC, cholesterol, Chol-DHA and 20 mol% DSPE-PEG2k and Zn-pyrophosphate bare particles to 500 ⁇ _ 30% (v/v) EtOH/H 2 O at 50°C. THF and EtOH were evaporated and the solution was allowed to cool to room temperature before use. The particle size and distribution were determined by DLS. See Table 2.
• A2780, A2780/CDDP, and SKOV-3 cells were seeded into 96-well plate at 2000 cells/well for 24 h. Afterwards cells were treated with free DHA or Zn/DHA at various concentrations and incubated for another 72 h. The cell viability was then determined via MTS assay by microplate reader.
• DHA showed high cytotoxicity, especially on A2780 cells.
• DHA showed IC 50 of 0.30 ⁇ 0.1 1 , 2.61 ⁇ 0.18, and 9.90 ⁇ 0.34 ⁇ on A2780, A2780/CDDP and SKOV-3, respectively.
• Zn/DHA showed IC 5 o values of 0.96 ⁇ 0.15, 3.71 ⁇ 0.55, and 1 1 .88 ⁇ 3.22 for A2780, A2780/CDDP and SKOV-3, respectively.
• Nanoscale Coordination Polymer Core-shell Nanoparticles Combine Cisplatin and Etoposide for the Treatment of Small Cell Lung Cancer Preparation and Characterization of NCP-1/ET and NCP-1/ET/siRNAs
• the solid core of NCP-1 was composed of 1 ,2-cis,cis,trans-
• DOPA dioleoyl-sn- glycero-3-phosphate
• NCP-1/ET was made by coating 84 ⁇ _ 1 ,2-dioleoyl-sn-glycero-3- phosphatidylcholine (DOPC) (5 mg/mL), 42 ⁇ _ Choi (5 mg/mL), 16 ⁇ _ Chol- ET (15 mg/mL), and 150 ⁇ _ 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol) 2 ooo] (DSPE-PEG2K) (5 mg/mL) onto 0.25 mg bare NCP particles via self-assembly to give a cisplatin to ET molar ratio of 1 : 1 .
• the Z-average diameter and polydispersity (PDI) of NCP-1/ET were found to be 106.8 ⁇ 1.05 nm and 0.121 ⁇ 0.01 , respectively, by dynamic light scattering (DLS) measurements.
• thiolated siRNA was conjugated to DSPE-SPDP to generate DSPE-siRNA.
• the disulfide bond in thiolated siRNA was placed on the 5' end of sense strand of siRNA duplexes in order to circumvent the potential inhibition on the antisense strand.
• DSPE- siRNA was incorporated into the outer lipid layer via self-assembly along with DOPC, Choi, Chol-ET, and DSPE-PEG 2K at a cisplatin to siRNA weight ratio of 4:1 to give NCP-1 /ET/siRNAs.
• Bcl-2, survivin, and ERCC-1 siRNAs were selected as a model for siRNA cocktails targeting multiple drug resistance pathways. Equal amounts of Bcl-2, survivin, and ERCC-1 siRNAs are present in NCP-1/ET/siRNAs. The Z-average diameter and PDI of NCP- 1 /ET/siRNAs were 100.2 ⁇ 1 .10 nm and 0.159 ⁇ 0.01 , respectively. See Table 3, below. Owing to the shielding effect of PEG, siRNA could be protected from nuclease degradation in physiological environments. Similar to Chol- ET, siRNA release can be triggered by cleaving the disulfide bond under reducing environment such as in the presence of GSH.
• SCLC cell lines H82 and H69 cells were seeded into 96-well plate at 2000 cells/well for 24 h. Afterwards, cells were treated by cisplatin, ET, cisplatin + ET, NCP-1 , NCP-1/ET, and NCP- 1/ET/siRNAs at various concentrations and incubated for another 72 h. The cell viability was then determined via MTS assay by microplate reader. The IC50 of NCP-1 /ET/siRNAs on SCLC cells was shown in Table 4, below.
• NCP-1/ET/siRNA The in vivo antitumor activity of NCP-1/ET/siRNA was evaluated on five tumor models, including two cispaltin-resistant ovarian cancers (A2780/CDDP and SKOV-3), two non-small cell lung cancers (H460 and A549) and one small cell lung cancer (H82).
• NCP-1/ET/siRNAs showed significantly inhibitory effect on all five tumor models, especially on SKOV-3, A549 and H82 tumor models. See Figures 1A-1 C. The tumors were completely inhibited at the first stage, but began to grow at the last stage after stopping the treatment.
• the bare NCP-1 comprised by cisplatin-bisphosphonates, Zn 2+ ions, and dioleoyl-sn-glycero-3-phosphate (DOPA), was prepared in a mixture of 0.3 M TritonX-100 and 1 .5 M hexanol in cyclohexane with DOPA sticking out in the medium, which provides a lipid layer on the bare NCP and facilitates the incorporation of other phospholipids or lipid-containing drugs.
• the bare NCP-1 particles are then mixed with twice amount of EtOH and centrifuged at 12000 rpm for 30 min to remove the solvent.
• NCP-1 /Chol-PTX 7.3% for Chol-PTX
• Cisplatin 3.3%, 300 g/mol
• the z- average size for NCP-1 /Chol-PTX is 94.1 ⁇ 1 .2 nm with PDI at 0.164 and neutral charge (-1 .5 mV) in PBS.
• the NCP-1 /Chol-PTX particles have been stable for 10 days in both PBS and PBS-containing 10% FBS solutions. The superior formulation stability could arise from the strong coordination bonding inside the core and the hydrophobic interactions between cholesterol and phospholipids.
• the bare NCP-1 comprised by cisplatin-bisphosphonates, Zn 2+ ions, and dioleoyl-sn-glycero-3-phosphate (DOPA), was prepared in a mixture of 0.3 M TritonX-100 and 1 .5 M hexanol in cyclohexane with DOPA sticking out in the medium, which provides a lipid layer on the bare NCP and facilitates the incorporation of other phospholipids or lipid-containing drugs.
• DOPA dioleoyl-sn-glycero-3-phosphate
• NCP- 1/OTS167 particles were obtained by adding a mixture of DOPC, Choi, Chol- OTS167, DSPE-PEG 2K (20 mol%), and bare NCP-1 particles to 30% (v/v) EtOH/H 2 O with vigorous stirring. THF and EtOH were evaporated and the solution was allowed to cool to r.t. prior to use. Particles with cisplatin to OTS167molar ratio of 1 : 1 , 5: 1 and 10: 1 were made. The z-average sizes for NCP-1/OTS167 were similar for different formulations with different molar ratios, which is 1 10-120 nm with PDI ranging from 0.15-0.18. See Table 5.
• NCP-1/OTS167 (1 :1) 1 19.7 ⁇ 2.54 0.175 ⁇ 0.02 144.4 ⁇ 2.48 73.48 ⁇ 7.47
• NCP-1/OTS167 (5:1) 1 17.7 ⁇ 1 .71 0.167 ⁇ 0.02 151 .9 ⁇ 2.24 74.83 ⁇ 7.58
• NCP-1/OTS167 (10:1) 109.3 ⁇ 1 .04 0.153 ⁇ 0.01 129.6 ⁇ 1 .05 66.55 ⁇ 1 .23
• NCP-1/OTS167 10:1) 109.3 ⁇ 1 .04 0.153 ⁇ 0.01 129.6 ⁇ 1 .05 66.55 ⁇ 1 .23
• the synergy between cisplatin and OTS167 was first examined against cisplatin-resistant ovarian cancer cell line A2780/CDDP by comparing free drug cytotoxicities alone or in combination.
• the IC50 of cisplatin was significantly decreased after combination with OTS167 at the molar ratio of 10: 1 and 100: 1 . See Table 6.
