Treating solid tumors with negatively charged particles

Abstract

The present disclosure relates generally to methods of treating solid tumors using negatively charged particles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 313,959, filed February 25, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates generally to methods of treating solid tumors using negatively charged particles. [Background technology]

[0003] Solid tumors are one of the leading causes of death in the United States and worldwide. Despite aggressive therapeutic interventions such as chemotherapy and immunotherapy, solid tumors constitute approximately 500,000 deaths per year in the United States and 9 million deaths per year worldwide. Current standard of care for solid tumors relies on multiple lines of treatment, including surgical tumor resection (when possible), radiation therapy, and monotherapy or combination therapy with cytotoxic agents, targeted therapies, and immunotherapies. A significant number of patients are unable to tolerate these intensive treatment regimens due to severe side effects (e.g., hepatotoxicity, cytopenias, gastrointestinal toxicity, and neurotoxicity). Of patients who undergo these treatment regimens, many patients fail to respond, develop treatment resistance after an initial response, or experience disease recurrence within a short period of time.

[0004] The immune system plays a key role in influencing disease progression, response to therapy, and treatment outcomes in solid tumors. Immunosuppression due to dysregulation of bone marrow-derived cells such as tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) during disease progression contributes to poor response to therapy, treatment resistance, and risk of relapse. These dysregulated bone marrow-derived cells are involved in tumor-promoting functions such as remodeling of the tumor microenvironment that promotes immune exclusion and contributes to immune exhaustion. At the same time, these activities promote tumor growth, metastasis, and mortality despite aggressive treatment. There is an urgent need for therapies that can overcome and / or reverse this immunosuppression and thus allow reactivation of endogenous antitumor immune functions to improve patient outcomes. Summary of the Invention

[0005] Negatively charged particles made from biodegradable materials and containing no attached (covalently and non-covalently), attached or embedded drugs (e.g., peptides, anionic moieties, and bioactive agents) are designed to have immunomodulatory properties due, at least in part, to particle diameter (size), surface charge (zeta potential), and size distribution. These negatively charged particles do not contain antigens or therapeutic agents. The physicochemical properties of the negatively charged particles are designed for targeted uptake by bone marrow-derived cells (e.g., monocytes, macrophages, and neutrophils). This targeted uptake results in downstream immunomodulation via induction of cell surface IL-15 expression in leukocytes, activation of antitumor T cells and NK cells, and production of antitumor cytokines / chemokines (e.g., TNF-α, MIP-1β, and RANTES (CCL5)). The immunomodulatory properties of the negatively charged particles relieve immunosuppression and reactivate endogenous antitumor immune function. In multiple preclinical solid tumor models representing treatment-responsive, treatment-resistant, and metastatic tumors, negatively charged particles have demonstrated efficacy in inhibiting tumor growth and metastasis leading to improved survival rates. Thus, the antitumor mechanism of action is highly relevant for the treatment of solid tumors.

[0006] Provided herein is a method of treating a solid tumor in a subject, comprising administering negatively charged particles to the subject, wherein the negatively charged particles are administered at a dose of about 0.1 to 15 mg / kg. Also provided herein is a method of inducing an anti-tumor or immune response in a subject suffering from a solid tumor, comprising administering negatively charged particles to the subject, wherein the negatively charged particles are administered at a dose of about 0.1 mg / kg to about 15 mg / kg. In various embodiments, the negatively charged particles are administered at a dose of about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 8 mg / kg, about 1.5 mg / kg to about 15 mg / kg, about 2 mg / kg to about 15 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 10 mg / kg, about 4 mg / kg to about 15 mg / kg, about 5 mg / kg to about 15 mg / kg, or about 2 mg / kg to about 8 mg / kg. In various embodiments, the negatively charged particles are administered at a dose of about 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, 12 mg / kg, or 15 mg / kg. In various embodiments, the negatively charged particles are administered in a dose of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 875 mg, 900 mg, 950 mg, 975 mg, or 1000 mg.

[0007] In various embodiments, the negatively charged particles are biodegradable.In embodiments, the negatively charged particles are polyglycolic acid (PGA) particles, polylactic acid (PLA) particles, polylactic-co-glycolic acid (PLGA) particles, polystyrene particles, diamond particles, or iron, zinc, cadmium, gold, or silver particles, or combinations thereof.

[0008] In some embodiments, the negatively charged particles are polylactic-co-glycolic acid (PLGA) particles. In various embodiments, the particles comprise PLGA in a copolymer ratio of polyglycolic acid:polylactic acid of about 100:0 to about 100, about 90:10 to about 10:90, about 80:20 to about 20:80, or about 50:50. In various embodiments, the particles comprise 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particles comprise polylactic acid:polyglycolic acid in a ratio of about 99:1 to about 1:99, e.g., about 99:1, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, and about 1:99, including all values ​​and ranges therebetween.

[0009] In various embodiments, the negatively charged particles are surface functionalized by carboxylation. In various embodiments, the negatively charged particles have a negative zeta potential. In some embodiments, the carboxylation creates a negative charge on the particles. In some embodiments, the carboxylation increases the negative charge of the negatively charged particles. In various embodiments, the negative zeta potential of the negatively charged particles is about -100 mV to about 0 mV. In various embodiments, the zeta potential of the particles is about -100 mV to about -25 mV, about -100 mV to about -30 mV, about -80 mV to about -30 mV, about -75 mV to about -30 mV, about -70 mV to about -30 mV, about -75 mV to about -35 mV, about -70 mV to about -25 mV, about -60 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -30 mV. In various embodiments, the zeta potential is about -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a negative zeta potential of about -30mV to -60mV.

[0010] In various embodiments, the size or diameter of the negatively charged particles is about 0.05 μm to about 10 μm. In various embodiments, the diameter of the negatively charged particles is about 0.1 μm to about 10 μm. In various embodiments, the diameter of the negatively charged particles is about 0.1 μm to about 5 μm. In various embodiments, the diameter of the negatively charged particles is 0.1 μm to about 3 μm. In various embodiments, the diameter of the negatively charged particles is 0.3 μm to about 5 μm. In various embodiments, the diameter of the negatively charged particles is 0.3 μm to about 3 μm. In various embodiments, the diameter of the negatively charged particles is about 0.3 μm to about 1 μm. In various embodiments, the diameter of the negatively charged particles is about 0.4 μm to about 1 μm. In various embodiments, the negatively charged particles have a diameter of about 100 nm to 10,000 nm, about 100 nm to 5,000 nm, about 100 nm to 3,000 nm, about 100 nm to 2,000 nm, about 100 nm to 1,500 nm, about 300 nm to 5,000 nm, about 300 nm to 3,000 nm, about 300 nm to 1,000 nm, about 300 nm to 800 nm, about 400 nm to 800 nm, or about 200 nm to 700 nm. In various embodiments, the negatively charged particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a diameter of 350 nm to 800 nm.

[0011] In various embodiments, the negatively charged particles have a homogeneous size distribution, in which at least 90% of the particles have a diameter of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, about 0.3 μm to about 3 μm (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 90% of the particles having a diameter of about 100 nm to 10,000 nm, about 100 nm to 5,000 nm, about 100 nm to 3,000 nm, about 100 nm to 2,000 nm, about 300 nm to 5,000 nm, about 300 nm to 3,000 nm, about 300 nm to 1,000 nm, about 300 nm to 800 nm, about 400 nm to 800 nm, or about 200 nm to 700 nm (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 50% of the particles having a diameter of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, and about 0.3 μm to about 3 μm (including all values ​​and ranges therein). In various embodiments, the particles have a homogenous size distribution, with at least 50% of the particles having a diameter of about 100 nm to about 10,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 3,000 nm, about 100 nm to about 2,000 nm, about 300 nm to about 5,000 nm, about 300 nm to about 3,000 nm, about 300 nm to about 1,000 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, or about 200 nm to about 700 nm (including all values ​​and ranges therein).In various embodiments, the negatively charged particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therein).

[0012] In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 10% of the particles having a diameter of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, or about 0.3 μm to about 3 μm (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 10% of the particles having a diameter of about 100 nm to 10,000 nm, about 100 nm to 5,000 nm, about 100 nm to 3,000 nm, about 100 nm to 2,000 nm, about 300 nm to 5,000 nm, about 300 nm to 3,000 nm, about 300 nm to 1,000 nm, about 300 nm to 800 nm, about 400 nm to 800 nm, or about 200 nm to 700 nm (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therein).

[0013] In various embodiments, the negatively charged particles are PLGA particles having a zeta potential in the range of about -80 mV to about -30 mV and a diameter in the range of about 200 nm to about 2000 nm.

[0014] In various embodiments, the negatively charged particles are administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, transdermally, intraocularly, or orally.

[0015] In various embodiments, the negatively charged particles are administered at a concentration of about 0.05 mg / mL to about 50 mg / mL. In various embodiments, the negatively charged particles are administered at a concentration of about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL (including all values ​​and ranges therein). In various embodiments, the negatively charged particles are administered via intravenous infusion lasting about 1, 2, 3, 4, 5, 6, 7, or 8 hours (including all values ​​and ranges therein).

[0016] In various embodiments, the negatively charged particles are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year. In various embodiments, the negatively charged surface functionalized particles and / or cancer therapeutic agent are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks (including all values ​​and ranges therein).

[0017] In various embodiments, the negatively charged particles are administered in a treatment cycle. In various embodiments, a treatment cycle is comprised of 1, 2, 3, 4, 5, 6, or 7 doses administered every week. In various embodiments, the subject is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 treatment cycles of the negatively charged particles.

[0018] In various embodiments, the subject has a cancer selected from the group consisting of brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, and testicular cancer.

[0019] In various embodiments, administering the negatively charged particles to a subject in need thereof ameliorates one or more symptoms associated with a solid tumor or cancer. In various embodiments, the one or more symptoms are selected from the group consisting of tumor size, tumor burden, and tumor metastasis. In various embodiments, administering the negatively charged particles to a subject improves survival.

[0020] In various embodiments, the efficacy of the negatively charged particles in ameliorating one or more symptoms of cancer in a subject is determined from an assay of one or more biological samples from the subject. In various embodiments, the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and bone marrow biopsy.

[0021] In various embodiments, the negatively charged particles are administered to a subject in need thereof, alone or in combination with one or more additional cancer therapeutics. In various embodiments, the negatively charged particles are administered prior to, in combination with, or after administration of one or more additional cancer therapeutics. In various embodiments, the negatively charged particles are administered as a neoadjuvant prior to surgical resection of a solid tumor. In various embodiments, the negatively charged particles are administered as an adjuvant after surgical resection of a solid tumor.

[0022] In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent selected from the group consisting of growth inhibitors, DNA replication inhibitors, kinase inhibitors, signal transduction cascade inhibitors, angiogenesis inhibitors, metabolic inhibitors, amino acid synthesis inhibitors, selective inhibitors of oncogenic proteins, metastasis inhibitors, inhibitors of anti-apoptotic factors, apoptosis inducers, nucleoside signal transduction inhibitors, enzyme inhibitors, proteasome inhibitors, and DNA damaging agents.

[0023] In various embodiments, the cancer therapeutic comprises one or more biologic agents selected from the group consisting of cytokines, angiogenesis inhibitors, immune checkpoint modulators, enzymes, and monoclonal antibodies.

[0024] In various embodiments, the cytokine is selected from the group consisting of transforming growth factors, tumor necrosis factors, interferons, and interleukins. Exemplary cytokines include, but are not limited to, IFN-alpha, IFN-beta, IFN-gamma, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, members of the transforming growth factor beta superfamily including TGF-β1, TGF-β2, and TGF-β3, tumor necrosis factor alpha, granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF).

[0025] In various embodiments, the cancer therapeutic comprises an enzyme. In various embodiments, the cancer therapeutic comprises an enzyme that targets T cells, B cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte-derived cells, tumor-associated stroma, cancer stem cells, mesenchymal stem cells, extracellular matrix, and amino acids. In various embodiments, the cancer therapeutic comprises an enzyme selected from the group including asparaginase, kynurinase, L-arginine deiminase, L-methionine-y-lyase, one or more amino acid degrading enzymes, and one or more nucleoside degrading enzymes.

[0026] In various embodiments, the monoclonal antibody is a monospecific, bispecific, trispecific, or bispecific T-cell engaging (BiTE) antibody.

[0027] In various embodiments, the monoclonal antibody is selected from the group including alemtuzumab, bevacizumab, brentuximab, cetuximab, denosumab, ibritumomab, trastuzumab, panitumumab, pertuzumab, and rituximab. In various embodiments, the monoclonal antibody targets a receptor tyrosine kinase, EGFR, VEGF, VEGFR, PDGF, PDGFR, TGF-β, TGF-β-LAP, SIRP-α, CD47, CD39, CD73, or fibroblast activation protein (FAP). In various embodiments, the immune checkpoint modulator targets programmed cell death protein 1 (PD1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain-containing-3 (TIM-3), lymphocyte activation gene 3 (LAG-3), and / or TIGIT (T-cell immunoreceptor with Ig and ITIM domains). In various aspects, the immune checkpoint modulator is an antibody selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemiplimab, and durvalumab. In various embodiments, the cancer therapeutic comprises one or more cell-based therapies selected from the group consisting of adoptive cell transfer, tumor infiltrating leukocyte therapy, chimeric antigen receptor T cells (CAR-T), NK cell therapy, and stem cell therapy.

[0028] In various embodiments, the cell-based therapy is the adoptive transfer of autologous patient-derived cells. In various embodiments, the cell-based therapy is the adoptive transfer of allogeneic donor-derived cells. In various embodiments, the cell-based therapy is the transfer of universal donor-derived or induced pluripotent stem cell-derived cells that are not patient-specific and suitable for long-term storage. Such therapies are also referred to as "off-the-shelf therapies."

[0029] In various embodiments, the cancer therapeutic is a hormone therapy. In various embodiments, the cancer therapeutic comprises one or more antibody-drug conjugates. In various embodiments, the cancer therapeutic comprises one or more cancer vaccines. In various embodiments, the cancer vaccine is a protein, polypeptide, and / or nucleic acid vaccine.

[0030] In various embodiments, the cancer therapeutic is an immunotherapy selected from the group including oncolytic viruses, bacteria, oncolytic bacteria or other bacterial populations, tumor cell lysates, bacterial cell lysates, lipopolysaccharide (LPS), Bacillus Calmette-Guerin (BCG), microbiota modulators, and / or toll-like receptor (TLR) agonists. In various embodiments, the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13 agonist. In various embodiments, the TLR agonist is derived from a virus, bacteria, and / or synthetically produced. In various embodiments, the immunotherapy is a STING pathway modulator.

[0031] In various embodiments, the cancer therapeutic comprises a viral or bacterial vector. In various embodiments, the viral vector is selected from the group comprising adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, potato virus x, comovirus, or cucumber mosaic virus. In various embodiments, the virus is an oncolytic virus. In various embodiments, the virus is a chimeric virus, a synthetic virus, a mosaic virus, or a pseudotype virus.

[0032] In various embodiments, the subject is a mammal, hi various embodiments, the subject is a human.

[0033] In various embodiments, the negatively charged particles are formulated in a composition that includes a pharma- ceutically acceptable excipient.

[0034] It is understood that each feature or embodiment or combination described herein is a non-limiting, illustrative example of any of the aspects of the invention and is therefore meant to be combinable with any other feature or embodiment or combination described herein. For example, when a feature is described with words such as "various embodiments," "one embodiment," "some embodiments," "certain embodiments," "further embodiments," "particular exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, and it is not necessary to list all possible combinations. Such features or combinations of features apply to any of the aspects of the present disclosure. When examples of values ​​falling within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, and any and all numerical values ​​between such endpoints are contemplated, and any and all combinations of upper and lower limits are envisioned. [Brief description of the drawings]

[0035] [Figure 1A-C] Figure 1 shows the efficacy of negatively charged particles (designated ONP-302) when administered at different dose levels and different dosing frequencies. MC38 tumor-bearing mice were administered the indicated therapeutic agents and the indicated dosing frequencies starting when tumor volumes were approximately 50 mm3. (A and C) Effect of the indicated therapeutic agents on tumor growth. (B) Effect of the indicated therapeutic agents on survival. The significance of tumor growth kinetics was analyzed using two-way repeated measures ANOVA and Bonferroni post-hoc analysis (*p=<0.05, **p<0.01, ****p<0.0001). [Figure 2A-B]Efficacy of ONP-302 when administered at different dose levels and different dosing frequencies in the B16-F10 tumor model. B16 tumor-bearing mice were administered the indicated therapeutics when tumor volumes were approximately 50 mm3. The effect of the indicated therapeutics on (A) tumor growth and (B) survival is shown. The significance of tumor growth kinetics was analyzed using two-way repeated measures ANOVA and Bonferroni post-hoc analysis (*p=<0.05, **p<0.01, ****p<0.0001). [Diagram 3] FIG. 1 shows the efficacy of ONP-302 in inhibiting primary tumor growth in the presence and absence of a functional adaptive immune system in an orthotopic 4T1 tumor model. 4T1 primary tumors were implanted into the mammary fat pad of female WT or Rag1- / - BALB / c mice. Animals were administered ONP-302 or saline (control) by intravenous tail vein injection one day after tumor inoculation once every three days. Tumor volumes are shown for mice in the indicated groups (N=10 per group). The significance of differences in tumor volumes was determined using multiple unpaired t-tests (*p<0.05). [Figure 4A-D] Figure 1 shows the efficacy of ONP-302 in inhibiting primary tumor metastasis to the lung in the presence and absence of a functional adaptive immune system in an orthotopic 4T1 tumor model. 4T1 primary tumors were implanted into the mammary fat pad of female WT or Rag1- / - BALB / c mice. On day 1 after tumor inoculation, animals were administered ONP-302 or saline (control) by intravenous tail vein injection once every 3 days. Mice were sacrificed on day 21 and lung metastases were assessed by assaying bioluminescence signals using IVIS®. Incidence of lung metastases in WT tumor-bearing mice treated with (A) saline or (B) ONP-302, and Rag1- / - tumor-bearing mice treated with (C) saline (control) or (D) ONP-302 is shown. [Figure 5A-B]Figure 1 shows the effect of ONP-302 on gene expression changes by scRNA-seq from lung tissue samples of orthotopic 4T1 tumor-bearing mice. Luciferase-expressing 4T1 tumor cells were orthotopically implanted in the mammary fat pad of 6-8 week-old female BALB / c mice. Mice were treated with ONP-302 or saline once every 3 days starting on day 1 after tumor implantation. Mice were sacrificed on day 21 after tumor implantation and lungs were harvested and processed for scRNA-seq. (A) Number of different cell types in the lungs of ONP-302- and saline-treated mice. (B) Phenotypic changes in the indicated cell populations as determined by gene set enrichment analysis (GSEA). [Figure 6A-C] Figure 1 shows the effect of ONP-302 on gene expression changes by scRNA-seq from naive mouse spleens. Naive female C57BL / 6 mice (n=3 per group) were treated with saline or ONP-302 (1.0 mg / dose in 200 mL saline) by intravenous injection once for 3 consecutive days (days 0, 1, and 2) or every 3 days (days 0, 3, and 6). 24 hours after the last injection, spleens were harvested to assess the transcriptome via single cell RNAseq. (A) The distribution of cells present within the spleen in response to each of the saline and ONP-302 dosing schemes is compared. (B) ONP-302-induced changes in gene expression by macrophages, monocytes, and neutrophils were compared. Red genes have log fold change >1 and p-value <0.05. (C) Signaling pathways induced by ONP-302 treatment compared to saline treatment alone as measured by gene set enrichment analysis (GSEA), NES=normalized enrichment score, *p<0.1, **p<0.01, ***p<0.001, #pathway not enriched in this sample. [Figure 7A-F]Figure 1 shows the effect of ONP-302 treatment on immune cells in the spleens of naive mice. Naive female C57BL / 6 mice (n=3 per group) were treated by intravenous injection with saline or ONP-302 (1.0 mg / dose in 200 mL saline) once for three consecutive days (days 0, 1, and 2) or every third day (days 0, 3, and 6). 24 hours after the last injection, spleens were harvested to assess the immune cell populations present. A gating schematic detailing how cells were identified is shown (A). Total cell numbers (B) and phenotypes of CD4+ T cells, CD8+ T cells, NK cells (C), monocytes and macrophages (D), and other myeloid cells (E) are shown as heat maps. The frequency of specific populations expressing cell markers of activation following treatment is shown in (F). Data are presented as mean percentage of cells + / - SEM. One representative experiment out of two is presented. Asterisks (*, **, ***) indicate statistically significant differences compared to saline-treated mice at p<0.05, <0.01, <0.001, and <0.0001, respectively. [Figure 8A-B] Figure 1 shows the effect of ONP-302 treatment on cytokine / chemokine expression in RAW macrophages and bone marrow derived myeloid cells. RAW cells with or lacking the cGAS gene (cGAS- / -) were treated with ONP-302 for 24 hours in the presence or absence of STING inhibitor (RU.521) or caspase inhibitor (ZVAD-FMK), and supernatants were collected to measure chemokine and cytokine levels (A). BMDMs from either WT C57BL / 6 mice or STING- / - mice were treated with ONP-302 for 24 hours, and supernatants were collected to measure chemokine and cytokine levels (B). Each dot represents a unique well of cells tested, and bars represent the mean + SEM. Asterisks (*, **, ***, ****) indicate statistically significant differences of p<0.05, <0.01, <0.001, and <0.0001, respectively, compared to untreated cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The present disclosure provides methods of administering negatively charged particles that do not contain an attached drug or bioactive agent, alone or in combination with one or more additional therapeutic agents, for the treatment of a solid tumor in a subject in need of such treatment.

[0037] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety, to the extent that it does not contradict this disclosure.

[0038] It is noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0039] "Particle" as used herein refers to any non-tissue derived composition and may be a spherical or globular entity, bead, or liposome. The terms "particle", "immunomodified particle", and "bead" may be used interchangeably depending on the context. The terms "negatively charged particle", "surface functionalized particle", and "negatively charged surface functionalized particle" may be used interchangeably depending on the context. Furthermore, the term "particle" may be used to encompass beads and spheres.

[0040] "Negatively charged particles", as used herein, refer to particles having a net surface charge of less than zero. In embodiments, the negatively charged particles are negatively charged surface-functionalized particles. "Surface-functionalized", as used herein, refers to particles having one or more functional groups on their surface. In some embodiments, surface functionalization occurs by introducing one or more functional groups to the surface of the particle. In embodiments, surface functionalization can be achieved by carboxylation (i.e., the addition of one or more carboxyl groups to the particle surface) or the addition of other chemical groups (e.g., other chemical groups that impart a negative surface charge).

[0041] "No therapeutic agent" or "no attached drug" refers to negatively charged particles that do not contain other therapeutic agents or drugs. In other words, no therapeutically active drugs, peptides, antigenic moieties, or bioactive agents are bound to, embedded in, or otherwise associated with the negatively charged particles described herein. This phrase is intended to distinguish negatively charged particles bound to a therapeutic agent from negatively charged particles described herein that are themselves therapeutic agents.

[0042] Zeta potential is the charge generated at the interface between a solid surface and its liquid medium. "Negative zeta potential" refers to a particle having a zeta potential at the particle surface, expressed in millivolts (mV), measured by devices known in the art for calculating zeta potential, such as a NanoBrook ZetaPlus zeta potential analyzer or a Malvern Zetasizer.

[0043] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particle that has been modified or surface functionalized to add one or more additional carboxyl groups to the particle surface. Carboxylation of the particle can be achieved using any compound that adds additional carboxyl groups, including but not limited to poly(ethylene-maleic anhydride) (PEMA). Carboxylation can also be achieved by forming the particle using a polymer that has native carboxyl groups (e.g., PLGA), where the manufacturing process results in additional carboxyl groups being located on the surface of the particle.

[0044] "Biodegradable" as used herein refers to particles that contain polymers that can be degraded, for example, as a result of functional groups reacting with water in solution. The term "degradation" as used herein refers to becoming soluble, either by reducing molecular weight or by converting hydrophobic groups to hydrophilic groups. Biodegradable particles do not persist in the body for long periods of time, and the time to completely degrade can be controlled. Biocompatible biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonate, amide, amino acid, orthoester, acetal, cyanoacrylate, and degradable urethane, as well as copolymers thereof with linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy or dicarboxylic acids. Additionally, biologically important amino acids having reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials to provide reactive groups for conjugation to antigenic peptides and proteins or binding moieties. Biodegradable materials suitable for the present invention include PLA, PGA, polypropylene sulfide, and PLGA polymers.

[0045] The term "subject" as used herein refers to a human or non-human animal, including a mammal or primate, to which a particle described herein is administered. Subjects can include animals such as dogs, cats, rats, mice, rabbits, horses, pigs, sheep, cows, as well as humans and other primates.

[0046] The term "therapeutic agent" refers to a moiety that, when administered in a therapeutically effective amount, can ameliorate or alleviate one or more symptoms or signs of the disease or disorder being treated. Non-limiting examples of therapeutic agents include peptide, protein, or other cancer therapeutics, including small molecule therapeutics.

[0047] The term "therapeutically effective amount" is used herein to indicate an amount of a target-specific composition of the present disclosure effective to ameliorate or alleviate one or more symptoms or signs of the disease or disorder being treated.

[0048] The terms "treat," "treated," "treating," and "treatment," as used in reference to the methods herein, refer to eliminating, reducing, inhibiting, or ameliorating, temporarily or permanently, partially or completely, one or more clinical symptoms, manifestations, or progression of an event, disease, or condition. Such treatment need not be absolute to be useful.

[0049] Negatively charged particles The present disclosure provides the use of negatively charged particles in the therapeutic methods described herein.In some embodiments, the negatively charged particles have a negative zeta potential.In some embodiments, the negative charge enhances the phagocyte / monocyte uptake of particles from circulation, for example, via receptor-mediated phagocytosis, including but not limited to, interaction with scavenger receptors such as MARCO.

[0050] Negatively charged particles can be formed from a wide range of materials. The particles are preferably composed of materials suitable for biological applications (e.g., pharma- ceutically acceptable materials). For example, the particles can be composed of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. In various embodiments, the particles can be composed of polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or bridged alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or bridged alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. Additionally, the particles may be or consist of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). Particles containing a mixture of ester and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) may also be used. For example, the particles may include materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), polylactic acid-glycolic acid copolymers (PLGA or PLG, the terms are interchangeable), polylactic acid-sebacic acid copolymers (PLSA), polyglycolic acid-sebacic acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable urethanes, and their copolymers with linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy or dicarboxylic acids.Additionally, biologically important amino acids having reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials to provide reactive groups for conjugation to antigenic peptides and proteins or binding moieties.

[0051] In an embodiment, the negatively charged particles comprise one or more biodegradable polymers or materials. Biodegradable materials suitable for the present invention include PLA, PGA, polypropylene sulfide, and PLGA polymers.

[0052] In various embodiments, it is contemplated that the particles comprise polyglycolic acid polymers (PGA), polylactic acid (PLA), polystyrene, copolymers of PLG and PLA (polylactic-co-glycolic acid, PLGA), liposomes, PEG, cyclodextran, or combinations thereof.

[0053] In various embodiments, the negatively charged particles are copolymers having a molar ratio of polyglycolic acid:polylactic acid of about 100:0 to about 0:100, about 99:1 to about 1:99, about 80:20 to about 20:80, or about 50:50 (including any value or range therebetween). In various embodiments, the negatively charged particles are copolymers having a molar ratio of polyglycolic acid:polyglycolic acid of about 50:50 or about 80:20 to about 99:1, or about 50:50 or about 80:20 to about 99:1. In some embodiments, the negatively charged surface functionalized particles are polylactic acid-glycolic acid copolymer particles. In various embodiments, the negatively charged particles include 50:50 polylactic acid:polyglycolic acid. In various embodiments, the negative particles comprise polylactic acid:polyglycolic acid in a ratio of about 99:1 to about 1:99, for example, about 99:1, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, and about 1:99 (including all values ​​and ranges therebetween).

[0054] In some embodiments, the zeta potential of the negatively charged particles is about -100 mV to about -1 mV. In some embodiments, the zeta potential of the negatively charged particles is about -100 mV to about -40 mV, about -80 mV to about -30 mV, about -75 mV to about -40 mV, about -70 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -40 mV (including any value or range therebetween). In various embodiments, the zeta potential is about -30 mV, about -35 mV, about -40 mV, about -45 mV, about -50 mV, about -55 mV, about -60 mV, about -65 mV, about -70 mV, about -75 mV, about -80 mV, about -85 mV, about -90 mV, about -95 mV, or about -100 mV (including all values ​​and subranges therebetween).

[0055] In some embodiments, the negatively charged particles have an average diameter of about 0.1 μm to about 10 μm. In some embodiments, the negatively charged particles have an average diameter of about 0.2 μm to about 2 μm. In some embodiments, the negatively charged particles have an average diameter of about 0.3 μm to about 5 μm. In some embodiments, the negatively charged particles have an average diameter of about 0.5 μm to about 3 μm. In some embodiments, the negatively charged particles have an average diameter of about 0.5 μm to about 1 μm. In some embodiments, the negatively charged particles have an average diameter of about 100 nm to 1500 nm, about 200 nm to 2000 nm, about 100 nm to 10000 nm, about 300 nm to 1000 nm, about 400 nm to 800 nm, or about 200 nm to 700 nm (including all values ​​and subranges therebetween).

[0056] In various embodiments, the negatively charged particles have a homogeneous size distribution, in which at least 90% of the negatively charged particles have an average diameter in the range of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, or about 0.3 μm to about 3 μm (including any value or range therebetween). In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 90% of the negatively charged particles having an average diameter of about 100 nm to about 10,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 3,000 nm, about 100 nm to about 2,000 nm, about 300 nm to about 5,000 nm, about 300 nm to about 3,000 nm, about 300 nm to about 1,000 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, or about 200 nm to about 700 nm (including any value or range therebetween). In various embodiments, the negatively charged particles have an average diameter of about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, about 1400 nm, about 1500 nm, or about 2000 nm, or any value or range therebetween. In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 50% of the negatively charged particles having an average diameter in the ranges of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, and about 0.3 μm to about 3 μm (including any value or range therebetween). In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 50% of the negatively charged particles having a diameter of about 100 nm to about 10,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 3,000 nm, about 100 nm to about 2,000 nm, about 300 nm to about 5,000 nm, about 300 nm to about 3,000 nm, about 300 nm to about 1,000 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, or about 200 nm to about 700 nm (including all values ​​and ranges therebetween).In various embodiments, the negatively charged particles have an average diameter of about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, about 1400 nm, about 1500 nm, or about 2000 nm (including any value or range therebetween). In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 10% of the negatively charged particles having an average diameter in the ranges of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, and about 0.3 μm to about 3 μm (including any value or range therebetween). In various embodiments, the negatively charged particles have a homogenous size distribution, with at least 10% of the negatively charged particles having an average diameter of about 100 nm to about 10,000 nm, about 100 nm to about 5,000 nm, about 100 nm to about 3,000 nm, about 100 nm to about 2,000 nm, about 300 nm to about 5,000 nm, about 300 nm to about 3,000 nm, about 300 nm to about 1,000 nm, about 300 nm to about 800 nm, about 400 nm to about 800 nm, or about 200 to about 700 nm (including any value or range therebetween). In various embodiments, the negatively charged particles have an average diameter of about 50 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, about 1400 nm, about 1500 nm, or about 2000 nm (including any value or range therebetween).

[0057] In embodiments, the negatively charged particles are about 1000 nm, about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm , about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, about 600 nm, about 610 nm, about 620 nm, about 630 nm, about 640 nm, about 650 nm, about 660 nm, about 670 nm, about 680 nm, about 690 nm, or about 700 nm, including any value or range therebetween. In embodiments, the negatively charged particles have a D50 of about 400 nm to about 600 nm, including about 400 nm, about 420 nm, about 440 nm, about 460 nm, about 480 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, about 550 nm, about 560 nm, about 570 nm, about 580 nm, about 590 nm, or about 600 nm (including any value or range therebetween). In an embodiment, the negatively charged particles have a D10 of about 300 nm to about 550 nm, including about 300 nm, about 320 nm, about 340 nm, about 360 nm, about 380 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, about 500 nm, about 510 nm, about 520 nm, about 530 nm, about 540 nm, or about 550 nm (including any value or range therebetween). In an embodiment, the negatively charged particles have a D90 of about 600 nm to about 700 nm. In an embodiment, the negatively charged particles have a D50 of about 550 nm to about 600 nm. In an embodiment, the negatively charged particles have a D10 of about 500 nm to about 550 nm.