• the bare NCP-1 comprised by cisplatin-bisphosphonates, Zn 2+ ions, and dioleoyl-sn-glycero-3-phosphate (DOPA), was prepared in a mixture of 0.3 M TritonX-100 and 1 .5 M hexanol in cyclohexane with DOPA sticking out in the medium, which provides a lipid layer on the bare NCP and facilitates the incorporation of other phospholipids or lipid-containing drugs.
• DOPA dioleoyl-sn-glycero-3-phosphate
• NCP- 1/OTSC41 particles were obtained by adding a mixture of DOPC, Choi, Chol-OTSC41 , DSPE-PEG 2K (20 mol%), and bare NCP-1 particles to 30% (v/v) EtOH/H 2 O with vigorous stirring. THF and EtOH were evaporated and the solution was allowed to cool to r.t. prior to use. Particles with cisplatin to OTSC41 molar ratio of 1 : 1 , 5: 1 and 10: 1 were made. The z-average sizes for NCP-1 /OTSC41 were similar for different formulations with different molar ratios, which is around 100 nm with PDI ranging from 0.14-0.16. See Table 8.
• NCp-l/OTSC41 108.1 ⁇ 0.21 0.137 ⁇ 0.01 126.3 ⁇ 1.15 72.93 ⁇ 5.68
• the synergy between cisplatin and OTSC41 was first examined against two ovarian cancer lines, including cisplatin-sensitive A2780 cells and cisplatin-resistant A2780/CDDP cells by comparing free drug cytotoxicities alone or in combination.
• the IC50 of cisplatin OTSC41 alone or in combination was shown in Table 9.
• Nanoscale Coordination Polymer Core-Shell Nanoparticles Combine Cisplatin and Dihydroartemisinin for the Treatment of Ovarian Cancer
• DHA dihydroartemisinin
• ROS free-radical reactive oxygen species
• NCP-1 particles carrying cisplatin prodrug were prepared as previously reported. Briefly, a mixture of Zn(NOs)2 and an cisplatin prodrug, 1 ,2-cis,cis,trans-[Pt(NH 3 )2CI 2 (OCONHP(0)(OH)2)2] (PtBp), with 1 ,2-dioleoyl- sn-glycero-3-phosphate sodium salt (DOPA) in the Triton X-100/1 - hexanol/cyclohexane/water reverse microemulsion was vigorously stirred at room temperature for 30 min to afford spherical DOPA-coated NCP-1 particles.
• NCP-1 has a cisplatin loading of 25 wt% as determined by inductively coupled plasma-mass spectrometry (ICP-MS).
• DOPA-NCP-1 particles were then coated with cholesterol, DOPC, Chol-DHA conjugates, and 20 mol% DSPE-PEG2k to afford a core-shell nanostructure with a solid core carrying cisplatin and a lipid layer containing DHA.
• Particles with different molar ratios of cisplatin to DHA were formulation by changing the amount of DHA during lipid coating. See Table 10.
• A2780, A2780/CDDP, and SKOV-3 cells were seeded into 96-well plate at 2000 cells/well for 24 h. Afterwards cells were treated with different formulations at various concentrations and incubated for another 72 h. The cell viability was then determined via MTS assay by microplate reader. After combination with DHA, the cytotoxicity of cisplatin decreased significantly, as shown by significantly decrease in IC50. The particles showed similar cytotoxicity, compared to free drugs. See Table 1 1 .
• the numbers in parenthesis refer to DHA concentrations.
• Nanoscale Coordination Polymer Core-Shell Nanoparticles Combine Oxaliplatin Analogue and Paclitaxel for the Treatment of Colorectal
• NCP-based core-shell nanoparticles carrying two chemotherapeutic inducers of immunogenic cell death are shown to exhibit substantial anticancer efficacy in the treatment of colorectal cancer.
• Effective anticancer therapy of NCP-2/PTX was demonstrated against two syngeneic murine colorectal cancer models, CT26 and MC38.
• Efficient anti-tumor immunity was evoked by NCP-2/PTX as demonstrated by early calreticulin (CRT) exposure on the cell surface, antitumor vaccination, and delayed tumor growth.
• CRT early calreticulin
• NCP-2/PTX nanoparticles were prepared by coating NCP-2 core with an asymmetric lipid bilayer containing chol-PTX and PEG. Particles containing either 1 :1 or 2: 1 molar ratio of Pt(dach)CI 2 : paclitaxel were prepared and characterized.
• a THF solution of 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), cholesterol, 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE- PEG2k), and chol-PTX (DOPC:cholesterol:chol-PTX:DSPE- PEG2k 2:2:0.26: 1 or 2:2:0.52: 1 for 2: 1 and 1 : 1 oxaliplatin:paclitaxel formulations, respectively), and DOPA-capped NCP-2 were added to 500 ⁇ _ of 30% (v/v) ethanol/water and kept stirring at 1700 rpm at 50°C for 1 min.
• DOPC 1,2-dioleoyl-sn-glycero-3- phosphocholine
• cholesterol 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-
• NCP-2/PTX possessed Z-average diameter, number-average diameter, and polydispersity index (PDI), of 121 .7 ⁇ 0.95 nm, 81 .98 ⁇ 4.45 nm, and 0.139 ⁇ 0.01 , respectively, for 1 : 1 molar ratio dispersed in phosphate buffered saline (PBS) by DLS measurement. 2: 1 particles showed similar characteristics: 1 12.0+0.51 nm, 74.26+3.60 nm, and 0.151 +0.01 respectively.
• PBS phosphate buffered saline
• NCP-2/PTX The cytotoxicity of NCP-2/PTX was evaluated against two colorectal cancer cells including murine colorectal adenocarcinoma CT26 and murine colorectal carcinoma MC38.
• oxaliplatin and paclitaxel were first examined by comparing free drug cytotoxicities alone or in combination.
• the IC 50 of oxaliplatin was substantially lower than that of paclitaxel by MTS assay, likely due to the hydrophobicity of paclitaxel.
• Paclitaxel was prepared in a stock solution of 3: 1 ethanol:water ratio at 1 mg/ml before subsequent dilution to prepare free drug doses.
• the combination index (CI50) for the two drugs was much more favorable for the 1 : 1 compared to 2: 1 molar ratio, with equal oxaliplatin doses.
• NCP formulations containing oxaliplatin analogue (NCP-2) or oxaliplatin analogue and paclitaxel at a 1 : 1 molar ratio showed similar cell killing effects to those of the free drugs. See Table 12.
• the numbers in parentheses refer to paclitaxel concentrations.
• the immunogenic cell death induced by NCP-2/PTX was evaluated by immunofluorescence and flow cytometry.
• CT26 cells were seeded at 5x10 5 cells per well in 6-well plates and further cultured for 24 h. The culture media were replaced by 2 ml_ of fresh culture media containing 10% FBS.
• Oxaliplatin, paclitaxel, chol-PTX, NCP-2, and NCP-2/PTX (1 : 1 ) were added to the cells, respectively, at an equivalent oxaliplatin and paclitaxel dose of 5 ⁇ . Cells incubated with PBS served as control.
• CT26 cells were seeded at 1 ⁇ 10 6 cells per well in 6-well plates and further cultured for 24 h. The culture media were replaced by 2 ml_ of fresh culture media containing 10% FBS.
• Oxaliplatin, paclitaxel, chol-PTX, NCP-2, and NCP-2/PTX (1 : 1 ) were added to the cells, respectively, at an equivalent Pt and/or paclitaxel dose of 5 ⁇ .
• Cells incubated with PBS served as control.
• the cells were collected, incubated with AlexaFluor 488-CRT antibody for 2 h, and stained with propidium iodide (PI).
• the samples were analyzed by flow cytometer (LSRII Orange, BD, Franklin Lakes, New Jersey, United States of America) to identify cell surface CRT. The fluorescence intensity of stained cells was gated on Pl-negative cells. See Figure 2.