[0058] For administration of the negatively charged particles described herein to humans or other mammals, the particles may be formulated in a sterile composition containing one or more sterile pharma- ceutically acceptable carriers. The phrase "pharmacologically or pharmacologically acceptable" refers to molecular entities and compositions that do not cause allergic or other adverse reactions when administered using routes well known in the art, as described below. "Pharmaceutically acceptable carriers" include clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc.

[0059] In the present specification, the pharmaceutical composition of the present disclosure containing negatively charged surface-functionalized particles may contain a sterile pharma- ceutically acceptable carrier or additive depending on the route of administration. Examples of such carriers or additives include water, pharma- ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacryl, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharma-ceutically acceptable surfactants, and the like. The additives used are appropriately selected from the above or combinations thereof depending on the dosage form of the present invention, but are not limited thereto. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles may contain various additives, preservatives, or fluid, nutrient or electrolyte replenishers. Various aqueous carriers, such as sterile phosphate buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, etc., are suitable, and may include other proteins to enhance stability, such as albumin, lipoproteins, globulins, etc., that have been mildly chemically modified.

[0060] Various aqueous carriers, e.g., sterile phosphate buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, may contain other proteins to enhance stability, such as albumin, lipoproteins, globulins, etc., that have been mildly chemically modified, etc.

[0061] Therapeutic formulations of the inhibitor are prepared for storage by mixing the inhibitor having the desired purity, in the form of a lyophilized formulation or aqueous solution, with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0062] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0063] Aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethylenoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, such as ethyl, or n-propyl, p-hydroxybenzoate.

[0064] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) humectants, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0065] It is contemplated that the particles may further comprise a surfactant and / or stabilizer. The surfactant and / or stabilizer may be anionic, cationic, or nonionic. Poloxamer and polaxamine family surfactants are commonly used in particle synthesis. Surfactants and / or stabilizers that may be used include, but are not limited to, polyethylene glycol (PEG), Tween-80, gelatin, dextran, Pluronic L-63, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methylcellulose, lecithin, dimethylaminobenzaldehyde (DMAB), and poly(ethylene-alt-maleic anhydride) (PEMA). Further included are biodegradable and biocompatible surfactants, including, but not limited to, vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers), and sulfate polymers. In certain embodiments, two surfactants are used. In certain embodiments, two stabilizers are used. In certain embodiments, a combination of two or more surfactants and stabilizers is used. For example, when the particles are produced by a double emulsion method, the two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion. For example, the stabilizer may be a compound that stabilizes the primary emulsion and / or secondary emulsion described herein by providing a physical or energy barrier between adjacent nanoparticle droplets in the emulsion, thereby reducing the probability of combining and forming larger nanoparticle droplets.

[0066] Emulsions occur in many processing forms. Oil-water (single) or water-oil-water (double) emulsions are methods that can produce negatively charged surface-functionalized PLGA particles. In summary, PLGA is dissolved in an organic phase (oil) that is emulsified with a surfactant or stabilizer (water). High-intensity homogenization (e.g., sonication bursts) promotes the formation of small polymer droplets. The resulting emulsion is added to a larger aqueous phase and stirred for several hours to allow the solvent to evaporate. The hardened nanoparticles are collected and washed by centrifugation. In certain embodiments, hardened emulsion particles can be obtained by evaporation of the oil phase.

[0067] "Water-in-oil-in-water" (W / O / W) emulsions are an example of double emulsions, a dispersion of small water droplets within larger oil droplets that are themselves dispersed in a continuous aqueous phase. Due to their compartmentalized internal structure, double emulsions can offer advantages over simple oil-in-water emulsions for encapsulation, such as the ability to carry both polar and non-polar cargo (pharmaceutical / biological agents, e.g., proteins), as well as improved control over the release of therapeutic molecules. The preparation of double emulsions typically requires a surfactant or mixtures thereof for stability. Surfactants stabilize the droplets exposed to extreme flows, leading to straightforward mass production of robust double nanoemulsions that are compatible with nanostructured encapsulation applications in various industries. In one example, the double emulsion process involves generating a primary emulsion by mixing an aqueous solution containing a polymer, resulting in a water-in-oil primary emulsion. The primary emulsion is then mixed with a solution containing one or more surfactants to form an oil-in-water secondary emulsion. The secondary emulsion is then hardened by evaporation to remove the solvent, and the hardened polymeric nanoparticles encapsulate the pharmaceutical / biological agent.

[0068] "Homogenization" as used herein refers to the operation of using a class of processing equipment called homogenizers, which are adapted to reduce the size of droplets in liquid-liquid dispersions. Factors that affect particle or droplet size include, but are not limited to, emulsifier type, emulsifier concentration, solution conditions, and mechanical devices (homogenization power, pressure, rotation speed, time). Non-limiting examples of homogenizers include high speed blenders, high pressure homogenizers, colloid mills, high shear dispersers, ultrasonic disintegrators, membrane homogenizers, and sonicators. Mechanical homogenizers, manual homogenizers, ultrasonicators, mixer mills, vortexers, etc. may be utilized for mechanical and physical disruption within the scope of the present disclosure.

[0069] How to use Provided herein is a method of treating a solid tumor in a subject, comprising administering negatively charged particles to the subject, wherein the negatively charged particles are administered at a dose of about 0.1 mg / kg to about 15 mg / kg. Also provided herein is a method of inducing an anti-tumor or immune response in a subject suffering from a solid tumor, comprising administering negatively charged particles to the subject, wherein the negatively charged particles are administered at a dose of about 0.1 mg / kg to about 15 mg / kg. In various embodiments, the negatively charged particles are administered at a dose of about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to 8 mg / kg, about 1.5 mg / kg to 15 mg / kg, about 2 mg / kg to 15 mg / kg, about 2 mg / kg to 10 mg / kg, about 3 mg / kg to 10 mg / kg, about 4 mg / kg to 15 mg / kg, about 5 mg / kg to 15 mg / kg, or about 2 mg / kg to 8 mg / kg. In various embodiments, the negatively charged particles are administered at a dose of about 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, 12 mg / kg, or 15 mg / kg. In various embodiments, the negatively charged particles are administered in a dose of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 875 mg, 900 mg, 950 mg, 975 mg, or 1000 mg.

[0070] In various embodiments, the negatively charged particles are administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

[0071] In various embodiments, the negatively charged particles are administered at a concentration of about 0.05 mg / mL to about 50 mg / mL. In various embodiments, the negatively charged particles are administered at a concentration of about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL. In various embodiments, the negatively charged particles are administered via intravenous infusion lasting about 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0072] In various embodiments, the negatively charged particles are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year. In various embodiments, the negatively charged surface functionalized particles and / or cancer therapeutic agent are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks.

[0073] In various embodiments, the negatively charged particles are administered in a treatment cycle. In various embodiments, a treatment cycle consists of 1, 2, 3, 4, 5, 6, or 7 doses administered every week. In various embodiments, the subject is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 treatment cycles of negatively charged particles.

[0074] Exemplary diseases, conditions, or disorders that may be treated using the methods herein include esophageal cancer, pancreatic cancer, metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas, bladder cancer, gastric cancer, fibrous carcinoma, glioma, malignant glioma, diffuse intrinsic pontine glioma, recurrent pediatric brain tumor renal cell carcinoma, clear cell metastatic renal cell carcinoma, kidney cancer, prostate cancer, metastatic castration-resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, melanoma, malignant melanoma, recurrent cutaneous melanoma, melanoma brain metastasis, stage IIIA cutaneous melanoma, stage IIIB cutaneous melanoma, stage IV ... Stage IIIB cutaneous melanoma, Stage IIIC cutaneous melanoma, Stage IV cutaneous melanoma, Head and neck melanoma, Lung cancer, Non-small cell lung cancer (NSCLC), Squamous non-small cell lung cancer, Breast cancer, Recurrent metastatic breast cancer, Hepatocellular carcinoma, Hodgkin lymphoma, Follicular lymphoma, Non-Hodgkin lymphoma, Progressive B-cell NHL, HL including diffuse large B-cell lymphoma (DLBCL), Multiple myeloma, Chronic myeloid leukemia, Remission phase adult acute myeloid leukemia, Adult acute myeloid leukemia with Inv(16)(p13.1 q22), CBFB-MYH11, Adult acute myeloid leukemia with t(16;16)(p13.1;q22), CBFB-MYH11, adult acute myeloid leukemia with t(8;21)(q22;q22), RIJNX1-RIJNX1T1, adult acute myeloid leukemia with t(9;11)(p22;q23), MLLT3-MLL, adult acute promyelocytic leukemia with t(15;17)(q22;q12), PML-RARA, alkylating agent-associated acute myeloid leukemia, chronic lymphocytic leukemia, Richter's syndrome, Waldenström's macroglobulinemia, adult glioblastoma, adult glioma, recurrent glioblastoma, recurrent childhood rhabdomyosarcoma, recurrent Ewing's sarcoma / peripheral primitive neuroectodermal tumor, recurrent neuroblastoma, recurrent osteosarcoma, colorectal cancer, MSI-positive colorectal cancer, These include MSI negative colorectal cancer, nasopharyngeal nonkeratinizing carcinoma, recurrent nasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma, cervical adenosquamous carcinoma, cervical squamous cell carcinoma, recurrent cervical cancer, stage IVA cervical cancer, stage IVB cervical cancer, anal canal squamous cell carcinoma, metastatic anal canal cancer, recurrent anal canal cancer, recurrent head and neck cancer, carcinoma, head and neck squamous cell carcinoma (HNSCC), ovarian cancer, colon cancer, gastric cancer, advanced GI cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, bone tumor, soft tissue sarcoma, osteosarcoma, thymic carcinoma, urothelial cell carcinoma, recurrent Merkel cell carcinoma, stage III Merkel cell carcinoma, stage IV Merkel cell carcinoma, myelodysplastic syndromes and recurrent fungoides and Sézary syndrome. In various embodiments, the cancer is selected from brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, and mesothelioma.

[0075] In various embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject a negatively charged particle, alone or in combination with a cancer therapeutic, wherein the subject has experienced disease progression after all prior standard lines of therapy or is unable to tolerate standard therapy. Standard therapy may include multiple lines of therapy, including surgery, cytotoxic agents, immunotherapy, and cell therapy. In various embodiments, the subject has received 1, 2, 3, 4, 5, or more prior lines of therapy and has experienced disease progression.

[0076] In various embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject negatively charged particles alone or in combination with a cancer therapeutic, the subject experiencing tumor recurrence after surgical resection of a primary tumor. In various embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject negatively charged particles alone or in combination with a cancer therapeutic, the subject having a tumor that cannot be surgically removed. In various embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject negatively charged particles alone or in combination with a cancer therapeutic, the subject having no available treatment options.

[0077] Several tools for the diagnosis of cancer in a subject have been described and are incorporated herein by reference. Examples of tools for detecting and diagnosing the presence of cancer in a subject include computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound, X-ray imaging, digital mammography, and sentinel node mapping. In various embodiments, the method for detecting and diagnosing cancer in a subject includes obtaining and characterizing one or more biological samples from the subject. In various embodiments, the biological sample is blood, tumor biopsy, tissue biopsy, cerebrospinal fluid, urine, stool, oral swab, nasal swab, lavage, and bone marrow biopsy.

[0078] Examples of approved diagnostics include FOUNDATIONONE® CDX, FOUNDATIONONE® LIQUID, FOUNDATIONONE® HEME, BRACAnalysis CDx, therascreen EGFR RGQ PCR kit, cobase EGFR Mutation Test v2, PD-L1 IHC 22C3 pharmDx, Abbott Real Time IDH1, MRDx BCR-AB L test, VENTANA ALK(D5F3)CDx Assay, Abbott RealTime IDH2, Praxis Extended RAS Panel, Oncomine Dx Target Test, LeukoStrat CDx FLT3 Mutation Assay, FoundationFocus CDxBRCA Assay, Vysis CLL FISH Probe Kit, KIT D816V Mutation Detection, PDGFRB FISH, cobas KRAS Mutation Test, therascreen KRAS RGQ PCR Kit, FerriScan, Dako c-KIT pharmDx, and INFORM Her-2 / neu, PathVysion HER-2 DNA Probe Kit, SPOT-LIGHT HER2 CISH Kit, Bond Oracle HER2 IHC System, HER2 CISH pharmDx Kit, INFORM HER2 DUAL ISH DNA Probe Cocktail, HercepTest, HER2 FISH pharmDx Kit, THXID BRAF Kit, Vysis ALK Break Apart FISH Probe Kit, cobas 4800 BRAF V600 Mutation Test, VENTANA PD-L1(SP142) Assay, therascreen FGFR RGQ RT-PCR Kit, and therascreen PIK3CA RGQ PCR Kit.

[0079] In various embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject negatively charged particles, alone or in combination with one or more cancer therapeutic agents.

[0080] In various embodiments, administration of the negatively charged particles reduces tumor size or tumor burden by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30% or more. In various embodiments, administration reduces tumor size or tumor burden by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% (including all values ​​and ranges between these values). Several criteria and definitions published in the literature can be used to determine the effect of one or more treatments on tumors in subjects suffering from cancer. Based on these criteria, tumors are defined as "responsive", "stable", or "progressing" if they improve, stay the same, or get worse during the course of treatment, respectively. Examples of the most commonly used criteria published in the literature include Remission Assessment Criteria in Solid Tumors (RECIST), modified Remission Assessment Criteria in Solid Tumors (mRECIST), PET Remission Criteria in Solid Tumors (PERCIST), Choi criteria, Lugano remission criteria, European Association for the Study of the Liver (EASL) criteria, Remission Assessment Criteria in Liver Cancer (RECICL), and WHO tumor remission criteria.

[0081] In various embodiments, administration of negatively charged particles in subjects suffering from cancer demonstrates therapeutic efficacy as defined by RECIST criteria.In various embodiments, therapeutic efficacy is calculated as Objective Response Rate (ORR), which is equal to the sum of complete responses (CR) and partial responses (PR) observed for each dose level, divided by the total number of subjects (N) dosed at that level.In various embodiments, tumor response criteria according to RECIST are defined as follows, based on the evaluation of target lesions: Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must be reduced to less than 1 cm in short axis. Partial Response (PR): At least a 30% reduction in the sum of the diameters of the target lesions compared to the baseline sum diameter. Progressive Disease (PD): The sum of the diameters of the target lesions increases by at least 20%, taking into account the smallest sum in the study (this includes the smallest baseline sum in the study) as a reference. In addition to the relative increase of 20%, the sum must also show an absolute increase of at least 0.5 cm. The appearance of one or more new lesions is also considered as progression. · Stable Disease (SD): Neither sufficient shrinkage to qualify as PR nor sufficient increase to qualify as PD, considering the smallest total diameter in the study as the criterion.

[0082] In various embodiments, tumor response criteria by RECIST are defined based on evaluation of non-target lesions as follows: Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker levels. All lymph nodes must be non-pathological in size (<1 cm in short axis). Non-complete remission / non-progressive disease: persistence of one or more non-target lesions and / or maintenance of tumor marker levels above normal limits. Progressive disease (PD): Definite progression of existing non-target lesions.

[0083] In various embodiments, a measurable lesion is defined as a lesion that can be accurately measured in at least one dimension (the longest diameter in the plane of measurement is recorded) with a minimum size of 1 cm by CT scan (CT scan slice thickness ≤ 0.5 cm), or a 1 cm caliper measurement by clinical examination (lesions that cannot be accurately measured with a caliper should be recorded as not measurable), or a minimum size of 2 cm by chest x-ray.

[0084] In various embodiments, non-measurable lesions are defined as all other lesions, including small lesions (pathological lymph nodes less than 1.0 cm in longest diameter or 10 to less than 1.5 cm in short axis) as well as truly non-measurable lesions. Lesions considered truly non-measurable include leptomeningeal disease, ascites, pleural or pericardial effusions, inflammatory breast disease, lymphangitic involvement of the skin or lungs, and abdominal masses / organomegaly identified by physical exam that are not measurable by reproducible imaging techniques.

[0085] In various embodiments, administration of the negatively charged particles improves survival. In various embodiments, administration of the negatively charged particles improves progression-free survival and / or overall survival. In various embodiments, administration of the negatively charged particles improves survival by 0.5 to 12 months. In various embodiments, administration of the negatively charged particles improves survival by 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (including all values ​​and ranges therebetween). In various embodiments, administration of the negatively charged particles improves survival by 1 to 100 years. In various embodiments, administration of the negatively charged particles improves survival by 1 to 50 years. In various embodiments, administration of the negatively charged particles improves survival by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 years (including all values ​​and ranges between these values).