• NCP-2/PTX pharmacokinetic and biodistribution study of NCP-2/PTX was carried out on CT26 tumor bearing BALB/c mice after intraperitoneal injection.
• the oxaliplatin distribution was quantified by ICP-MS.
• the pharmacokinetics and biodistribution of a NCP particle comprising a zinc and oxaliplatin analogue core and a lipid coating layer comprising cholesterol-modified paclitaxel (NCP-2/PTX) after intravenous (i.v.) injection in CT26 murine colorectal adenocarcinoma tumor bearing mice at a dose of 1 milligram per kilogram (mg/kg) showed greater presence in the blood compared to liver, lung, spleen, kidney, bladder, and tumor.
• Platinum (Pt) concentration was analyzed via inductively coupled plasma-mass spectrometry (ICP-MS) at 5 minutes, and at 1 , 3, 5, 8, 24, and 48 hours, and is expressed in micrograms g). Results of the pharmacokinetics and biodistribution were expressed as percentage of the initial dose (% ID). See Figure 3.
• CT26 or MC38 tumor bearing mice were treated by intraperitoneal injection at equivalent oxaliplatin doses of 1 mg/kg and paclitaxel doses of 2.24 mg/kg (1 ) PBS, (2) NCP-2/PTX, or (3) NCP-2/PTX + PD-L1 antibody on the indicated days for a total of 6-10 doses. Consistent, low oxaliplatin analogue doses of NCP-2/PTX significantly delayed tumor growth in both CT26 and MC38 murine mouse models.
• NCP-2/PTX 5 x 10 6 CT26 cells were implanted into the right flank regions of immunocompromised athymic nude mice deficient in mature T cells, or Rag-/- BALB/c mice deficient in both T and B cells. See Figure 7.
• MC38 tumor bearing mice were treated by intraperitoneal injection at equivalent oxaliplatin analogue doses of 2 mg/kg and antibody doses of 75 g for (1 ) PBS, (2) NCP-2, (3) NCP-2/PTX (1 : 1 ), (4) NCP-2/PTX (2: 1 ), (5) NCP-2/PTX (1 : 1 ) + PD-L1 antibody, or (6) NCP-2/PTX (2: 1 ) + PD-L1 antibody every four days for a total of 5 doses. See Figure 8.
• NCP-2/PTX antitumor vaccination capability of NCP-2/PTX was further evaluated.
• PBS or NCP-2/PTX were inoculated subcutaneously to the right flank region of BALB/c mice.
• mice were re-challenged by injecting 1 10 5 CT26 cells on the contralateral flank.
• the animals were checked daily for tumor development using calipers and body weight evolution. See Figures 9A and 9B. All mice were sacrificed when the right tumor size of PBS group exceeded 2 cm 3 .
• Nanoscale Coordination Polymer Core-Shell Nanoparticles Combine Oxaliplatin Analogue and Mitoxantrone for the Treatment of Colorectal
• NCP-based core-shell nanoparticles carrying two chemotherapeutic inducers of immunogenic cell death are shown to exhibit substantial anticancer efficacy in the treatment of colorectal cancer.
• NCP-2 particles carrying an oxaliplatin prodrug were prepared as previously reported.
• NCP-2/MTX nanoparticles were prepared by coating NCP-2 core with an asymmetric lipid bilayer containing chol-MTX and PEG.
• Particles containing a 2: 1 or 1 : 1 molar ratio of oxaliplatin: mitoxantrone were prepared and characterized.
• a THF solution of 1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), cholesterol, 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE- PEG2k), and chol-MTX (DOPC:cholesterol:chol-MTX:DSPE- PEG2k 2:2:0.30: 1 or 2:2:0.60: 1 for 2: 1 and 1 : 1 oxaliplatin analogue: mitoxantrone molar ratios, respectively), and DOPA-capped NCP-2 were added to 500 ⁇ _ of 30% (v/v) ethanol/water and kept stirring at 1700 rpm at 50 °C for 1 min.
• the THF and ethanol in the nanoparticle suspension was completely evaporated before subsequent use in in vitro and in vivo experiments. The particle size and distribution were determined by DLS. See Table 13.
• NCP-2/MTX The cytotoxicity of NCP-2/MTX was evaluated against two syngeneic cancer cell lines:colorectal adenocarcinoma CT26 and colorectal carcinoma MC38.
• NCP-2/MTX elicited substantial anticancer efficacy owing to synergy between the drugs.
• the synergy between oxaliplatin and mitoxantrone were first examined by comparing free drug cytotoxicities alone or in combination.
• the combination index (CI50) for the two drugs was much more favorable for the 2: 1 compared to 1 : 1 molar ratio, with equal Pt doses.
• NCP formulations containing oxaliplatin analogue (NCP-2) or oxaliplatin analogue and mitoxantrone at a 1 : 1 molar ratio (NCP-2/MTX) showed similar cell killing effects to those of the free drugs. See Table 14.
• a colorectal mouse model of murine colorectal adenocarcinoma CT26 was employed to assess the in vivo anticancer activity of NCP-2/MTX.
• 5 x 10 6 CT26 cells were injected into the right flank and treatment began on day 7, after all tumors had reached 100-150mm 3 in size.
• CT26 tumor bearing mice were treated by intraperitoneal injection at equivalent oxaliplatin analogue doses of 1 mg/kg and paclitaxel doses of 0.58 mg/kg (1 ) PBS, (2) NCP-2/MTX (2: 1 ), or (3) NCP-2/MTX (2: 1 ) + 75 ⁇ g PD-L1 antibody every four days for a total of 6 doses. Consistent, low doses of chemotherapeutics in NCP-2/MTX significantly delayed tumor growth in CT26 murine mouse models and synergized with checkpoint blockade immunotherapy to sustain tumor growth inhibition. See Figure 10.
• a colorectal mouse model of murine colorectal carcinoma MC38 was employed to assess the in vivo anticancer activity of NCP-2/MTX.
• 1 x 10 6 MC38 cells were injected into the right flank and treatment began on day 12, after all tumors had reached 50-70mm 3 in size.
• MC38 tumor bearing mice were treated by intraperitoneal injection at equivalent oxaliplatin doses of 2 mg/kg and mitoxantrone doses of 1 .16 mg/kg (1 ) PBS, (2) NCP-2/MTX (2: 1 ), or (3) NCP-2/MTX (2: 1 ) + 75 ⁇ g PD-L1 antibody every four days for a total of 5 doses. See Figure 1 1 .
• the bare NCP-2 comprised by oxaliplatin-bisphosphonates, Zn 2+ ions, and dioleoyl-sn-glycero-3-phosphate (DOPA), was prepared in a mixture of 0.3 M TritonX-100 and 1 .5 M hexanol in cyclohexane with DOPA sticking out in the medium, which provides a lipid layer on the bare NCP and facilitates the incorporation of other phospholipids or lipid-containing drugs.
• the bare NCP-2 particles are then mixed with twice amount of EtOH and centrifuged at 12000 rpm for 30 min to remove the solvent.
• the particle pellet was further washed once with 50% EtOH/cyclohexane and twice with 50% EtOH/THF to remove excess amounts of DOPA and re-dispersed in THF. Finally, the bare NCP-2 particles were filtered via 200 nm syringe filter prior to any use.
• the NCP-2 particle has a Z-average diameter and PDI of 78.56 ⁇ 1 .03 nm and 0.147 ⁇ 0.01 , respectively.
• NCP-2/DHA was formulated by adding a mixture of DOPC, Choi, Chol-DHA, DSPE-PEG 2K and bare NCP-2 particles to 30% (v/v) EtOH/H 2 O with vigorous stirring.
• the NCP- 2/DHA particle has a number-averaged dimeter, Z-averaged diameter, and PDI of 51 .90 ⁇ 1 .06 nm, 80.74 nm ⁇ 0.79 nm, and 0.152, respectively.
• NCP-2/DHA The cytotoxicity of NCP-2/DHA was evaluated against two colon cancer cell lines CT26 and MC38.
• the synergy between oxaliplatin and DHA was first examined by comparing free drug cytotoxicities alone or in combination.
• the combination index (CI50) for the two drugs was lower than 1 in some drug effect levels, indicating the syngestic effect between two drugs.
• NCP formulations containing oxaliplatin analogue (NCP-2), DHA or oxaliplatin and DHA at a 1 : 1 molar ratio showed similar cell killing effects to those of the free drugs. See Table 15.
• CT26 5.77 ⁇ 7.77 ⁇ 8.1 1 ⁇ 1 .17 1 1 .03 ⁇ 2.58 8.51 ⁇ 6.74 ⁇ 1 .24
• the immunogenic cell death induced by DHA was evaluated by immunofluorescence and flow cytometry.
• CT26 and MC38 cells were seeded at 5x10 5 cells per well in 6-well plates and further cultured for 24 h. The culture media were replaced by 2 mL of fresh culture media containing 10% FBS. DHA was added to the cells at a dose of 1 ⁇ . Cells incubated with PBS served as control.
• CT26 and MC38 cells were seeded at 1 10 6 cells per well in 6-well plates and further cultured for 24 h. The culture media were replaced by 2 mL of fresh culture media containing 10% FBS. DHA was added to the cells at a dose of 1 ⁇ . Cells incubated with PBS served as control.
• a colorectal mouse model of murine colorectal adenocarcinoma CT26 was employed to assess the in vivo anticancer activity of NCP-2/DHA. 5 x 10 6 CT26 cells were injected into the right flank and treatment began on day 7, after all tumors had reached 100-150 mm 3 in size. CT26 tumor bearing mice were treated by intraperitoneal injection of NCP-2/DHA at an oxaliplatin analogue dose of 2 mg/kg. NCP-2/DHA particles inhibited the growth of CT26 tumor models. See Figure 13.
• PC-DHA phosphocoline-conjugated DHA
• NCP-3 particles carrying an oxaliplatin prodrug carrying an oxaliplatin prodrug.
• NCP-3 has an oxaliplatin loading of 28.2 wt% as determined by inductively coupled plasma-mass spectrometry (ICP-MS).
• NCP-3/DHA-OA-DHA refers to a 1 : 1 mixture of chol- S-S-DHA and OA-DHA conjugates. Characterization of the particles is described in Table 16. Table 16. Particle size and PDI of NCP-2/DHA.
• NCP-3/DHA The cytotoxicity of NCP-3/DHA was evaluated against two colon cancer cell lines CT26 and MC38. DHA, chol-S-S-DHA, OA-DHA, and PC- DHA showed fairly similar IC50 values.
• the synergy between oxaliplatin and DHA were first examined by comparing free drug cytotoxicities alone or in combination. NCP formulations containing oxaliplatin (NCP-3), DHA or oxaliplatin and DHA at a 1 : 1 molar ratio showed similar cell killing effects to those of the free drugs. See Table 17 and Figures 14 and 15.
• CT26 cells seeded in 24-well plates at 1 10 5 cells/well were first incubated with NCP-3/DHA or appropriate controls for 24 h at an oxaliplatin concentration of 10 ⁇ .
• the floating and adherent cells were collected and stained with Alexa Fluor 488 AnnexinV/dead cell apoptosis kit (Invitrogen, USA) according to manufacturer's instructions.
• the apoptosis and necrosis were examined by a flow cytometry (LSRII Blue, BD, Franklin Lakes, New Jersey, United States of America). Both oxaliplatin and DHA individually caused necrosis, but the combination therapy evoked a high level of apoptosis. See Figure 16.
• the immunogenic cell death induced by NCP-3/DHA was evaluated by flow cytometry and ELISA.
• flow cytometry analysis CT26 cells were seeded at 1 *10 cells per well in 6-well plates and further cultured for 24 h. The culture media were replaced by 2 mL of fresh culture media containing 10% FBS. Oxaliplatin, DHA, chol-DHA, oxaliplatin + DHA, NCP-3, Zn/DHA and NCP-3/DHA were added to the cells, respectively, at equivalent oxaliplatin and/or DHA dose of 10 ⁇ and 20 ⁇ , respectively. Cells incubated with PBS served as control.
• the cells were collected, incubated with AlexaFluor 488-CRT antibody for 2 h, and stained with PI.
• the samples were analyzed by flow cytometer (LSRII Orange, BD, Franklin Lakes, New Jersey, United States of America) to identify cell surface CRT.
• the fluorescence intensity of stained cells was gated on Pl-negative cells. Both oxaliplatin and DHA individually caused cell surface CRT expression with slightly increased CRT exposure in cells treated with NCP-3/DHA compared to Zn/DHA.
• HMGB-1 high mobility group box-1
• CT26 cells were seeded at 1 *10 6 cells per well in 6-well plates and further cultured for 24 h. The culture media were replaced by 2 mL of fresh culture media containing 10% FBS. Oxaliplatin, DHA, chol- DHA, oxaliplatin + DHA, NCP-3, Zn/DHA, NCP-3/DHA, and NCP-3/OA-DHA were added to the cells, respectively, at equivalent oxaliplatin and/or DHA dose of 10 ⁇ and 20 ⁇ , respectively. Cells incubated with PBS served as control.
• NCP-3/DHA A pK study of NCP-3/DHA was carried out on SD/CD rats.
• the oxaliplatin distribution was quantified by ICP-MS and the chol-DHA concentration in the blood was quantified by LC-MS after extraction by THF.
• Both oxaliplatin and chol-DHA concentrations in blood versus time were fitted by a one-compartment model.
• Blood circulation half-lives of oxaliplatin and chol-DHA were determined to be (1 1 .8 ⁇ 0.6) and (13.3 ⁇ 1 .2) h, respectively, and did not exhibit statistically significant difference. See Figures 18A and 18B.
• One-compartment model fit of NCP-3/OA-DHA exhibited a blood circulation half-life of 18.1 +3.1 h for oxaliplatin.
• NCP-3/DHA was carried out on CT26 tumor bearing BALB/c mice. See Figure 19.
• the oxaliplatin distribution was quantified by ICP-MS.
• NCP-3/DHA exhibited low uptake by the mononuclear phagocyte system (MPS) as evidenced by the low % ID/g (percent injected dose per gram tissue) in liver ( ⁇ 2.7 ⁇ 0.3%), spleen ( ⁇ 7.6 ⁇ 2.2%), and kidney ( ⁇ 3.0 ⁇ 1 .0%).
• MPS mononuclear phagocyte system
• NCP-3/DHA by i.p. injection were performed on BALB/c and C57BL/6 mice.
• Single dose injections of NCP-3/DHA at 50 and 60 mg oxaliplatin/kg showed minimal weight loss in BALB/c and C57BL/6 mice, respectively.
• a once-weekly repeated dose of NCP-3/DHA at 60 mg oxaliplatin/kg on C57BL/6 showed mice could tolerate at least 4 repeated doses without significant toxicity.
• NCP-3/OA-DHA intraperitoneally injected into C57BL/6 mice at an oxaliplatin dose of 24 mg/kg once every 3 days for a total of 8 doses also showed minimal body weight loss and toxicity.
• CT26 and MC38 were employed to assess the in vivo anticancer activity of NCP-3/DHA.
• 1 x 10 6 CT26 or MC38 cells were injected into the right flank and treatment began on day 12, after all tumors had reached 80-100 mm 3 in size.