[0086] In various embodiments, administration of negatively charged particles reduces the number of circulating tumor cells (CTCs) in the blood. In various embodiments, administration of negatively charged particles reduces the number of CTCs by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30% or more. In various embodiments, administration reduces the number of CTCs by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% (including all values ​​and ranges between these values). In various embodiments, administration of negatively charged particles reduces the number of CTC clusters. In various embodiments, administration of negatively charged particles reduces the number of CTC clusters in blood by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or more. In various embodiments, administration reduces the number of CTC clusters by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, including all values ​​and ranges between these values. In various embodiments, administration of negatively charged particles reduces the number of CTC-leukocyte clusters. In various embodiments, administration of negatively charged particles reduces the number of CTC-leukocyte clusters in blood by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, or more. In various embodiments, administration reduces the number of CTC-leukocyte clusters by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% (including all values ​​and ranges between these values). In various embodiments, the level of CTCs and / or CTC-leukocyte clusters in the blood is reduced to 20 or less, 15 or less, 10 or less, 5 or less, 1 or less, or 0 per 7.5 mL of blood (including all values ​​and ranges between these values). In various embodiments, the leukocytes in the CTC-leukocyte clusters are bone marrow-derived cells. In various embodiments, the bone marrow-derived cells are monocytes, macrophages, neutrophils, or dendritic cells. In various embodiments, the number of CTCs and CTC-leukocyte clusters in the blood is enumerated per mL of blood.In various embodiments, the number of CTCs and CTC-leukocyte clusters in blood is determined using commercially available size-based and / or tumor marker-based microfluidic capture devices.

[0087] In various embodiments, administration of the negatively charged particles to a subject in need thereof induces an anti-tumor immune response. In various embodiments, the anti-tumor immune response is a T cell response, a B cell response, a NK cell response, a NKT cell response, and / or a myeloid cell response. In various embodiments, the myeloid cells are selected from the group consisting of monocytes, macrophages, dendritic cells, and neutrophils. In various embodiments, the anti-tumor response is a cytokine and / or chemokine response. In various embodiments, the induction of an anti-tumor response is assayed from one or more biological samples obtained from the subject before (baseline), during, and / or after completion of treatment. In various embodiments, the biological sample is selected from the group consisting of whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, and / or bone marrow biopsy.

[0088] In various embodiments, administration of the negatively charged particles to a subject in need thereof increases the level of activated anti-tumor immune cells. In various embodiments, the level of activated anti-tumor immune cells is increased by 5-100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to one or more blood and / or tumor samples taken from the subject prior to treatment. In various embodiments, the activated pro-inflammatory cells are dendritic cells (DCs), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and iNK cells. In various embodiments, the frequency of pro-inflammatory immune cells is increased to 10-50% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% (including all values ​​and ranges therebetween)) of all leukocytes analyzed from one or more blood samples taken from the subject. In various embodiments, the activated pro-inflammatory immune cells are identified by assaying cell surface protein expression.

[0089] In various embodiments, administration of the negatively charged particles to a subject in need thereof reduces the level of tumor-promoting cells. In various embodiments, the level of tumor-promoting immune cells is reduced by 5-100% (e.g., reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to one or more blood and / or tumor samples taken from the subject prior to treatment. In various embodiments, the levels of immunosuppressive cells are reduced by about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween)) as compared to one or more blood samples taken from the subject prior to treatment. In various embodiments, the immunosuppressive cells are identified by assays of cell surface protein expression. In various embodiments, the tumor promoting cells are myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs), neutrophils, T reg Cells, and B reg In various embodiments, the MDSC is a monocytic MDSC (M-MDSC) and a polymorphonuclear MDSC (PMN-MDSC). In various embodiments, the TAM is an M2 TAM. In various embodiments, the immunosuppressive cell is a CAF.

[0090] In various embodiments, the cell surface protein is a receptor tyrosine kinase (RTK), CD1C, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11B, CD11C, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32B, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41B, CD42A, CD42B, CD43, CD44, CD45 , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66B, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120A, CD120B, CD123, CD125, CD134, CD135, CD140A, CD141, CD154, CD155, CD160, CD163, CD172A, XCR1, CD203C, CD204, CD206, CD207CD226、CD244、CD267、CD268、CD269、CD355、CD358、NKG2A、NKG2B、NKG2C、NKG2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL5A、K IR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、KIR2DS5、DAP12、KIR3DS、NKP44、NKP46、TCR、BCR、inテグリン、FCβεRI、MHC-I、MHC-II、IL-1R、IL-2Rα、IL-2Rβ、IL-2Rγ、IL-3Rα、CSF2RB、IL-4R、IL-5Rα、CSF2RB、IL-6Rα、GP130、IL-7Rα、IL-9R、IL-12Rβ1、I L-12Rβ2、IL-13Rα1、IL-13Rα2、IL-15Rα、IL-21R、IL23R、IL-27Rα、IL-31Rα、OSMR、CSF-1R、CELLIL-15、IL-10Rα、IL-10Rβ、IL-20Rα、IL-20Rβ、IL -22Rα1、IL-22Rα2、IL-22Rβ、IL-28RA、PD-1、PD-1H、BTLA、CTLA-4、PD-L1、PD-L2、2B4、B7-1、B7-2、B7-H1、B7-H4、B7-DC、DR3、LIGHT、LAIR、LTα1 β2、LTβR、TIM-1、TIM-3、TIM-4、TIGIT、LAG-3、ICOS、ICOS-L、SLAM、SLAMF2、OX-40、OX-40L、GITR、GITRL、TL1A、HVEM、41-BB、41BB-L、TL-1A、TRAF 1、TRAF2、TRAF3、TRAF5、BAFF、BAFF-R、APRIL、TRAIL、RANK、AITR、TRAMP、CCR1、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、CCR8、CCR9、CCR10、CCR11、CX CR1、CXCR2、CXCR3、CXCR4、CXCR5、CXCR6、CXCR7、CLECL9A、DC-SIGN、IGSF4A、SIGLEC、EGFR、PDGFR、VEGFR、FAP、α-SMA、ビメンチン、ラミニン、FAS、FAS-L、F c, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, ULI16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ, and ε, A 1 R,A 2A R,A 2B R and A 3 R, H60A, H60B, and H60C. In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε, and ζ, TCR. Several methods for assaying cell surface protein expression have been described in the literature, including flow cytometry and mass cytometry (CyTOF). The presence or abundance of one or more of these cell surface proteins indicates that the patient will respond to treatment with the methods disclosed herein.

[0091] In various embodiments, administering the negatively charged particles to a subject in need thereof increases the level of a tumor-inhibiting, anti-tumor, and / or pro-inflammatory protein in one or more blood and / or tumor samples taken from the subject. In various embodiments, the tumor-inhibiting, anti-tumor, and / or pro-inflammatory protein is selected from the group consisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-71, IL-72, IL 4, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, cell surface IL-15, CXCL2 (MCP-1 ), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, and TNF-α. In various embodiments, the levels of anti-tumor proteins and / or pro-inflammatory proteins are increased by 5-100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more blood samples taken prior to treatment. In various embodiments, the levels of anti-tumor and / or pro-inflammatory proteins are increased 2-100 fold compared to one or more blood samples taken from the subject prior to treatment (e.g., increased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment). Several methods for assaying proteins from blood samples have been described in the literature, including Western blot and ELISA.

[0092] In various embodiments, administering the negatively charged particles to a subject in need thereof reduces the levels of tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins. In various embodiments, the tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins include CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, MMP29, MMP30, MMP31, MMP32, MMP33, MMP34, MMP35, MMP36, MMP37, MMP38, MMP39, MMP40, MMP41, MMP42, MMP43, MMP44, MMP45, MMP46, MMP47, MMP48, MMP49, MMP50, MMP51, MMP52, MMP53, MMP54, MMP55, MMP56, MMP57, MMP58, MMP59, MMP60, MMP61, MMP62, MMP63, MMP64, MMP65, MMP66, MMP67, MMP68, MMP69, MMP70, MMP71, MMP72, MMP73, MMP74, MMP75, MMP76, MMP77, MMP78, MMP79, MMP80, MMP81, MMP82, MMP83, MMP84, MMP85, MMP86, MMP87, MMP88, MMP89, MMP90, MMP91, MMP9 27 and MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2, and TGF-β3, arginase, asparaginase, kynurellinase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the level of a tumor-promoting, anti-inflammatory, and / or immunosuppressive protein in one or more blood and / or tumor samples from the subject is reduced by 5-100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 9 ... In various embodiments, the level of a tumor-promoting, anti-inflammatory, and / or immunosuppressive protein in one or more blood and / or tumor samples from the subject is decreased by 2-100 fold compared to one or more blood samples taken from the subject prior to treatment (e.g., decreased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment).

[0093] In various embodiments, administering the negatively charged particles to a subject in need thereof increases the levels of tumor-inhibiting, anti-tumor, and / or pro-inflammatory genes in one or more samples taken from the subject. In one or more embodiments, expression of tumor-inhibiting, anti-tumor, and / or pro-inflammatory genes is increased by 5-100% (e.g., increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more blood and / or tumor samples taken prior to treatment. In various embodiments, expression of tumor-inhibiting, anti-tumor, and / or pro-inflammatory genes is increased by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more blood and / or tumor samples taken from the subject prior to treatment. In various embodiments, administering negatively charged particles to a subject in need thereof reduces the levels of tumor-inhibiting and / or anti-inflammatory genes in one or more samples taken from the subject. In some embodiments, expression of tumor-promoting and / or anti-inflammatory genes is increased by 5-100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% (including all values ​​and ranges therebetween) compared to one or more blood and / or tumor samples taken prior to treatment.In various embodiments, expression of tumor-promoting and / or anti-inflammatory genes is increased by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges between these values)) compared to one or more blood and / or tumor samples taken from the subject prior to treatment. In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0094] In various embodiments, administering negatively charged particles to a subject in need thereof reduces the level of neutrophil extracellular traps (NETs) in one or more blood and / or tumor samples taken from the subject. In various embodiments, the level of NETs in one or more blood and / or tumor samples is reduced by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken prior to treatment. In various embodiments, the level of NETs in one or more blood and / or tumor samples is reduced 2-100 fold compared to one or more blood and / or tumor samples taken from the subject prior to treatment (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment). Several methods for assaying NETs from tumor samples have been described in the literature, including Western blot, ELISA, and flow cytometry.

[0095] In various embodiments, administering negatively charged particles to a subject in need thereof reduces the neutrophil-to-lymphocyte (NLR) in one or more blood samples from high to moderate, or from high to low, hi various embodiments, the NLR is reduced to a level of 1 to 2 (e.g., between 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2 (including all values ​​and ranges therebetween)).

[0096] In various embodiments, administering the negatively charged particles to a subject in need thereof alters the level of one or more metabolites associated with tumor growth and progression. In various embodiments, administering the negatively charged particles to a subject in need thereof increases the level of one or more anti-tumor metabolites. In various embodiments, the anti-metabolite is an inflammatory metabolite. In various embodiments, examples of inflammatory metabolites include acids, lipids, sugars, amino acids, lactic acid, trimethylamine N-oxide, O-acetylcreatine, L-carnitine, choline, succinate, glutamine, fatty acids, cholesterol, 3-hydroxybutyrate, 3'-sialyllactose, arachidonic acid, prostaglandins (G2 and H2), PGD2, PGE2, PGF2a, PGI2, TXA2, leukotrienes (A4, B4, C4, D4, E4), lipoxin A4, and lipoxin B4. In various embodiments, the level of the anti-tumor metabolite in one or more blood and / or tumor samples is increased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken prior to treatment. In various embodiments, the level of the anti-tumor metabolite in one or more blood and / or tumor samples is increased by 2-100 fold compared to one or more blood and / or tumor samples taken from the subject prior to treatment (e.g., is decreased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment).

[0097] In various embodiments, administering negatively charged particles to a subject in need thereof reduces the level of one or more tumor-promoting metabolites.In various embodiments, the tumor-promoting metabolite is an anti-inflammatory metabolite.In various embodiments, examples of anti-inflammatory metabolites include kynurenine, 3-hydroxykynurenine, 2-amino-3-carboxymucon 6-semialdehyde, picolinic acid, anthranilic acid, 3-hydroxyanthranilic acid, glutaryl co-A, NAD+, quinolinic acid, arginine, butyrate, and adenosine. In various embodiments, the level of the tumor-promoting metabolite in one or more blood and / or tumor samples is decreased by 5-100% (e.g., by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken prior to treatment. In various embodiments, the level of the tumor-promoting metabolite in one or more blood and / or tumor samples is reduced by between 2 and 100 fold compared to one or more blood and / or tumor samples taken from the subject prior to treatment (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment).

[0098] Lists of human metabolites that can be assayed from biological samples can be found in (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6, 2007), and references contained in the Human Metabalome Database (HMDB), which are incorporated herein by reference.

[0099] In various embodiments, the therapeutic efficacy of the negatively charged particles administered to a subject with cancer is determined based on successful demonstration of at least one of the following parameters: Reduction in tumor burden as determined by RECIST. A reduction in the number of circulating tumor cells (CTCs) in the blood after and / or during treatment with the negatively charged particles compared to one or more samples taken before treatment with the negatively charged particles. an increase in the frequency of T cells and NK cells in the blood and / or tumor after and / or during treatment with negatively charged particles compared to one or more samples taken before treatment with negatively charged particles. -An increase in the frequency of activated T cells and NK cells in the blood and / or tumor after and / or during treatment with negatively charged particles compared to one or more samples taken before treatment with negatively charged particles. an increase in IL-15 gene expression levels in APCs in the blood and / or tumor following and / or during treatment with the negatively charged particles compared to one or more samples taken prior to treatment with the negatively charged particles. A reduction in the level of neutrophil extracellular traps (NETs) in the blood after and / or during treatment with the negatively charged particles compared to one or more samples taken before treatment with the negatively charged particles. A decrease in the neutrophil-to-lymphocyte ratio (NLR) in the blood after and / or during treatment with negatively charged particles compared to one or more samples taken before treatment with negatively charged particles. an increase in the levels of one or more anti-tumor cytokines and chemokines in the blood and / or tumor after and / or during treatment with the negatively charged particles compared to one or more samples taken before treatment with the negatively charged particles. an increase in the frequency of activated APCs in the blood and / or tumor after and / or during treatment with the negatively charged particles compared to one or more samples taken before treatment with the negatively charged particles.

[0100] Administration and Dosing Contemplated herein is a method for treating a subject suffering from cancer comprising administering a composition comprising the negatively charged surface-functionalized particles described herein, alone or in combination with one or more cancer therapeutic agents.

[0101] The methods of the present disclosure are carried out using any medically acceptable means for directly or indirectly introducing a therapeutic agent into a mammalian subject, including, but not limited to, injection, ingestion, intranasal, topical, transdermal, parenteral, inhalation spray, vaginal, or rectal administration. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal, and intracisternal injections, as well as catheter or infusion techniques. In various embodiments, the particles are administered intravenously, but may also be administered via other routes of administration, such as, but not limited to, intradermal, subcutaneous, epidermal, oral, intraarticular, and intrathecal.

[0102] In various embodiments, the composition is administered to the site of a tumor and the negatively charged particles are administered at a dose of about 0.1 mg / kg to 15 mg / kg. In various embodiments, the negatively charged particles are administered at a dose of about 0.5 mg / kg to 10 mg / kg, about 1 mg / kg to 8 mg / kg, about 1.5 mg / kg to 15 mg / kg, about 2 mg / kg to 15 mg / kg, about 2 mg / kg to 10 mg / kg, about 3 mg / kg to 10 mg / kg, about 4 mg / kg to 15 mg / kg, about 5 mg / kg to 15 mg / kg, or about 2 mg / kg to 8 mg / kg. In various embodiments, the negatively charged particles are administered at a dose of about 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, 12 mg / kg, or 15 mg / kg (including any value or range therebetween). In various embodiments, the negatively charged particles are administered at a dose of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 875 mg, 900 mg, 950 mg, 975 mg, or 1000 mg (including any value or range therebetween). These dose levels can be administered as a single dose or multiple doses.

[0103] In various embodiments, the negatively charged particles are administered at a concentration of about 0.05 mg / mL and about 50 mg / mL. In various embodiments, the negatively charged particles are administered at a concentration of about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL (including any value or range therebetween). In various embodiments, the negatively charged particles are administered via intravenous infusion lasting about 1, 2, 3, 4, 5, 6, 7, or 8 hours.