• CT26 or MC38 tumor bearing mice were treated by intraperitoneal injection of NCP-3/DHA and appropriate controls.
• NCP-3/DHA particles inhibited the growth of both CT26 and MC38 tumor models. See Figures 20A and 20B.
• NCP-3/DHA could effectively eradicate all mice with CT26 tumor models at an oxaliplatin dose of 8 mg/kg.
• the spleens were harvested and ground into single-cell suspensions.
• the splenocytes were treated with ACK lysis buffer and then analyzed by Enzyme-Linked ImmunoSpot (ELISPOT, eBioscience, San Diego, California, United States of America).
• ELISPOT Enzyme-Linked ImmunoSpot
• Chemotherapy and immunotherapy alone increased tumor-specific T cells, but NCP-3/DHA and NCP-3/DHA + PD-L1 showed the greatest increase in tumor-specific immune response. See Figure 21 .
• LL/2 tumor model were also used to evaluate the in vivo efficacy of NCP- 3/DHA in combination with immunotherapy. See Figures 22A and 22B. 2 x 10 6 4T1 cells or 1.5 x 10 6 LL/2 cells were injected into the right flank and treatment began on day 10 or day 12, respectively, after all tumors had reached 80-100mm 3 in size. Mice were treated by intraperitoneal injection at equivalent oxaliplatin doses of 8 mg/kg. Repeated doses of NCP-3/DHA significantly inhibited tumor growth in both the 4T1 and LL/2 tumor models. NCP-3/OA-DHA at equivalent oxaliplatin and DHA doses was also able to cause tumor growth inhibition on mice bearing LL/2 tumors.
• the hydroxyl group in cholesterol was converted to acyl chloride by mixing cholesterol (1 eq.), triphosgene (0.35 eq.), and DMAP (4 eq.) in DCM for 20 min with stirring at r.t.
• the resultant Chol-COCI (1 eq.) was then added dropwise into OH-S-S-OH (2 eq.) in DCM and the reaction mixture was stirred overnight to produce Chol-S-S-OH, which was purified by column chromatography with ethyl acetate/hexane (1 :2, v/v).
• Chol-NLG919 (1.7 eq.) in DCM to generate Chol-NLG919, which was subsequently subject to column chromatography with methanol/DCM (3:97, v/v) with yield of 62.2%.
• the identity of Chol-NLG919 was confirmed by 1 H-NMR, 13 C-NMR, and ESI-MS.
• the active NLG919 will be readily released following the cleavage of the disulfide bond inside cells by GSH and/or cysteine.
• the bare NCP-2 comprised by oxaliplatin-bisphosphonates, Zn 2+ ions, and dioleoyl-sn-glycero-3-phosphate (DOPA), was prepared in a mixture of 0.3 M TritonX-100 and 1 .5 M hexanol in cyclohexane with DOPA sticking out in the medium, which provides a lipid layer on the bare NCP and facilitates the incorporation of other phospholipids or lipid-containing drugs.
• the bare NCP-2 particles are then mixed with twice amount of EtOH and centrifuged at 12000 rpm for 30 min to remove the solvent.
• NCP-2/Chol-NLG919 was formulated by adding a mixture of DOPC (0.5 mg), Choi (0.25 mg) and Chol-NLG919 (62 pg), DSPE-PEG 2K (20 mol%) (0.9 mg) and bare NCP-2 particles (0.25 mg) to 30% (v/v) EtOH/H 2 O with vigorous stirring.
• Oxaliplatin/NLG919 2.18: 1 (molar ratio); Drug loading: NLG919 (1 .02%, 282.17 g/mol, 3.16% for Chol-NLG919); Oxaliplatin: (3.13%, 397.3 g/mol). Small sized particle (85.2 nm ⁇ 0.8) with PDI of 0.152 and near neutral charge (-2.1 mV) were observed. Besides, the NCP-2/Chol-NLG919 particles have been stable for 14 days in both PBS and PBS-containing 10% FBS solutions with no noticeable size, PDI, and zeta potential changes. The enhanced formulation stability is likely to stem from the strong coordination bonding inside the core and the hydrophobic interactions between cholesterol and phospholipids. EXAMPLE 15
• OA-CPT was synthesized as follows. First, oleic acid (OA, 1 eq.) reacted with OH-S-S-OH (2 eq.) in DCM for overnight to generate OA-S-S- OH, which was purified by ethyl acetate/hexane (2:3, v/v). In parallel, hydroxyl group in CPT was converted to acyl chloride by mixing CPT (1 eq.), triphosgene (0.35 eq.), and DMAP (4 eq.) in DCM for 20 min with stirring at r.t.
• the bare NCP-2 comprised by oxaliplatin-bisphosphonates, Zn 2+ ions, and dioleoyl-sn-glycero-3-phosphate (DOPA), was prepared in a mixture of 0.3 M TritonX-100 and 1 .5 M hexanol in cyclohexane with DOPA sticking out in the medium, which provides a lipid layer on the bare NCP and facilitates the incorporation of other phospholipids or lipid-containing drugs.
• the bare NCP-2 particles are then mixed with twice amount of EtOH and centrifuged at 12000 rpm for 30 min to remove the solvent.
• NCP-1/OA-CPT/Chol-PTX was formulated by adding a mixture of DOPC (0.5 mg), Choi (0.25 mg), OA-CPT (57.2 pg), and Chol- PTX (106.2 pg), DSPE-PEG 2K (20 mol%) (0.9 mg) and bare NCP-1 particles (0.25 mg) to 30% (v/v) EtOH/H 2 0 with vigorous stirring.
• NCP-1 /OA-CPT/Chol-ET was prepared by adding a mixture of DOPC (0.5 mg), Choi (0.25mg), OA-CPT (57.2 pg), and Chol-ET (109.8 pg), DSPE- PEG 2K (20 mol%) (0.9 mg) and bare NCP-1 particles (0.25 mg) to 30% (v/v) EtOH/H 2 O with vigorous stirring. THF and EtOH were evaporated and the solution was allowed to cool to r.t prior to use. In this formulation, Cisplatin loading is at 3.38%; CPT loading is at 1 .25%, and 2.86% for OA-CPT; ET loading is at 2.14% and 5.49% for Chol-ET.
• the particle was found to be 56.4 ⁇ 0.7 nm in size with PDI at 0.152 and zeta potential at 0.12 mV in PBS. Spherical and mon-dispersed nanoparticles were confirmed in TEM.
• the NCP-1 /OA-CPT/Chol-ET has been stable for 10 days in both PBS and PBS- containing 10% FBS solutions with no significant size, PDI, and zeta potential changes.
• NCP-1 /OA-CPT/Chol-ET/Chol-PTX was formulated by adding a mixture of DOPC (0.5 mg), Choi (0.25 mg), OA-CPT (57.2 pg), Chol-ET (109.8 pg), and Chol-PTX (106.2 pg), DSPE-PEG 2K (20 mol%) (0.9 mg) and bare NCP-1 particles (0.25 mg) to 30% (v/v) EtOH/H 2 O with vigorous stirring.
• Cisplatin loading is at 3.38%; CPT loading is at 1 .25%, and 2.86% for OA-CPT; ET loading is at 2.14% and 5.49% for Chol-ET; PTX loading is at 3.13%, and 5.31 % for Chol-PTX.
• the particle was found to be 64.2 ⁇ 0.9 nm in size with PDI at 0.183 and zeta potential at 1 .02 mV in PBS. Spherical and mon-dispersed nanoparticles were confirmed in TEM.
• the NCP-1/OA-CPT/Chol-ET/Chol-PTX particles are stable for 7 days in both PBS and PBS-containing 10% FBS solutions with no significant size, PDI, and zeta potential changes.