[0104] In various embodiments, the negatively charged particles are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year. In various embodiments, the negatively charged surface functionalized particles and / or cancer therapeutic agent are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks.

[0105] In various embodiments, the negatively charged particles are administered in a treatment cycle. In various embodiments, a treatment cycle consists of 1, 2, 3, 4, 5, 6, or 7 doses administered every week. In various embodiments, the subject is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 treatment cycles of negatively charged particles.

[0106] The present disclosure further contemplates a sterile pharmaceutical composition comprising a particle described herein, a cancer therapeutic agent, and a pharma- ceutically acceptable carrier.

[0107] The present disclosure further contemplates a sterile pharmaceutical composition comprising the discrete particles described herein and a pharma- ceutically acceptable carrier.

[0108] The present disclosure further contemplates a sterile pharmaceutical composition comprising a separate cancer therapeutic agent and a pharma- ceutically acceptable carrier.

[0109] Syringes, e.g., single-use or pre-filled syringes, sterile sealed containers, e.g., vials, bottles, reservoirs, and / or kits or packages containing any of the foregoing antibodies or compositions, optionally along with suitable instructions for use, are also contemplated.

[0110] Combination therapy It is contemplated that the particles described herein are administered in combination with a cancer therapeutic agent for treating cancer or a proliferative disorder. In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent, a biologic agent, a cell-based therapy, a hormonal therapy, an antibody-drug conjugate, an oncolytic virus, or a cancer vaccine.

[0111] Hormonal therapy drugs include tamoxifen for breast cancer, zoladex for breast and prostate cancer, aromatase inhibitors (e.g., anastrazole, letrozole, exemestane). Antibody drug conjugates include brentuximab vedotin (anti-CD30 mAB+monomethyl auristatin E) for lymphoma, adotrastuzumab entansine (anti-Her2 / Neu+maytansinoid) for breast cancer, and inotuzumab ozagamycin (anti-CD22+calicheamicin) for ALL. Oncolytic viruses include Imlygic (Amgen®). Cancer vaccines include Sipuleucel-T for prostate cancer. Several cancer vaccines are in development, including but not limited to protein, polypeptide, and nucleic acid vaccines.

[0112] In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent selected from the group consisting of growth inhibitors, cytotoxic agents, DNA replication inhibitors, kinase inhibitors, signal transduction cascade inhibitors, angiogenesis inhibitors, metabolic inhibitors, amino acid synthesis inhibitors, selective inhibitors of oncogenic proteins, metastasis inhibitors, inhibitors of anti-apoptotic factors, apoptosis inducers, nucleoside signal transduction inhibitors, enzyme inhibitors, and DNA damaging agents.

[0113] A cytotoxic agent refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioisotopes (e.g., I131, I125, Y90 and Re 186), chemotherapeutic agents, and toxins such as enzymatically active or synthetic toxins of bacterial, fungal, plant or animal origin, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and / or does not cause destruction of cells. Non-cytotoxic agents can include agents that can be activated to be cytotoxic.

[0114] Chemotherapeutic agents contemplated for use in the methods of the present disclosure include, but are not limited to, those listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0115] It is also contemplated that the cancer therapeutic agent may include one or more biologic agents, such as cytokines, angiogenesis inhibitors, immune checkpoint modulators, and monoclonal antibodies.

[0116] Cytokines include interferons (IFNs) and interleukins (ILs), such as IFN-alpha, IFN-beta, IFN-gamma, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, members of the transforming growth factor beta superfamily, including TGF-β1, TGF-β2, and TGF-β3, tumor necrosis factor alpha, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0117] In various embodiments, the cancer therapeutic comprises an enzyme. In various embodiments, the cancer therapeutic comprises an enzyme that targets T cells, B cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte-derived cells, tumor-associated stroma, cancer stem cells, mesenchymal stem cells, extracellular matrix, and amino acids. In various embodiments, the cancer therapeutic comprises an enzyme selected from the group including asparaginase, kynurinase, L-arginine deiminase, L-methionine-gamma-lyase, one or more amino acid degrading enzymes, and one or more nucleoside degrading enzymes.

[0118] Biologic agents such as immune checkpoint modulators target PD1, PD-L1, CTLA-4, TIMP-3, LAG-3 and / or TIGIT (T cell immunoreceptor with Ig and ITIM domains). In various embodiments, the immune checkpoint modulator is an antibody specific for PD-1, PD-L1, or CTLA-4. Antibodies specific for checkpoint proteins include ipilimumab (YERVOY®, Bristol-Myers Squibb Company), and tremelimumab, which bind to CTLA-4; antibodies against PD-1, such as pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp), dostallimab (Jemperli, GSK), and nivolumab (OPDIVO®, Bristol-Myers Squibb); and antibodies targeting PD-L1, such as atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®) (approved for the treatment of urothelial skin cancer and non-small cell lung cancer), cemiplimab (LIBTAYO®) (approved for cutaneous squamous cell carcinoma).

[0119] In various embodiments, the monoclonal antibody is a monospecific, bispecific, trispecific, or bispecific T-cell engaging (BiTE) antibody.

[0120] In various embodiments, the monoclonal antibody is an immune cell costimulatory molecule agonist that induces an anti-tumor immune response. Exemplary costimulatory molecules include, but are not limited to, ICOS (inducible T cell costimulator) (CD278), OX40 (CD134), GITR (glucocorticoid-inducible tumor necrosis factor receptor), CD40, and CD27.

[0121] In various embodiments, the monoclonal antibodies useful in the methods are selected from the group including alemtuzumab, bevacizumab, brentuximab, cetuximab, denosumab, ibritumomab, trastuzumab, panitumumab, pertuzumab, and rituximab. In various embodiments, the monoclonal antibodies useful in the methods target receptor tyrosine kinases, EGFR, VEGF, VEGFR, PDGF, PDGFR, TGF-β, TGF-β-LAP, SIRP-α, CD47, CD39, CD73, and fibroblast activation protein (FAP).

[0122] Biologic agents include monoclonal antibodies that are monospecific, bispecific, trispecific, or bispecific T cell engagers (BiTEs). Monoclonal antibodies useful in the treatment of cancer include bevacizumab (AVASTIN®, Genentech), an antibody against VEGF-A; erlotinib (TARCEVA®, Genentech and OSI Pharmaceuticals), a tyrosine kinase inhibitor that acts on EGFR; dasatinib (SPRYCEL®, Bristol-Myers Squibb Company), an oral Bcr-Abl tyrosine kinase inhibitor; IL-21; pegylated IFN-α2β; axitinib (INLYTA®, Pfizer, Inc.), a tyrosine kinase inhibitor; and trametinib (MEKINIST®, GlaxoSmithKline), a MEK inhibitor (Philips and Atkins, Int Immunol., 27(1):39-46 (2015), incorporated herein by reference). Bispecific antibodies useful for treating cancer are described in Krishnamurthy et al., (Pharmacol Ther. 2018 May;185:122-134), and Yu et al., (J. Hematol Oncol 2017,10:155), and include blinatumomab and catumaxomab.

[0123] The method also provides that the cancer therapeutic agent comprises one or more cell-based therapies, including adoptive cell transfer, tumor infiltrating leukocyte therapy, chimeric antigen receptor T cell (CAR-T) therapy, NK cell therapy, invariant NKT cell therapy, and stem cell therapy.

[0124] In various embodiments, the cell-based therapy is the adoptive transfer of autologous patient-derived cells. In various embodiments, the cell-based therapy is the adoptive transfer of allogeneic donor-derived cells.

[0125] In various embodiments, the cell-based therapy is the transfer of universal donor-derived or induced pluripotent stem cell-derived cells that are not patient-specific and suitable for long-term storage. Such therapies are also referred to as "off-the-shelf therapies."

[0126] In various embodiments, the cancer therapeutic is a hormone therapy. In various embodiments, the cancer therapeutic comprises one or more antibody-drug conjugates. In various embodiments, the cancer therapeutic comprises one or more cancer vaccines. In various embodiments, the cancer vaccine is a protein, polypeptide, and / or nucleic acid vaccine.

[0127] In various embodiments, the cancer therapeutic is an immunotherapy selected from the group including oncolytic viruses, bacteria, oncolytic bacteria or other bacterial populations, Bacillus Calmette-Guerin (BCG), microbiota modulators, and / or toll-like receptor (TLR) agonists. In various embodiments, the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13 agonist. In various embodiments, the TLR agonist is derived from a virus, bacteria, and / or synthetically produced. In various embodiments, the immunotherapy is a STING pathway modulator.

[0128] In various embodiments, the cancer therapeutic comprises a viral or bacterial vector. In various embodiments, the viral vector is selected from the group comprising adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, potato virus x, comovirus, or cucumber mosaic virus. In various embodiments, the virus is an oncolytic virus. In various embodiments, the virus is a chimeric virus, a synthetic virus, a mosaic virus, or a pseudotype virus.

[0129] It is contemplated that the particles and the cancer therapeutic agent can be administered contemporaneously, simultaneously, or sequentially. Contemporaneous administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, so long as there is an overlap in the period during which the agents exert their therapeutic effect. Simultaneous or sequential administration is contemplated, as well as administration on different days or weeks.

[0130] It is contemplated that the particles and the cancer therapeutic agent may be given simultaneously in the same formulation. It is further contemplated that the agents may be given in separate formulations and given contemporaneously, where contemporaneous refers to agents given within 30 minutes of each other.

[0131] In another embodiment, the cancer therapeutic agent is administered before the administration of the particle composition.Pre-administration refers to administration of the cancer therapeutic agent within a range of 1 week before the treatment with particles to 30 minutes before the administration of particles.It is further contemplated that the cancer therapeutic agent is administered after the administration of the particle composition.Subsequent administration is intended to describe administration from 30 minutes after the particle treatment to 1 week after the administration.

[0132] In various embodiments, the particles and / or cancer therapeutic agent are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

[0133] In various embodiments, the particles and / or cancer therapeutics are administered intravenously, orally, intranasally, intramuscularly, intraocularly, transdermally, or subcutaneously.

[0134] In various embodiments, the subject is a mammal, hi various embodiments, the subject is a human.

[0135] kit As an additional aspect, the present disclosure includes a kit that includes one or more compounds or compositions packaged in a manner that facilitates their use to carry out the methods of the present disclosure. In one embodiment, such a kit includes a compound or composition described herein (e.g., particles alone or in combination with a cancer therapeutic agent, or compositions thereof) packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes the use of the compound or composition in carrying out the method. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration or for carrying out a screening assay. Preferably, the kit includes a label that describes the use of the inhibitor composition.

[0136] Manufacturing method In various embodiments, the present disclosure also provides a process for producing a composition comprising negatively charged particles. In various embodiments, the method includes: (a) generating a primary emulsion by mixing an aqueous solution with an oil phase comprising a polymer; (b) mixing the primary emulsion with a solution comprising one or more surfactants and / or stabilizers to form a secondary emulsion; (c) hardening the secondary emulsion by evaporation to remove the solvent and provide hardened polymer nanoparticles; (d) filtering, washing, and concentrating the nanoparticles; and (e) freeze-drying the nanoparticles. In various embodiments, the primary emulsion in step (a) is a water-in-oil emulsion. In various embodiments, the secondary emulsion in step (b) is an oil-in-water emulsion.

[0137] In an embodiment, the method includes (a) generating a primary emulsion by mixing an aqueous solution with an oil phase comprising a polymer; (b) mixing the primary emulsion with a solution comprising one or more surfactants and / or stabilizers to form a secondary emulsion; (c) removing the solvent to form crude negatively charged particles; and (d) filtering the crude particles to form a composition comprising the negatively charged particles, the composition comprising less than 0.5 wt.% (e.g., less than 0.4 wt.%, less than 0.3 wt.%, less than 0.2 wt.%, less than 0.1 wt.%, less than 0.05 wt.%, less than 0.01 wt.%) of one or more surfactants and less than 1 wt.% (e.g., less than 0.9 wt.%, less than 0.8 wt.%, less than 0.7 wt.%, less than 0.5 wt.%, less than 0.4 wt.%, less than 0.3 wt.%, less than 0.2 wt.%, less than 0.1 wt.%, less than 0.05 wt.%, less than 0.01 wt.%) of one or more stabilizers. In embodiments, the coarse particles are filtered through a filter having a pore size of about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, or about 50 μm (including any value or range therebetween). In embodiments, the filter has a pore size of about 10 μm, about 20 μm.

[0138] In various embodiments, the manufacturing process is designed to obtain negatively charged particles having an average diameter of about 0.1 μm to about 10 μm, or about 0.1 μm to about 5 μm, or about 0.1 μm to about 3 μm, or about 0.3 μm to about 5 μm, or about 0.3 μm to about 3 μm, or about 0.3 μm to about 1 μm (including all values ​​and ranges therein). In various embodiments, the manufacturing process is designed to obtain negatively charged particles having a negative zeta potential of about -100 mV to about -10 mV, or about -100 mV to about -25 mV, or about -100 to about -30 mV, or about -80 mV to about -30 mV, or about -75 mV to about -30 mV, or about -70 mV to about -30 mV, or about -60 mV to about -30 mV, or about -75 to about -35 mV, or about -70 to about -25 mV (including all values ​​and ranges therein).

[0139] In various embodiments, the aqueous solution of step (a) comprises a solvent. In various embodiments, the solvent is an organic solvent. In various embodiments, the solvent is an inorganic solvent. In various embodiments, the organic solvent is dichloromethane, acetone, ethanol, methylene chloride, dimethylsulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the solvent in the aqueous solution is at a concentration of 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% (v / v). In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 mM. In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 M. In various embodiments, the choice of solvent in the manufacturing process is designed to obtain negatively charged particles having a diameter of about 0.1 μm to about 10 μm, or about 0.1 μm to about 5 μm, or about 0.1 μm to about 3 μm, or about 0.3 μm to about 5 μm, or about 0.3 μm to about 3 μm, or about 0.3 μm to about 1 μm (including all values ​​and ranges therein). In various embodiments, the selection of solvent in the manufacturing process is designed to obtain negatively charged particles having a negative zeta potential of about -100 mV to about -10 mV, or about -100 mV to about -25 mV, or about -100 to about -30 mV, or about -80 mV to about -30 mV, or about -75 mV to about -30 mV, or about -70 mV to about -30 mV, or about -60 mV to about -30 mV, or about -75 to about -35 mV, or about -70 to about -25 mV (including all values ​​and ranges therein).

[0140] In various embodiments, the surfactant and / or stabilizer solution of step (b) comprises a solvent. In various embodiments, the solvent is an organic solvent. In various embodiments, the solvent is an inorganic solvent. In various embodiments, the organic solvent is dichloromethane, acetone, ethanol, methylene chloride, dimethylsulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid. In various embodiments, the inorganic solvent is water, ammonia, sulfuric acid, carbon disulfide, bromine trifluoride, phosphorus oxychloride, hydrogen fluoride, and sulfur dioxide. In various embodiments, the solvent in the aqueous solution is at a concentration of 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% (v / v). In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 mM. In various embodiments, the solvent in the aqueous solution is at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 10.0 mM. In various embodiments, the solvent in the solutions of step (a) and step (b) is the same. In various embodiments, the solvent in the solutions of step (a) and step (b) is different.

[0141] In various embodiments, the polymer in step (a) is a biodegradable polymer. In various embodiments, the biodegradable polymer is polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), polylactic-glycolic acid copolymer (PLGA), polylactic-sebacic acid copolymer (PLSA), polyglycolic-sebacic acid copolymer (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, polysaccharide, or lipid. In various embodiments, the polymer is a copolymer. In various embodiments, the copolymer has various molar ratios of the constituent polymers. In various embodiments, the molar ratio is 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0.

[0142] In various embodiments, the polymer in step (a) is PLGA. In various embodiments, the molar ratio of the copolymer of PLGA is 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In various embodiments, the PLGA has a high molecular weight. In various embodiments, the PLGA has a low molecular weight. In various embodiments, the PLGA has a molecular weight of about 10 to 10,000 kDa (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 kDa, including all values ​​within this range). In various embodiments, the amount of PLGA in the solution in step (a) is about 0.05-100% by weight (e.g., between 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, including all values ​​within this range).

[0143] In various embodiments, the surfactant and / or stabilizer used in step (b) is anionic, cationic, or nonionic. In various embodiments, the surfactant and / or stabilizer is poloxamer, polyamine, PEG, Tween-80, gelatin, dextran, Pluronic L-63, Pluronic F-68, Pluronic 188, Pluronic F-127, PVA, PAA, methylcellulose, lecithin, DMAB, PEMA, Vitamin E TPGS (Da-tocopheryl polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers), methylcellulose, hydroxyethylcellulose, hydroxyprolylcellulose, hydroxypropylmethylcellulose, gelatin, carbomer, or sulfate polymers (e.g., heparin sulfate, chondroitin sulfate, ulvan, and carrageenan). In various embodiments, the amount of surfactant and / or stabilizer present in the solution in step (b) is about 0.0005% to 100% by weight or volume (e.g., between 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, including all values ​​within this range). In various embodiments, the surfactant and / or stabilizer has a molecular weight of about 0.1 kDa to 10,000 kDa (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 kDa, including all values ​​within this range).

[0144] In various embodiments, the water-in-oil primary emulsion of step (a) is obtained by homogenizing the aqueous solution with the oil phase containing the polymer. In various embodiments, the homogenization is carried out for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, 600, 700, 800, 900, or 1000 seconds. In various embodiments, the oil-in-water secondary emulsion of step (b) is obtained by homogenizing the primary emulsion with a solution containing one or more surfactants and / or stabilizers. In various embodiments, homogenization is carried out for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, 600, 700, 800, 900, or 1000 seconds. In various embodiments, the water-in-oil primary emulsion of step (a) is obtained by ultrasonication of an aqueous solution with an oil phase containing the polymer. In various embodiments, sonication is performed for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, or 600 seconds.

[0145] In various embodiments, the solution comprising one or more surfactants and / or stabilizers that form the oil-in-water secondary emulsion in step (b) has a pH less than or equal to 4.0. In various embodiments, the oil-in-water secondary emulsion has a pH of about pH 1 to about pH 4, about pH 2 to about pH 4, about pH 3 to about pH 4, or about pH 1, about pH 1.5, about pH 2, about pH 2.5, about pH 3, about pH 3.5, or about pH 4 (including all values ​​and ranges therein).

[0146] In various embodiments, the oil-in-water secondary emulsion of step (b) is obtained by sonicating the primary emulsion with a solution comprising one or more surfactants and / or stabilizers, hi various embodiments, the sonication is carried out for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, or 600 seconds.

[0147] In various embodiments, the secondary emulsion is hardened by evaporation. In various embodiments, the evaporation is active evaporation. In various embodiments, the evaporation is passive evaporation. In various embodiments, the active evaporation is vacuum driven evaporation. In various embodiments, the evaporation is carried out for 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, or 96 hours. In various embodiments, the secondary emulsion is hardened by evaporation. In various embodiments, the evaporation is active evaporation. In various embodiments, the evaporation is passive evaporation. In various embodiments, the active evaporation is carried out using agitation or under vacuum. In various embodiments, the active evaporation is carried out under high pressure vacuum. In various embodiments, the active evaporation is carried out under low pressure vacuum. In various embodiments, evaporation is carried out for 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, or 96 hours. In various embodiments, evaporation is carried out at a pressure between 0.01 kDa and 1000 mBar (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mBar (including all values ​​within this range)). In various embodiments, the evaporation step is designed to remove the process solvent such that the level of residual process solvent in the final nanoparticle formulation is about 0.0005% to 100% by weight or volume (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, including all values ​​within this range).In various embodiments, the evaporation step is designed to remove the process solvent such that the level of residual process solvent in the final nanoparticle formulation is 0.0005 to 500,000 ppm (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, 100000, 200000, 400000, or 500000 ppm (including all values ​​within this range)). In various embodiments, the evaporation step is designed to remove the process solvent such that the level of residual process solvent in the final nanoparticle formulation is between 0.0005 and 500,000 ppb (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, 100000, 200000, 400000, or 500000 ppb, inclusive of all values ​​within this range).

[0148] In various embodiments, the filtering, washing, and concentrating of the particles in step (d) is carried out by gel filtration, membrane filtration, dialysis, centrifugation, chromatography, density gradient centrifugation, or combinations thereof. In various embodiments, this step is designed to remove process solvents, surfactants, and / or stabilizers such that the level of residual process solvents, surfactants, and / or stabilizers in the final nanoparticle formulation is 0.0005-100% (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (including all values ​​within this range)) by weight or volume. In various embodiments, this step is designed to remove process solvents, surfactants, and / or stabilizers such that the level of residual process solvents, surfactants, and / or stabilizers in the final nanoparticle formulation is 0.0005 to 500,000 ppm (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, 100000, 200000, 400000, or 500000 ppm (including all values ​​within this range)). In various embodiments, this step is designed to remove process solvents, surfactants, and / or stabilizers such that the level of residual process solvents, surfactants, and / or stabilizers in the final nanoparticle formulation is between 0.0005 and 500,000 ppb (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, 100000, 200000, 400000, or 500000 ppb, inclusive of all values ​​within this range).

[0149] In various embodiments, an excipient is added to the nanoparticle composition prior to lyophilization in step (e). In various embodiments, the excipient is a buffer and / or a cryoprotectant. In various embodiments, the excipient is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, dextran, ficoll, dextran 70k, sodium citrate, lactose, L-arginine, or glycine. In various embodiments, the amount of excipient added to the nanoparticle composition prior to lyophilization is 0.05-100% by weight or volume (e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (including all values ​​within this range)). As used herein, when an excipient is present at 100% by weight or volume, the excipient is present in an equal amount relative to the negatively charged particles disclosed herein. In various embodiments, the amount of excipient added to the nanoparticle composition prior to lyophilization is 0.01-500 g (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 g) per gram of nanoparticles. In various embodiments, excipients used in the process for manufacturing negatively charged particles are selected to achieve a pH of about 1.0 to 14.0 (e.g., between 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, or 14.0) (including all values ​​and ranges therein) for the final formulation.In various embodiments, the excipients used in the process for manufacturing the negatively charged particles provide a pH for the final formulation of about 1.0-7.0, or about 2.0-7.0, or about 3.0-7.0, or about 4.0-7.0, or about 5.0-7.0, or about 6.0-7.0, or about 2.0-6.0, or about 3.0-6.0, or about 4.0-6.0, or about 1.0-5.0, or about 2.0-5.0, or about 3.0-5.0, or about 4.0-5.0, or about 1.0-4.0, or about 2.0-2.0, or about 3.0-4.0 (including all values ​​and ranges therein).

[0150] In various embodiments, the lyophilization process is carried out for about 1 hour to about 2000 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 hours), including all ranges and values ​​therein. In various embodiments, the lyophilization step used in the process for producing negatively charged particles is designed to result in a moisture level in the final formulation of 0.0005 to 25% (e.g., 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25%) by weight or volume, including all values ​​and ranges therein.

[0151] In various embodiments, the process for producing negatively charged particles comprises a process for producing negatively charged particles having a molecular weight of about 10 to 10 99 particles (e.g., 10, 10 per vial) 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 20 , 10 30 , 10 40 , 10 50 , 1060 , 10 70 , 10 80 , 10 90 , or 10 99 particles) (including all values ​​and ranges therein).

[0152] In various embodiments, the production batch size of the negatively charged particles can be scaled up or down. In various embodiments, the production batch size is 0.01g to 100kg. In various embodiments, the batch size is 0.01g, 0.1g, 10g, 20g, 40g, 60g, 80g, 100g, 160g, 240g, 320g, 400g, 480g, 560g, 640g, 720g, 800g, 1000g, 5kg, 10kg, 50kg, or 100kg (including all values ​​and ranges therebetween).

[0153] "Batch size" as used herein refers to the scale of production according to the weight of particles in the final product. The manufacturing process can be modified, scaled up or scaled down. The manufacturing process can be modified, scaled up or scaled down by changing the amount or volume of solvent, antigen / protein, polymer, surfactant, stabilizer, cryoprotectant or excipient. The manufacturing process can be scaled up or scaled down by changing the time of homogenization, sonication, evaporation, filtration, concentration, washing or lyophilization.

[0154] In certain embodiments, particles encapsulating antigens are produced by nanoprecipitation, co-precipitation, inert gas condensation, sputtering, microemulsion, sol-gel techniques, layer-by-layer techniques, or ionic gelation. Several methods for producing nanoparticles have been described in the literature and are incorporated herein by reference.

[0155] Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative and not limiting. EXAMPLES

[0156] Example 1: Phase Ib / 2a clinical trial of negatively charged particles of the present disclosure in adult subjects with metastatic or advanced solid tumors This example describes a Phase 1b / 2a open-label clinical trial to evaluate the safety, tolerability, pharmacodynamics, and efficacy of a negatively charged particle of the present disclosure (designated ONP-302) in adult subjects with refractory metastatic or progressive solid tumors.

[0157] The negatively charged particles comprise PLGA nanoparticles that contain no other drugs. The negatively charged particles have a mean diameter of 350-800 nm and a negative zeta potential of -30 mV to -60 mV. The negatively charged particles are supplied as a lyophilized formulation. The negatively charged particles are reconstituted in sterile water for injection and diluted in sterile saline (0.9% sodium chloride, USP) prior to administration. Adult subjects aged 18 years or older are screened prior to enrollment. Subjects who meet all inclusion criteria and no exclusion criteria will be enrolled in an open cohort at the time of receiving ONP-302. The study consists of two parts: Part I (dose escalation) and Part II (dose expansion). Part I follows a classical 3+3 design to aid in escalation decisions. Three dose levels are defined for escalation: Dose level 1: 2mg / kg ONP-302 Dose level 2: 4mg / kg ONP-302 Dose level 3: 8mg / kg ONP-302 (up to 650mg)

[0158] In Part I, each subject will receive the negatively charged particles as a cycle of four doses administered once weekly. The negatively charged particles will be administered via an intravenous infusion lasting approximately 3-4 hours unless safety concerns require early infusion discontinuation in the subject. Subjects will be observed for AEs and SAEs, including infusion reactions (IR), for up to 2 hours after infusion on each dosing day. The first subject in the first cohort at each dose level will be followed through two doses (study day 8) before additional subjects are enrolled in that cohort. Enrollment of successive subjects into the cohort will proceed if the previous subject has received two doses of the negatively charged particles without a grade ≥2 AE that does not resolve to baseline within 48 hours and no relevant SAEs and / or DLTs have been reported after the second dose. The initial cohort at each dose level will consist of three patients. Once initial enrollment of a cohort is complete, further enrollment decisions will be made according to the DMC's decision to escalate to the next dose level, add an additional set of 3 patients at the current dose level (3+3), or if escalation is complete. Part I will end with either identification of the MTD or completion of the planned dose levels without reaching the MTD. The MTD is defined as the highest dose at which 2 / 6 or fewer patients experience DLT during the initial cycle of treatment.

[0159] Once Part I is completed, one or more dose levels below the MTD will be selected for expanded enrollment in Part II. The decision regarding which dose level to enroll in Part II may include indications of pharmacodynamic and / or antitumor response from Part I in addition to safety data. Subjects may be added to the expanded dose levels (up to a total of 12 subjects per dose level) to further evaluate safety, tolerability, and pharmacodynamic effects and prospectively evaluate antitumor activity. Approximately 12 subjects are expected to be included in Part I, and up to 18 additional subjects may be included in Part II, for a total of up to 30 subjects. Subjects who tolerate the negatively charged particles without signs of progression may be eligible to receive additional treatment cycles at the same dose level for a total of up to 6 cycles, at the discretion of the investigator.

[0160] The safety assessment includes: Physical examination at screening, then symptom-based physical examination on each dose of the first cycle, then on the fourth dose of each additional cycle. Vital signs at screening, then on each dosing day of the first cycle, then on the fourth dosing day of each additional cycle. A single 12-lead ECG at screening, then on each dosing day of the first cycle only -AEs and SAEs throughout the subject's participation in the study. Safety labs (chemistry, hematology, coagulation, urinalysis) at screening, on each dose day of the first cycle, and then on the fourth dose day of each additional cycle. Complement (C3a, C5a, sC5b9, and C1q) and cytokines / chemokines (IL-1b, IL-2, IL-6, IL-7, IL-10, IP-10, MCP-1, and GM-CSF) in case of IR (samples collected will be analyzed in case of infusion reactions or other related infusion reaction adverse events with presumed complement and cytokine / chemokine involvement). Samples will be collected pre-dose on Day 1 to establish a baseline. Samples will be collected approximately 15 minutes after onset, 2 hours (+ / - 5 minutes) after onset, and approximately 24 hours (+ / - 2 hours) after the infusion reaction or related adverse event. Patients will remain in the unit for up to 8 hours after any infusion related AE to monitor for recovery.

[0161] Pharmacodynamic (PD) assessments included the following: Frequencies of T cells and NK cells (total T cells / total white blood cells and total NK cells / total white blood cells) in the blood before dosing (baseline) and 24 hours after dosing on each dosing day of cycle 1, then 22 days before dosing and 24 hours after dosing on day 4 of each additional cycle. Frequencies of activated T cells and NK cells (activated T cells / total T cells and activated NK cells / total NK cells) in the blood before dosing (baseline) and 24 hours after dosing on each dosing day of the first cycle, then 22 days before dosing and 24 hours after dosing on the fourth dosing day of each additional cycle. CTC counts (per mL of blood) at screening (baseline) and then 24 hours after every 4th dose of each cycle. -IL-15 gene expression levels in APCs in blood pre-dose (baseline) and 24 hours post-dose on each dosing day of the first cycle, then 22 days pre-dose and 24 hours post-dose on the fourth dosing day of each additional cycle. Levels of NETs in the blood pre-dose (baseline) and 24 hours post-dose on each dosing day of the first cycle, then 22 days pre-dose and 24 hours post-dose on the fourth dosing day of each additional cycle. Neutrophil-to-lymphocyte ratio (NLR) pre-dose at screening (baseline) and then 24 hours after every fourth dose of each cycle. Serum levels of cytokines and chemokines (soluble IL-15, IL-17, TNF-α, IFN-γ, MIP-1α, MIP-1β, RANTES, TGF-β1, TGF-β2, VEGF, and M-CSF) pre-dose (baseline) and 24 hours post-dose on each dosing day of the first cycle, then 22 days pre-dose and 24 hours post-dose on the fourth dosing day of each additional cycle.

[0162] Clinical evaluation includes the following: Tumor response by RECIST v1. 18 weeks after treatment initiation and every 8 weeks during the study.

[0163] Pharmacokinetic (PK) assessments included: Plasma concentrations of the negatively charged particle PD cytokine biomarker IL-8 pre-dose and at 0.5, 2 (+ / - 5 minutes), and 24 hours (+ / - 2 hours) after IV administration of negatively charged particles on Day 1.

[0164] Selection criteria: Men aged 18 or older and non-pregnant women. ECOG performance status 0–2. Signed, written informed consent obtained prior to any study procedures. Recurrent / refractory metastatic or advanced solid tumors for which no standard therapy exists. Measurable disease by RECIST v1.1 criteria. Adequate organ function as defined by: Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 × institutional ULN (for patients without synchronous liver metastases) or ≤ 5.0 × institutional ULN (for patients with synchronous liver metastases). Total bilirubin ≤ 1.5 x ULN, except for patients with confirmed Gilbert's syndrome who should have a total bilirubin ≤ 3 x ULN. - Hemoglobin 9g / dL or higher without red blood cell transfusions for at least 2 weeks. ○LVEF 45% or higher. ○ANC10×10 3 / μL or more and platelet count 100×10 3 / μL or more, without myeloid growth factor support or infusions, respectively, for at least 1 week. Men and women of childbearing potential (WOCBP) must be willing to practice highly effective methods of contraception, including but not limited to abstinence, sexual activity with a person of the same sex, being in a monogamous relationship with a vasectomized partner, vasectomy, hysterectomy, bilateral tubal ligation, approved hormonal therapy, or an intrauterine device (IUD). · Willingness to comply with all study procedures for the duration of the study.