• cells were treated by cisplatin, ET and Chol-ET, PTX and Chol- PTX, CPT and OA-CPT, NCP-1 , NCP-1/OA-CPT/Chol-PTX, NCP-1 /OA- CPT/Chol-ET, and NCP-1 /OA-CPT/Chol-ET/Chol-PTX at various concentrations and incubated for another 72 h.
• the cell viability was then determined via MTS assay by microplate reader.
• Nanoscale Coordination Polymer Core-Shell Nanoparticles Combine Chemotherapy and Photodynamic Therapy to Elicit Potent Anti-Tumor
• Nanoscale coordination polymers can comprise hybrid nanomaterials constructed from metal-connecting points and organic bridging ligands via self-assembly.
• NCPs possess several advantages over existing nanocarriers in biomedical applications such as the high tunability of composition and structure, versatility of combining multiple therapeutic agents or modalities in one nanoplatform, and intrinsic biodegradability due to the labile metal-organic ligand bonds.
• NCP-based core-shell nanoparticles carrying high payloads of cisplatin and the photosensitizer pyrolipid, NCP@pyrolipid were described for combined chemotherapy and photodynamic therapy (PDT).
• NCP@pyrolipid showed superior potency and efficacy in tumor regression in the cisplatin-resistant human head and neck cancer SQ20B xenograft mouse model.
• Photodynamic therapy is an FDA-approved anticancer modality that has been shown to enhance antitumor immunity.
• Kroemer and coworkers have demonstrated the immunogenic cell death induced by oxaliplatin in murine colorectal cancer models.
• Recent studies also suggested that immune response elicited against the residual cancer cells can contribute to the complete eradication of micrometastases and cancer stem cells.
• the combination therapy of oxaliplatin and PDT could be particularly efficient by simultaneously killing the tumor cells and stimulating an immune response against tumor cells. This can cause tumor inhibition/regression not only in the primary tumor site but also in the distant metastatic tumors.
• NCP-based core-shell nanoparticles carrying oxaliplatin and photosensitizer pyroiipid are shown herein to provide enhanced anti-tumor immunity for achieving superior anticancer efficacy in colorectal cancers and can be used in the treatment of metastatic colorectal cancer.
• NCP-2@pyrolipid combines two therapeutic modalities, oxaliplatin and PDT, which not only kill the cancer cells but also elicit strong immunogenic response for the control and eradication of metastatic tumor nodules.
• NCP- 2@pyrolipid Effective anticancer therapy with NCP- 2@pyrolipid is demonstrated against two colorectal cancer models including syngeneic CT26 murine colorectal cancer model and HT29 human colorectal cancer xenografts. Efficient anti-tumor immunity evoked by NCP-2@pyrolipid such as early calreticulin (CRT) exposure on the cell surface, successful antitumor vaccination, and abscopal effect, is also demonstrated.
• CTR early calreticulin
• NCP-2 has an oxaliplatin analogue loading of 27.6 wt% as determined by ICP-MS.
• NCP-2@pyrolipid nanoparticles were prepared by coating NCP-
• THF tetrahydrofuran
• NCP-2@pyrolipid is a core-shell nanostructure with DOPA-capped coordination polymer NCP-2 carrying an oxaliplatin analogue as a solid core and a self-assembled and asymmetric lipid bilayer as a shell.
• the NCP-2 cores were constructed from the coordination between Zn 2+ and phosphate groups of the oxaliplatin analogue prodrug, which were further capped with a monolayer of DOPA via Zn-phosphate interactions between NCPs and DOPA molecules and hydrophobic-hydrophobic interactions among DOPA molecules.
• the lipid shell contains pyrolipid as photosensitizer for PDT and 20 mol% of PEG- coating to minimize mononuclear phagocyte system (MPS) uptake and prolong blood circulation after systemic injection.
• This core-shell structured NCP-2@pyrolipid can take advantages of efficient cancer cell killing induced by chemotherapy and PDT as well as the anti-tumor immunity evoked by oxaliplatin analogue and PDT to enable the effective treatment of both primary and metastatic colorectal cancers.
• TEM images of NCP-2@pyrolipid demonstrated the formation of uniform and spherical nanoparticles.
• DLS measurements gave a Z-average diameter, number-average diameter, PDI, and zeta potential of 83.00 ⁇ 0.98 nm, 51.19 ⁇ 0.1 1 nm, 0.143 ⁇ 0.01 1 , and -3.67 ⁇ 0.85 mV, respectively, of NCP- 2@pyrolipid dispersed in PBS.
• the small sizes and near neutral surface charge of NCP-2@pyrolipid suggests their potential in in vivo applications.
• the lipid bilayer of NCP-2@pyrolipid dissolved and pyrolipid shows a broad Soret band around 400 nm and a distinct Q-band at 669 nm.
• Porphysome was prepared by following the procedure reported by
• pyrolipid can incorporate into the highly oriented and asymmetric lipid bilayer with a high loading.
• the pyrolipid excited states are highly quenched, and therefore no energy transfer to triplet oxygen was observed as evidenced by the low amount of 1 0 2 generated determined by the singlet oxygen sensor green (SOSG) reagent.
• SOSG singlet oxygen sensor green
• the time-dependent cellular uptake of NCP-2@pyrolipid was evaluated in CT26 cells with an incubation time ranging from 1 h to 24 h.
• Free oxaliplatin, porphysome, and the original NCP-2 carrying a cisplatin prodrug and coated with 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, and DSPE-PEG2k served as comparisons.
• the Pt and pyrolipid concentrations in the cells after incubating with NCP particles, oxaliplatin, or porphysome were determined by ICP-MS and fluorimetry, respectively.
• NCP-2@pyrolipid The cytotoxicity of NCP-2@pyrolipid was evaluated against two colorectal cancer cells including murine colorectal adenocarcinoma CT26 and human colorectal adenocarcinoma HT29 cells.
• NCP-2@pyrolipid induces apoptosis/necrosis as well as elicit immunogenic cell death upon LED light irradiation.
• oxaliplatin IC50 of free oxaliplatin, NCP-2, and NCP-2@pyrolipid in dark showed no significant difference in both cell lines, suggesting, without wishing to be bound by theory, pyrolipid does not cause cytotoxicity without light activation.
• the oxaliplatin IC50 of NCP-2@pyrolipid was decreased by ⁇ 4-fold and ⁇ 5-fold in CT26 and HT29 cells, respectively.
• the pyrolipid IC 50 values also significantly dropped for NCP-2@pyrolipid with irradiation accordingly. No toxicity was observed for porphysome under light and dark in both cell lines within the tested pyrolipid concentration range.
• Calreticulin is a distinct biomarker exposed on the surface of cells undergoing immunogenic cell death (ICD).
• ICD immunogenic cell death
• the immunogenic cell death induced by NCP-2@pyrolipid was evaluated by immunofluorescence and flow cytometry.
• CT26 cells were seeded at 5x10 5 cells per well in 6-well plates and further cultured for 24 h. The culture media were replaced by 2 ml_ of fresh culture media containing 10% FBS.
• Oxaliplatin, NCP-2, NCP-2@pyrolipid, and porphysome were added to the cells, respectively, at an equivalent Pt dose of 5 ⁇ and pyrolipid dose of 1 .6 ⁇ . Cells incubated with PBS served as control.
• the cells were irradiated with LED light (670 nm) at 100 mW/cm 2 for 15 min (equal to 90 J/cm 2 ).
• the cells were washed with PBS three times, fixed with 4% paraformaldehyde, incubated with AlexaFluor 488- CRT antibody for 2 h, stained with DAPI, and observed under CLSM using 405 nm and 488 nm lasers for visualizing nuclei and CRT expression on the cell membrane, respectively.
• CT26 cells were seeded at 1 10 6 cells per well in 6-well plates and further cultured for 24 h.
• the culture media were replaced by 2 ml_ of fresh culture media containing 10% FBS.