[0165] Exclusion criteria: Primary CNS malignancy or known CNS involvement. Previous unrelated malignancies within 3 years of the study procedure, except for basal cell carcinoma or cancer ·Original position. - Course of standard therapy or investigational drug within 28 days or 5 half-lives, whichever is shorter, prior to initiation of investigational treatment. -Comorbid conditions requiring immunosuppressive therapy. ·Known or suspected pregnancy or breastfeeding. Clinically significant, active, and uncontrolled infection, including HIV, Hepatitis B, or Hepatitis C infection. ·PT, PTT>1.5×ULN. Clinically significant coagulopathy or uncontrolled hypertension at the investigator's discretion. History of Grade 3 or greater immune-related AE that has not resolved to < Grade 2. Known sensitivity to any component of negatively charged particles. Other known comorbidities that, in the opinion of the investigator, pose an undue safety risk. Potential co-morbid conditions including: - Subjects with a history of cerebrovascular disease. o Subjects with abnormal QTc (>450 ms in men, >470 ms in women). Subjects with known prior myocardial infarction (occurring at any time in the past) defined by any of the following criteria: ■ The occurrence of known new pathologic Q waves, with or without symptoms. ■ Imaging evidence of lost areas of viable myocardium that thin and do not contract, in the absence of nonischemic causes. ■ Pathological findings of healed or healing myocardial infarction. At least 60mL / min / 1.73m 2 Subjects with chronic kidney disease as defined by a 3-month glomerular filtration rate (GFR) of less than 0.05 mg / kg / day, with GFR calculated based on the following CKD-EPI equation: ■ GFR = 141 × min(Scr / κ,1)α × max(Scr / κ,1) - 1.209 × 0.993 age × 1.018 [for women] × 1.159 [for blacks] Where: Scr is serum creatinine in mg / dL Kappa is 0.7 for women and 0.9 for men · α is -0.329 for women and -0.411 for men ·Min indicates the minimum value of Scr / α or 1 ·Max indicates the maximum value of Scr / α or 1 Subjects who have received a live vaccine (except intranasal influenza) within 28 days prior to screening or a subunit vaccine within 14 days prior to screening, or who are scheduled to receive a vaccination at any time during the study. Subjects who have received any COVID-19 vaccination within 14 days prior to screening. Subjects who have received the first dose of any COVID-19 vaccine cannot be screened for the study until 14 days after their last vaccine dose, if applicable.

[0166] Example 2: Determination of optimal dose levels and dosing frequency of negatively charged particles to effectively inhibit tumor growth using the MC38 mouse tumor model The objective of this study was to identify the dose level and dose frequency at which optimal antitumor efficacy was observed using a syngeneic MC38 colon adenocarcinoma mouse tumor model. MC38 tumor cells (1.0 × 10 6 The tumors were subcutaneously implanted into 6- to 8-week-old C57BL / 6 mice with an average volume of 50 mm. 3 Once tumor size reached 100%, mice were treated with saline (control) or different dose levels of negatively charged particles (5 mg / kg, 25 mg / kg, 50 mg / kg, and 125 mg / kg mouse doses: 0.4 mg / kg, 2 mg / kg, 4 mg / kg, or 10 mg / kg HED, respectively). Mice were monitored for tumor growth and survival.

[0167] As shown in FIG. 1A, treatment with negatively charged particles (designated ONP-302) at a 50 mg / kg mouse dose (4 mg / kg HED) resulted in a statistically significant 2.9-fold reduction in tumor growth compared to the control treatment. In comparison, treatment with negatively charged particles at the lower dose levels tested (5 mg / kg and 25 mg / kg mouse doses, respectively; 0.4 mg / kg and 2 mg / kg HED) did not show the same tumor growth inhibition efficacy as observed at the 50 mg / kg mouse dose (4 mg / kg HED). At the highest dose level of 125 mg / kg mouse dose (10 mg / kg HED), the negatively charged particles did not show improved tumor growth inhibition efficacy compared to the 50 mg / kg mouse dose (4 mg / kg HED). As shown in FIG. 1B, treatment with negatively charged particles at a 50 mg / kg mouse dose (4 mg / kg HED) improved survival compared to the control treatment. In comparison, treatment with negatively charged particles at lower dose levels (5 mg / kg and 25 mg / kg mouse doses; 0.4 mg / kg and 2 mg / kg HED, respectively) showed no efficacy in improving survival. At the highest 125 mg / kg mouse dose level (10 mg / kg HED), the survival efficacy of negatively charged particles was similar to that at the 50 mg / kg mouse dose (4 mg / kg HED) level. In a separate experiment, the efficacy of negatively charged particles at a 50 mg / kg mouse dose (4 mg / kg HED) administered once every 3 days was compared to the same dose level administered once every 6 days. As shown in FIG. 1C, negatively charged particles administered less frequently (once every 6 days) resulted in a 1.5-fold reduction in suboptimal tumor growth compared to administration once every 3 days, which resulted in a 4.4-fold reduction in tumor growth (N=5 per group).

[0168] In conclusion, a bell-shaped dose-response was observed: a therapeutic dose of 50 mg / kg mouse (4 mg / kg HED) of negatively charged particles administered once every 3 days was optimal to inhibit tumor growth and improve survival in the MC38 mouse tumor model.

[0169] Example 3: Determination of optimal dose levels and dosing frequency of negatively charged particles to effectively inhibit tumor growth using a B16F10 mouse tumor model The objective of this study was to identify the dose level and dose frequency of negatively charged particles at which optimal antitumor efficacy was observed using a syngeneic B16.F10 (B16) melanoma mouse tumor model. B16 tumor cells (0.5 × 10 6 The tumors were subcutaneously implanted into 6- to 8-week-old C57BL / 6 mice with an average volume of 50 mm. 3 Once this was reached, mice were treated once every 3 days with saline (control) or different dose levels of ONP-302 (25 mg / kg, 50 mg / kg, and 125 mg / kg mouse doses: 2 mg / kg, 4 mg / kg, or 10 mg / kg HED, respectively).

[0170] As shown in FIG. 2A, treatment with negatively charged particles (designated ONP-302) at a dose of 50 mg / kg mice (4 mg / kg HED) administered once every 3 days resulted in a statistically significant 3-fold reduction in tumor growth compared to the control treatment (p<0.05). Increasing the dose level of negatively charged particles to 125 mg / kg (10 mg / kg HED) did not show improved efficacy in tumor growth inhibition compared to the 50 mg / kg mouse dose (4 mg / kg HED). As shown in FIG. 2B, treatment with negatively charged particles at a dose of 50 mg / kg mice (4 mg / kg HED) administered once every 3 days resulted in improved survival compared to the control treatment (p<0.05). In comparison, treatment with negatively charged particles at a lower dose level of 25 mg / kg (2 mg / kg HED) did not show efficacy in improving survival. Increasing the dose level of negatively charged particles to 125 mg / kg mouse dose (10 mg / kg HED) did not show any improvement in survival efficacy compared to the 50 mg / kg mouse dose (4 mg / kg HED). In the same experiment, the efficacy of ONP-302 at a 50 mg / kg mouse dose (4 mg / kg HED) administered once every 3 days was compared to the same dose level administered at a less frequent dosing frequency (once every 6 days). As shown in Figure 2A, administration of negatively charged particles once every 6 days resulted in a 1.5-fold reduction in suboptimal tumor growth compared to administration once every 3 days, which resulted in a 3-fold reduction in tumor growth. Similarly, administration of negatively charged particles once every 6 days showed suboptimal survival efficacy compared to administration once every 3 days, as shown in Figure 2B.

[0171] In conclusion, a bell-shaped dose-response was observed: ONP-302 at a therapeutic dose of 50 mg / kg mouse (4 mg / kg HED) administered once every 3 days was optimal for inhibiting tumor growth and improving survival in the B16 mouse tumor model.

[0172] Example 4: Antitumor efficacy of negatively charged ONP-302 nanoparticles depends on a functional adaptive immune system. The purpose of this study was to evaluate the efficacy of ONP-302 in inhibiting primary tumor growth and metastasis in the presence and absence of a functional adaptive immune response using the 4T1 orthotopic tumor model. The efficacy of ONP-302 in inhibiting primary tumor growth and metastasis was demonstrated in Rag1 knockout (Rag1 - / - ) mice and was evaluated using the 4T1 orthotopic tumor model.

[0173] Approximately 0.1 x 10 6 4T1 tumor cells were cultured in 6- to 8-week-old female WT or Rag1 mice. - / - Tumors were orthotopically implanted into the mammary fat pad of BALB / c mice. Mice were randomized into treatment groups and administered saline (control) or ONP-302 (50 mg / kg mouse dose: 4 mg / kg HED) starting 1 day after tumor implantation. ONP-302 or saline was administered by intravenous tail vein injection once every 3 days. Tumor growth was monitored by measuring tumor volume. On day 21 after tumor inoculation, mice were euthanized and primary tumor metastasis to the lungs was evaluated by assaying bioluminescent signals from 4T1 tumor cells using an IVIS® imaging system.

[0174] As shown in Figure 3, treatment with 50 mg / kg (4 mg / kg HED) ONP-302 in WT tumor-bearing mice resulted in a statistically significant 1.6-fold reduction in tumor growth (p<0.05) compared to WT tumor-bearing mice treated with saline (control). In contrast, ONP-302 treatment at the same dose level significantly reduced Rag1 - / - ONP-302 did not show any efficacy in tumor-bearing mice. As shown in Figure 4, 9 / 10 (90%) mice in the saline (control) group showed lung metastasis, whereas only 1 / 10 (10%) mice in the ONP-302 group showed lung metastasis. In contrast, ONP-302 did not show any efficacy in tumor-bearing mice. - / - As shown in Figures 4C and 4D, saline (control) and ONP-302 (50 mg / kg mouse dose; 4 mg / kg HED)-treated Rag1 mice did not show any efficacy in inhibiting primary tumor metastasis to the lungs in tumor-bearing mice.- / - In both tumor-bearing mice, 10 / 10 (100%) mice showed evidence of lung metastasis. In conclusion, the results of this study demonstrate that the efficacy of ONP-302 (50 mg / kg mouse dose; 4 mg / kg HED) in inhibiting tumor growth and metastasis in an orthotopic 4T1 breast cancer model is dependent on the presence of a functional adaptive immune system. These results support an ONP-302 antitumor mechanism of action that overcomes immune suppression via activation of antitumor T and NK cells.

[0175] Example 5: Negatively charged ONP-302 particles induce gene expression changes in pulmonary immune cells associated with inhibition of primary orthotopic 4T1 tumor metastasis to the lung To understand ONP-302-induced immunological changes associated with inhibition of primary orthotopic 4T1 tumor metastasis to the lung, we performed single-cell RNA sequencing (scRNA-seq) from lung tissue of 4T1 tumor-bearing mice administered ONP-302 or saline.

[0176] Approximately 0.1 x 10 6 4T1 tumor cells were orthotopically implanted into the mammary fat pad of 6-8 week old female BALB / c mice. Mice were treated with ONP-302 or saline every 3 days starting on day 1 after tumor implantation. Mice were sacrificed on day 21 after tumor implantation and lungs were harvested and processed for scRNA-seq.

[0177] As shown in Figure 5A, treatment with ONP-302 resulted in a decrease in the number of neutrophils and an increase in the number of endothelial cells present in the lung tissue. Phenotypic analysis of cell populations in the lung using gene set enrichment analysis (GSEA) revealed that ONP-302 treatment induced the greatest phenotypic changes in endothelial cells, dendritic cells, monocytes, and neutrophils. As shown in Figure 5B, ONP-302 treatment resulted in the induction of interferon, TNF, and antigen-related gene expression signatures indicative of a shift to an anti-tumor environment in the lung tissue associated with the suppression of primary tumor metastasis to the lung.

[0178] In conclusion, these results indicate that the anti-metastatic mechanism of ONP-302 is likely via inducing a shift from immunosuppression to a more anti- or pro-inflammatory environment at the metastatic site.

[0179] Example 6: Negatively charged ONP-302 nanoparticles induce activation of the cGAS / STING pathway in myeloid cells The cGAS / STING pathway is essential for the control of both bacterial and viral pathogens and can also be induced by host genomic DNA in the presence of tumor cells. As a result, the use of cGAS / STING pathway agonists has been suggested as a potential therapeutic agent for the treatment of cancer. cGAS / STING-mediated dendritic cell sensing of tumor DNA is associated with increased expression of tumor NK cells and CD8 + It has been shown to be essential for T cell regulation.

[0180] The aim of this study was to evaluate the effect of negatively charged ONP-302 nanoparticles on bone marrow cells. To that end, single cell RNA sequencing (scRNAseq) was performed on splenic leukocytes from naive C57BL / 6 mice intravenously injected with saline, one dose of ONP-302, three doses of ONP-302 on three consecutive days, or three doses of ONP-302 administered every three days. Treatment with three doses of ONP-302 (1 mg / dose), either on consecutive days or every three days, induced a significant shift in cell population clustering compared to saline treatment alone. Treating mice for three consecutive days induced an increase in the proportion of B cells, whereas treating mice every three days induced an increase in monocytes, macrophages, and neutrophils (Figure 6A).

[0181] To determine the transcripts and pathways that were altered by three doses of ONP-302 treatment compared to saline treatment alone, differential transcript expression was analyzed for macrophages, monocytes, and neutrophils. The data show that three doses of ONP-302 induced significant changes in transcripts expressed by macrophages, monocytes, and neutrophils (Figure 6B). Further analysis of the differentially expressed transcripts identified a significant increase in transcripts associated with cellular pathways related to IFN-g, IFN-a, activation of the innate immune response, and antiviral immune responses (Figure 6C). The findings suggest that treatment of C57BL / 6 mice with three doses of ONP-302 every three days activates cellular pathways in splenic bone marrow cells that may be favorable for immune cell control of tumor growth.

[0182] To further confirm and validate the scRNAseq results, the gating scheme shown in Figure 7A was used to screen CD4 + T cells, CD8 + Both the numbers (FIG. 7B) and phenotypes of T cells and NK cells (FIG. 7C), monocytes and macrophages (FIG. 7D), and other myeloid populations (FIG. 7E) were assessed. Notably, repeated dosing of ONP-302 every 72 hours significantly upregulated costimulatory molecules such as CD40 and CD86 on myeloid populations, and also induced a more activated phenotype on NK cells, significantly upregulating CD244 (2B4), PD-1, and NKG2D (FIG. 7F). Taken together, these data indicate that multiple treatments of ONP-302 result in an increase in a pro-inflammatory myeloid cell signature that leads to a downstream increase in NK cell activation and possible function.

[0183] To test whether cGAS / STING signaling is involved in the transcriptional signature identified after ONP-302 treatment, RAW cells were cultured in the presence of ONP-302 for 24 hours. After culture, supernatants were collected to determine the levels of secreted cytokines and chemokines. Significant increases in the levels of TNF-a, MIP-1a, MIP-1b, and IL-15 were observed. It was found that wild-type RAW cells cultured in the presence of the STING inhibitor, RU.521, or cGAS-deficient RAW cells stimulated with ONP-302 failed to upregulate the expression of TNF-a, MIP-1a, MIP-1b, and IL-15 compared to controls (Figure 8A). Furthermore, the expression of wild-type vs. STING - / - GM-CSF-driven bone marrow-derived myeloid cells from mice were tested. Bone marrow cells from both lines were cultured in the presence of 25 ng / mL GM-CSF for 7 days, at which point cell phenotype was analyzed by flow cytometry. The remaining cells were cultured in the presence of LPS (1 mg / mL) or ONP-302 (2.5 mg / mL) for 24 hours and the levels of secreted cytokines / chemokines were analyzed. Lipopolysaccharide (LPS) increased the secretion of TNF-a, MIP-1a, MIP-1b, and IL-15 in wild-type and STING-derived cells. - / - Although induced by both myeloid cells, cytokine secretion in response to ONP-302 was lost in the absence of STING expression (Fig. 8B).

[0184] Collectively, these data indicate that phagocytosis of ONP-302 by myeloid cells results in activation of the cGAS / STING pathway leading to the secretion of TNF-a, MIP-1a, MIP-1b, and IL-15.

[0185] Example 7: Method for producing negatively charged ONP-302 nanoparticles One method to fabricate negatively charged surface-functionalized PLGA particles (ONP-302) is to use a double emulsion process, which uses PLGA, polyvinyl alcohol (PVA), polyacrylic acid (PAA), and ethyl acetate (EtOAc).

[0186] The PLGA used in the manufacture is a 50:50 copolymer of lactic and glycolic acid (±5 mol%) with a molecular weight of 10-75 kDa, as a result of ring-opening polymerization from lactide and glycolide monomers.