• Oxaliplatin, NCP-2, NCP-2@pyrolipid, and porphysome were added to the cells, respectively, at an equivalent oxaliplatin dose of 5 ⁇ and pyrolipid dose of 1.6 ⁇ .
• Cells incubated with PBS served as control. After 24-h incubation, the cells were irradiated with LED light (650 nm) at 100 mW/cm 2 for 15 min (equal to 90 J/cm 2 ). Following further incubation of 4 h, the cells were collected, incubated with AlexaFluor 488-CRT antibody for 2 h, and stained with PI.
• CT26 murine colorectal adenocarcinoma cells were treated with phosphate buffered saline (PBS, free oxaliplatin, a NCP comprising zinc (Zn) and an oxaliplatin analogue (NCP-2, porphysome, or NCP-2 comprising pyrolipid in a lipid coating layer (NCP-2@pyrolipid with or without light irradiation.
• PBS phosphate buffered saline
• NCP free oxaliplatin
• NCP-2 oxaliplatin analogue
• NCP-2 porphysome
• NCP-2 pyrolipid in a lipid coating layer
• NCP- 2@pyrolipid exhibited low uptake by the MPS as evidenced by the low % ID/g (percent injected dose per gram tissue) in liver ( ⁇ 7.1 ⁇ 2.5%), spleen ( ⁇ 10.4 ⁇ 4.3%), and kidney ( ⁇ 9.1 ⁇ 2.5%).
• CT26 or HT29 tumor bearing mice were treated by intravenous injection of (1 ) PBS, (2) NCP-2 at an oxaliplatin analogue dose of 2 mg/kg, (3) and (4) NCP-2@pyrolipid at an oxaliplatin dose of 2 mg/kg every four days for a total of two treatments for CT26 model and four treatments for HT29 model.
• mice in group (1 )-(3) were anesthetized with 2% (v/v) isoflurane and tumors were irradiated with a 670 nm LED at an irradiance of 100 mW/cm 2 for 30 min.
• NCP-2@pyrolipid combined with light irradiation led to efficient tumor inhibition on both CT26 and HT29 subcutaneous tumor mouse models.
• mice bearing CT26 tumors receiving NCP-2@pyrolipid and PDT treatment blood was collected on Day 7, 8, 9, and 10, and the serum TNF-a, IFN- ⁇ , and IL-6 production was determined by ELISA (R&D Systems, Minneapolis, Minnesota, United States of America) to evaluate the immunogenic response evoked by the treatment.
• the significantly elevated TNF-a, IFN- ⁇ , and IL-6 levels in the serum of mice treated with NCP-2@pyrolipid and PDT confirmed the robust immunogenic response evoked by the treatment. See Figures 28A-28C.
• mice treated with NCP-2 (+) and NCP-2@pyrolipid (-) could be due to the immunogenic response induced by oxaliplatin analogue.
• NCP- 2@pyrolipid was administered to the right flank region of 6-week-old male BALB/c mice.
• mice were re-challenged by injecting 1 ⁇ 1 0 5 CT26 cells on the contralateral flank. The animals were checked daily for tumor development using calipers and body weight evolution.
• mice were sacrificed when the right tumor size of PBS group exceeded 2 cm 3 As shown in Figures 29A-29C, NCP-2@pyrolipid and PDT achieved 100% success in antitumor vaccination against the re-challenge of healthy tumor cells.
• NCP-2@pyrolipid upon light irradiation was evaluated against subcutaneous CT26 tumor bearing BALB/c mice.
• Tumor bearing mice were established by subcutaneous inoculation of CT26 cell suspension (2x 10 6 cells per mouse) into the right flank region and CT26 cell suspension (4x 10 5 cells per mouse) into the left flank region of the same mouse.
• Four groups were included for comparison: (1 ) PBS + irradiation (2) NCP-2@pyrolipid + irradiation (3) NCP-1 @pyrolipid (NCP carrying cisplatin and pyrolipid) + irradiation (4) NCP-2@pyrolipid dark control.
• NCPs were intratumorally injected at an oxaliplatin analogue dose of 2 mg/kg. Twelve hour post-injection, mice were anesthetized with 2% (v/v) isoflurane and tumors were irradiated with LED light at 100 mW/cm 2 for 30 min (180 J/cm 2 ). The NCPs were injected every three day for a total two injections. LED light irradiation was performed daily on six consecutive days. To evaluate the therapeutic efficacy, tumor growth was monitored. The tumor size was measured with a digital caliper every day. Tumor volumes were calculated as follows: (width 2 ⁇ length)/2.
• NCP-2@pyrolipid plus LED light irradiation not only led to tumor regression/eradication of the treated right tumors, but also inhibited the growth of the distant left tumors, suggesting, without wishing to be bound by any one theory, that the combination therapy successfully evokes immunoresponse in immunocompotent mouse models of colorectal cancer.
• the treatment of NCP-1 @pyrolipid successfully suppressed/eradicated the right tumors, however showed limited inhibition on the growth of the left tumors. See Figures 30A and 30B. This result indicates that the combined immunogenic response evoked by oxaliplatin analogue and PDT elicits an abscopal effect.
• Nanoscale Coordination Polymer Core-Shell Nanoparticles Combine Chemotherapy, Photodynamic Therapy, and PD-L1 antibody for Anti- Tumor Immunity
• NCP- 2/pyrolipid in the presence of light activation, we add an immune checkpoint inhibitor in the treatment regimen.
• the abscopal effect of NCP-2@pyrolipid plus light irradiation in combination with checkpoint blockade PD-L1 antibody was evaluated against subcutaneous MC38 tumor bearing mice. C57BL/6 mice were injected s.c. with 5 ⁇ 10 5 MC38 cells into the right flank (primary tumor) and 1 ⁇ 10 5 MC38 cells into the left flank (secondary tumor).
• NCP@pyrolipid was i.p. injected into animals at an oxaliplatin analogue dose of 2 mg/kg every three days for a total of three injections.
• mice Twenty-four hours after injection, mice were anesthetized with 2% (v/v) isoflurane, and primary tumors were irradiated with a 670 nm LED at a light dose of 180 J/cm 2 given at 100 mW/cm 2 . After irradiation, mice were immediately i.p. injected with PD-L1 antibody at a dose of 50 ⁇ g/mouse. Primary and secondary tumor sizes were measured with a digital caliper and calculated as follows: (width 2 ⁇ length)/2.
• Zn-pyrophosphate Core-Shell Nanoparticles Combine Photodynamic therapy and PD-L1 antibody for Anti-Tumor Immunity Preparation of pyrolipid-loaded Zn-pyrophosphate particles (Zn@pyrolipid)
• Zn@pyrolipid was obtained by adding a 80 ⁇ _ THF solution of DSPC, cholesterol, pyrolipid (molar ratio 1 : 1 :0.5), DSPE-PEG2k (20 mol%) and Zn- pyrophosphate bare particles to 500 ⁇ _ 30% (v/v) EtOH/H 2 0 at 60 °C. THF and EtOH were evaporated and the solution was allowed to cool to room temperature before use. The particle size and distribution were determined by DLS. See Table 19.
• the pyrolipid loading amount was quantified with a UV-Vis spectrophotomer (UV-2401 PC, Shimadzu, Japan). After lipid coating, Zn@pyrolipid was centrifuged at 13000 rpm for 30 min. The precipitate of Zn@pyrolipid was redispersed in tetrahydrofuran (THF), and the pyrolipid amount in the nanoparticle suspension was determined by UV-Vis absorption at 669 nm. The pyrolipid loading was determined to be 10.6 ⁇ 0.8 wt.%.
• the fluorescence of Zn@pyrolipid with intact or disrupted lipid layer was measured to calculate the fluorescence quenching efficiency.