[0187] The double emulsion is initiated by first generating a water-in-oil primary emulsion, where a PLGA solution (5% in ethyl acetate) is rapidly mixed with sterile water for injection (WFI). The primary emulsion is rapidly mixed with a surfactant and stabilizer solution (4% PVA and PAA (Sigma Aldrich, 100 kDa, 35% by weight)) in ethyl acetate such that the ratio of PAA / PVA / EtOAc in the emulsion is approximately 1:4:12.5% ​​by weight, forming an oil-in-water secondary emulsion. The composition of the PVA / PAA / ethyl acetate blend is maintained at a pH of less than 4.0. Mixing of the primary and secondary emulsions is performed by homogenization.

[0188] The nanoparticles formed in the secondary emulsion are then hardened by removing the solvent via evaporation under vacuum (25-35 mBar, for at least 4 hours), followed by passive evaporation for 12-72 hours. The hardened nanoparticles are passed through a 20 μm filter before starting tangential flow filtration (TFF). The nanoparticles are then filtered, washed, and concentrated via tangential flow filtration (TFF) to remove residual surfactant and solvent. The concentrated nanoparticles are sampled for in-process analysis to confirm a size of 300 nm or greater and a zeta potential of less than -20 mV.

[0189] Cryoprotectants and buffer excipients sucrose, mannitol, and sodium citrate are then added to the concentrated nanoparticles. The nanoparticles are then filled into vials and lyophilized. The physicochemical properties of the final ONP-302 nanoparticle formulation are summarized in Table 2 below. [Table 2]

Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising administering a dose of negatively charged particles ranging from about 0.1 mg / kg to about 15 mg / kg, thereby treating said cancer, wherein said negatively charged particles comprise: a) a zeta potential in the range of about -10 mV to -100 mV; b) an average diameter ranging from about 100 nm to about 1500 nm; wherein the negatively charged particles do not include a therapeutic agent.

2. The method of claim 1 , wherein the negatively charged particles do not include small molecules.

3. The method of claim 1 , wherein the negatively charged particles do not contain peptides.

4. 10. The method of any one of the preceding claims, wherein the negatively charged particles are administered by injection.

5. 13. The method of any one of the preceding claims, wherein the negatively charged particles are present in the pharmaceutical composition at a concentration ranging from about 0.05 mg / mL to about 50 mg / mL.

6. 13. The method of any one of the preceding claims, wherein the negatively charged particles are administered at a dose ranging from about 1.5 mg / kg to about 2.5 mg / kg, 3.5 mg / kg to about 4.5 mg / kg, or 7.5 mg / kg to about 8.5 mg / kg.

7. 13. The method of any one of the preceding claims, wherein the negatively charged particles are administered in a dose ranging from about 10 mg to about 1000 mg.

8. 13. The method of any one of the preceding claims, wherein the negatively charged particles are administered in a dose of about 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 875 mg, 900 mg, 950 mg, 975 mg, or 1000 mg.

9. The method of any one of claims 1 to 8, wherein the negatively charged particles are administered in multiple doses.

10. The method of any one of claims 1 to 8, wherein the negatively charged particles are administered in a single dose.

11. 9. The method of any one of claims 1 to 8, wherein the negatively charged particles are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

12. 13. The method of any one of the preceding claims, wherein the negatively charged particles are administered in treatment cycles.

13. 13. The method of claim 12, wherein each said treatment cycle is one week, one month, or one year in duration.

14. 14. The method of claim 13, wherein the treatment cycle is one week in duration and the negatively charged particles are administered once a week.

15. 15. The method of claim 13 or 14, wherein the treatment cycle is of one week duration and the negatively charged particles are administered for four treatment cycles.

16. 16. The method of claims 12-15, wherein the negatively charged particles are administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 treatment cycles.

17. 10. The method of claim 1, wherein the negatively charged particles are administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally.

18. 13. The method of any one of the preceding claims, wherein the negatively charged particles are administered alone or in combination with one or more additional cancer therapeutic agents.

19. 20. The method of claim 18, wherein the negatively charged particles are administered prior to, simultaneously with, or after the administration of the one or more additional cancer therapeutic agents.

20. 10. The method of any one of the preceding claims, wherein the negatively charged particles are administered before or after surgical resection of a tumor.

21. 13. The method of any one of the preceding claims, wherein administering the negatively charged particles alone or in combination with one or more additional cancer therapeutic agents reduces one or more symptoms of cancer.

22. 22. The method of claim 21, wherein the administration of the negatively charged particles reduces tumor size and / or tumor burden in the subject in need thereof.

23. 23. The method of claim 22, wherein the tumor size and / or tumor burden in the subject is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%.

24. 23. The method of claim 22, wherein the change in tumor size and / or tumor burden is determined using Response Evaluation Criteria in Solid Tumors (RECIST).

25. 22. The method of claim 21, wherein administration of the negatively charged particles reduces the number of circulating tumor cells (CTCs) in the subject in need thereof.

26. 26. The method of claim 25, wherein the CTCs are reduced by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the CTCs before treatment.

27. 22. The method of claim 21, wherein the administration of the negatively charged particles reduces the number of CTC clusters in the subject in need thereof.

28. 28. The method of claim 27, wherein the number of CTC clusters is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% compared to the CTC clusters before treatment.

29. 22. The method of claim 21, wherein said administration of said negatively charged particles reduces the number of CTC-leukocyte clusters in said subject in need thereof.

30. 30. The method of claim 29, wherein the number of CTC-leukocyte clusters is reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the number of CTC-leukocyte clusters before treatment.

31. 31. The method of claim 29 or 30, wherein the white blood cells are bone marrow-derived cells.

32. The method of any one of claims 29 to 31, wherein the bone marrow-derived cells are monocytes, macrophages, neutrophils, or dendritic cells.

33. 13. The method of any one of the preceding claims, wherein said administering extends survival of said subject in need thereof.

34. 34. The method of claim 33, wherein the survival time is increased by about 1 year to about 50 years.

35. 2. The method of any one of the preceding claims, wherein said administration of negatively charged particles induces an anti-cancer immune response.

36. 36. The method of claim 35, wherein the anti-cancer immune response comprises an increase in the number of anti-cancer T cells, NK cells, B cells, and / or bone marrow-derived cells.

37. 36. The method of claim 35, wherein the anti-cancer immune response comprises an increase in the number of activated anti-tumor T cells, NK cells, B cells, and / or bone marrow-derived cells.

38. 38. The method of claim 36 or 37, wherein the bone marrow-derived cells are monocytes, macrophages, neutrophils, or dendritic cells.

39. 36. The method of claim 35, wherein the anti-cancer immune response comprises an increase in the concentration of one or more anti-cancer cytokines and / or chemokines.

40. 40. The method of claim 39, wherein the one or more anti-cancer cytokines and / or chemokines are soluble IL-15, IL-17, TNF-α, IFN-γ, MIP-1α, MIP-1β, RANTES, TGF-β1, TGF-β2, VEGF, or M-CSF.

41. 36. The method of claim 35, wherein the anti-cancer immune response comprises increased anti-tumor gene expression levels in T cells, NK cells, B cells, and / or bone marrow-derived cells.

42. 42. The method of claim 41, wherein the bone marrow-derived cell is a monocyte, macrophage, neutrophil, or dendritic cell.

43. 36. The method of claim 35, wherein the anti-cancer immune response is detected in one or more of a tumor, blood, spleen, or lymph nodes.

44. 36. The method of claim 35, wherein the anti-cancer immune response is detected in one or more metastatic sites.

45. 36. The method of claim 35, wherein the anti-cancer immune response is measured in one or more biological samples including whole blood, peripheral blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, cerebrospinal fluid (CSF), stool, tissue biopsy, or bone marrow biopsy.

46. 13. The method of any one of the preceding claims, wherein the administration of the negatively charged particles reduces levels of neutrophil extracellular traps (NETs).

47. 10. The method of any one of the preceding claims, wherein the administration of the negatively charged particles reduces the neutrophil to lymphocyte ratio (NLR).

48. 13. The method of any one of the preceding claims, wherein the cancer is a metastatic cancer.

49. 10. The method of any one of the preceding claims, wherein the cancer is a solid cancer.

50. 13. The method of any one of the preceding claims, wherein the cancer is brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, or testicular cancer.

51. 1. A method for preparing a composition comprising negatively charged particles, comprising: (a) forming a primary emulsion by mixing an aqueous solution with an oil phase containing a polymer; (b) mixing the primary emulsion with a solution containing one or more surfactants and / or stabilizers to form a secondary emulsion; (c) removing the solvent to form crude negatively charged particles; (d) filtering the crude particles to form a composition comprising negatively charged particles.

52. 52. The method of claim 51 , wherein the solution of step (a) comprises a solvent.

53. 52. The method of claim 51 , wherein the solution of step (b) comprises a solvent.

54. The method of any one of claims 51 to 53, wherein the solvent is an organic solvent.

55. 55. The method of any one of claims 51 to 54, wherein the solutions of step (a) and step (b) comprise the same solvent.

56. 55. The method of any one of claims 51 to 54, wherein the solutions of step (a) and step (b) comprise different solvents.

57. 55. The method of claim 54, wherein the organic solvent is dichloromethane, acetone, ethanol, methylene chloride, dimethylsulfoxide (DMSO), ethyl acetate, dimethylformamide, tetrahydrofuran, chloroform, and acetic acid.

58. 58. The method of any one of claims 51 to 57, wherein the emulsion obtained from step (a) is a water-in-oil emulsion.

59. 59. The method of any one of claims 51 to 58, wherein the emulsion resulting from step (b) is an oil-in-water emulsion.

60. 60. The method of any one of claims 51 to 59, wherein the polymer in step (a) is a biodegradable polymer.

61. 61. The method of claim 60, wherein the biodegradable polymer is polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), polylactic-glycolic acid copolymer (PLGA), polylactic-sebacic acid copolymer (PLSA), polyglycolic-sebacic acid copolymer (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, a polysaccharide, or a lipid.

62. 62. The method of any one of claims 51 to 61, wherein the surfactant or stabilizer of step (b) is anionic.

63. 63. The method of claim 62, wherein the surfactant and / or stabilizer is a poloxamer, a polyamine, PEG, Tween-80, gelatin, dextran, Pluronic L-63, Pluronic F-68, Pluronic 188, Pluronic F-127, PVA, PAA, methylcellulose, lecithin, DMAB, PEMA, Vitamin E TPGS (Da-tocopheryl polyethylene glycol 1000 succinate), hyaluronic acid, polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or enantiomers thereof), methylcellulose, hydroxyethylcellulose, hydroxyprolylcellulose, hydroxypropylmethylcellulose, gelatin, sodium cholate, carbomer, or a sulfate polymer.

64. 64. The method according to any one of claims 51 to 63, wherein the primary emulsion of step (a) is obtained by homogenization.

65. 64. The method according to any one of claims 51 to 63, wherein the primary emulsion of step (a) is obtained by ultrasonic treatment.

66. 66. The method of any one of claims 51 to 65, wherein the secondary emulsion of step (b) is obtained by homogenization.

67. 66. The method according to any one of claims 51 to 65, wherein the secondary emulsion of step (b) is obtained by ultrasonic treatment.

68. 68. The method of any one of claims 51 to 67, wherein the pH of the secondary emulsion of step (b) is less than pH 4.

69. 67. The method of claim 64 or 66, wherein homogenization is carried out for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, or 600 seconds.

70. 68. The method of claim 65 or 67, wherein sonication is carried out for 5, 10, 15, 20, 25, 30, 30, 40, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 450, 480, 510, 540, 570, or 600 seconds.

71. The method according to any one of claims 51 to 70, wherein the removing in step (c) is carried out by evaporation of the solvent.

72. 72. The method of claim 71, wherein the evaporation is active evaporation or passive evaporation.

73. 73. The method of claim 72, wherein the active evaporation is vacuum driven evaporation.

74. 74. The method of claim 73, wherein the vacuum driven evaporation is carried out under high or low pressure.

75. 73. The method of claim 72, wherein passive evaporation is performed by stirring.

76. 76. The method of claim 75, wherein the evaporation is carried out for 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 48, 72, or 96 hours.

77. 77. The method according to any one of claims 51 to 76, wherein the filtering, washing and concentrating of the nanoparticles in step (d) is carried out by filtration, gel conversion, membrane filtration, dialysis, centrifugation, chromatography, density gradient centrifugation, or a combination thereof.

78. 78. The method of any one of claims 51 to 77, wherein the particles have a negative zeta potential.

79. 79. The method of claim 78, wherein the zeta potential of the particles is from about -1 mV to about -100 mV.

80. 80. The method of claim 79, wherein the zeta potential of the particles is from about -30 mV to about -80 mV.

81. 81. The method of any one of claims 51 to 80, wherein the particles have an average diameter of from about 0.3 μm to about 3 μm.

82. 82. The method of claim 81, wherein the particles have an average diameter of about 0.3 μm to about 1 μm.

83. 83. The method of claim 82, wherein the particles have an average diameter of about 0.4 μm to about 1 μm.

84. 84. The method of any one of claims 51 to 83, wherein at least 90% of the particles have an average diameter of from about 0.3 μm to about 3 μm.

85. 85. The method of claim 84, wherein at least 90% of the particles have an average diameter of from about 0.3 μm to about 1 μm.

86. 86. The method of claim 85, wherein at least 90% of the particles have an average diameter of from about 0.4 μm to about 1 μm.

87. 87. The method of any one of claims 51 to 86, wherein at least 50% of the particles have an average diameter of from about 0.3 μm to about 3 μm.

88. 88. The method of claim 87, wherein at least 50% of the particles have an average diameter of about 0.3 μm to 1 μm.

89. 89. The method of claim 88, wherein at least 50% of the particles have an average diameter of about 0.4 μm to 1 μm.

90. 90. The method of any one of claims 51 to 89, wherein at least 10% of the particles have an average diameter of about 0.3 μm to 3 μm.

91. 91. The method of claim 90, wherein at least 10% of the particles have an average diameter of about 0.3 μm to 1 μm.

92. 13. The method according to any one of the preceding claims, wherein the production batch size is from about 0.01 g to 100 kg.

93. 93. The method of claim 92, wherein the production batch size is 0.01 g, 0.1 g, 10 g, 20 g, 40 g, 60 g, 80 g, 100 g, 160 g, 240 g, 320 g, 400 g, 480 g, 560 g, 640 g, 720 g, 800 g, 1000 g, 5 kg, 10 kg, 50 kg, or 100 kg.

94. A composition comprising particles produced by the method of any one of claims 51 to 93.

95. 95. The composition of claim 94, further comprising a pharma- ceutically acceptable excipient.

96. 96. The composition of claim 95, wherein the excipients include sucrose, mannitol, and sodium citrate.

97. A pharmaceutical composition comprising negatively charged particles, sucrose, mannitol, and sodium citrate.

98. 98. A method of treating a subject having cancer, comprising administering a composition according to any one of claims 94 to 97.

99. 10. The method of claim 1, wherein the negatively charged particles are administered at a dose of about 2 mg / kg.

100. 10. The method of claim 1, wherein the negatively charged particles are administered at a dose of about 4 mg / kg.

101. 10. The method of claim 1, wherein the negatively charged particles are administered at a dose of about 10 mg / kg.

102. 1. A method of treating a solid tumor in a subject in need thereof, comprising administering a dose of negatively charged particles ranging from about 0.4 mg / kg to about 10 mg / kg, wherein the negatively charged particles comprise: a) a zeta potential of about -10 mV to about -100 mV; b) an average diameter of about 100 nm to about 1500 nm; wherein the negatively charged particles do not comprise a therapeutic agent.

103. 103. The method of claim 102, wherein the negatively charged particles are administered at a dose of about 2 mg / kg.

104. 103. The method of claim 102, wherein the negatively charged particles are administered at a dose of about 4 mg / kg.

105. 105. The method of any one of claims 102 to 104, wherein the cancer is selected from the group consisting of brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, or testicular cancer.

106. 13. The method of any one of the preceding claims, wherein the negatively charged particles have a D90 of about 600 nm to about 700 nm.

107. 13. The method of any one of the preceding claims, wherein the negatively charged particles have a D50 of about 550 nm to about 600 nm.

108. 13. The method of any one of the preceding claims, wherein the negatively charged particles have a D10 of about 500 nm to about 550 nm.

109. 56. The method of claim 55, wherein the coarse particles of step (d) are filtered through a filter having a pore size of about 10 μm to about 20 μm.

110. 56. The method of claim 55, wherein step (d) further comprises washing the filtered crude particles.

111. 56. The method of claim 55, wherein step (d) further comprises concentrating the filtered crude particles.

112. 52. The method of claim 51, wherein the composition comprises less than 0.5% by weight of the one or more surfactants and less than 1% by weight of the one or more stabilizers.

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