• Zn@pyrolipid was diluted in PBS as intact samples or PBS containing 0.5% Triton X-100 to disrupt the lipid layer.
• the samples were subjected to spectrofluorophotometer (RF-5301 PC, Shimadzu, Kyoto, Japan) for fluorescence measurement (excitation: 427 nm, emission: 600-750 nm).
• the fluorescence intensity at 675 nm for Zn@pyrolipid with intact lipid layer was normalized to Zn@pyrolipid with disrupted lipid layer to calculate the quenching efficiency.
• the fluorescence intensity of intact Zn@pyrolipid was 2.7% of that of Zn@pyrolipid with disrupted lipid layer.
• the singlet oxygen sensor green (SOSG) reagent (Life Technologies, USA) was employed for the detection of singlet oxygen generated by Zn@pyrolipid. After lipid coating, Zn@pyrolipid was centrifuged at 13000 rpm for 30 min. The supernatant was discarded and the pellet was re- suspended with PBS. Five microliter of freshly prepared SOSG solution in methanol (5 mM) was mixed with 2 ml_ of Zn@pyrolipid intact in PBS or disrupted with 0.5% Triton X-100. Free pyrolipid at the same pyrolipid concentration as Zn@pyrolipid served as a control.
• SOSG singlet oxygen sensor green
• 4T1 triple negative breast cancer cells and B16F10 murine melanoma cells were seeded on 24-well plates at a density of 1 10 5 cells/well and incubated for 24 h.
• Zn@pyrolipid and free pyrolipid were added to the cells at a pyrolipid dose of 2 ⁇ , respectively.
• cells were collected, washed with PBS three times, and counted with a hemocytometer. The cells were centrifuged at 3,000 rpm for 5 min and the cell pellet was lysed with 0.5% (w/v) SDS (pH 8.0).
• the cellular uptake of Zn@pyrolipid in both cell lines was rapid and mostly completed within 1 h, as indicated by the stable uptake amounts of pyrolipid over time up to 24 h.
• the cell viability was detected by (3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay (Promega, Madison, Wisconsin, United States of America) and the IC 50 values were calculated accordingly.
• Zn particles with or without irradiation did not present any toxicity on both cell lines, suggesting that Zn particles can serve as a safe and reliable nanocarrier for drug delivery.
• Zn@pyrolpid and free pyrolipid without irradiation also induced no cytotoxicity on both cells, indicating that Zn@pyrolipid is safe, and the particle can be injected without worries regarding toxicity.
• Zn@pyrolipid and free pyrolpid exhibited very high cytotoxicity, as shown by significantly decrease in the IC50 values. See Table 20.
• Table 20 IC50 values of Zn@pyrolipid and free pyrolipid under light and dark in 4T1 and B16F10 cells after 72 h incubation.
• Zn@pyrolipid and free pyrolipid did not induce any apoptosis without irradiation, compared to blank control, but evoked high level of apoptosis under irradiation.
• Zn@pyrolipid and free pyrolipid after irradiation induced 71 .61 % and 90.18% apoptosis on 4T1 cells, and 63.72% and 85.79% apoptosis on B16F10 cells, respectively.
• the 4T1 tumor model was generated by an orthotopic injection of 5 x10 4 cells into the mammary fat pad of the Balb/c female mice and tumors were allowed to grow until 100 mm 3 before they received intravenous administration of Zn@pyrolipid at a pyrolipid dose of 6 mg/kg.
• Zn@pyrolipid was intravenously (i.v.) injected to animals at a pyrolipid dose of 6 mg/kg every two days for a total three injections.
• mice Twenty four hours after injection, mice were anesthetized with 2% (v/v) isoflurane and tumors were irradiated with a 670 nm LED at a light dose of 180 J/cm 2 given at 100 mW/cm 2
• mice were intraperitoneally (i.p.) injected immediately with PD-L1 antibody at a dose of 50 ⁇ g/mouse.
• the tumor size was measured every day with a digital caliper and calculated as follows: (width 2 ⁇ length)/2.
• mice were sacrificed, and tumors were excised, weighed (see Figure 33B) and photographed.
• Lungs were also harvested, sectioned at 10 ⁇ thickness and stained with (H&E), and observed for histological examination of metastases with light microscopy.
• the primary tumor disappeared at day 13 after treatment by Zn@pyrolipid (+) + PD-L1 antibody, and did not recur.
• the growth of tumor treated with Zn@pyrolipid plus irradiation was inhibited at the first several days, but tumors began to grow fast at the last several days.
• the tumor volume treated with PD-L1 antibody showed a little bit of decrease, but did not have significant difference compared to PBS control group.
• the metastasis to the lungs was examined by the gross appearance of tumor nodules. Many tumor nodules in lungs treated with PBS, Zn@pyrolipid with irradiation, and Zn@pyrolipid without irradiation but plus PD-L1 antibody were observed. For Zn@pyrolipid with irradiation plus PD- L1 antibody treated group, only 1 or 2 tumor nodules were observed, indicating that the combination of PDT with PD-L1 antibody can significantly prevent the lung metastasis of breast cancer. The lungs were further sectioned and stained with H&E, and the percentage of metastasis area in total lung area was calculated.
• mice were injected s.c. with 5 ⁇ 10 4 4T1 cells or 1 ⁇ 10 6 TUBO cells in the right flank (primary tumor) and 1 ⁇ 10 4 4T1 cells or 2 ⁇ 10 5 TUBO cells in the left flank (secondary tumor).
• Zn@pyrolipid was i.v.
• mice were anesthetized with 2% (v/v) isoflurane and primary tumors were irradiated with a 670 nm LED at a light dose of 180 J/cm 2 given at 100 mW/cm 2
• mice were i.p. injected immediately with PD-L1 antibody at a dose of 50 ⁇ g/mouse.
• the primary and secondary tumor sizes were measured every day with a digital caliper and calculated as follows: (width 2 x length)/2. All mice were sacrificed when the primary tumor size of control group exceeded 2 cm 3 , and the excised tumors were photographed and weighed.
• Blood was collected daily from TUBO tumor-bearing mice from Day 10 after tumor inoculation, when the mice received the first Zn@pyrolipid injections, to Day 13.
• the serum was separated and analyzed by ELISA to determine cytokine production of IL-6, TNF-a, and IFN- ⁇ .
• tumor-draining lymph nodes were harvested and ground using the rubber end of a syringe.
• Tumors were harvested, treated with 1 mg/mL collagenase I (GIBCOTM, Thermo Fisher Scientific, Waltham, Massachusetts, United States of America) for 1 h, and ground using the rubber end of a syringe.
• Cells were filtered through nylon mesh filters and washed with PBS. The single-cell suspension was incubated with anti-CD16/32 (clone 93; eBiosciences, San Diego, California, United States of America) to reduce nonspecific binding to FcRs.
• Cells were further stained with the following fluorochrome-conjugated antibodies: CD45 (30-F1 1 ), CD3e (145-2C1 1 ), CD4 (GK1.5), CD8 (53-6.7), Foxp3 (FJK-16s),
• CD1 1 b M1/70
• Ly6C HK1.4
• Ly6G RB6-8C5
• F4/80 BM8
• B220 RA3-
• Zn@pyrolipid with irradiation in combination with anti-PD-L1 significantly increased the proportion of infiltrating CD8 + T cells in the left tumor, an essential step to induce the abscopal effect.
• the percentage of infiltrating CD45 + leukocytes, CD4 + T cells, and B cells were also significantly increased. See Figures 37A-37D.
• CD8 + and CD4 + T cells in lymph nodes decreased at day 12 after first treatment (see Figures 38A and 38B), possibly because the CD8 + and CD4 + T cells migrated from lymph nodes to the tumor site to kill the tumor cells. All these results indicated that the immune system was activated by the combination treatment of PDT with PD-L1 blockade.

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