Use of a hydrogel-nanoparticle system for the delivery of protein kinase inhibitors and chemotherapeutics

Abstract

Provided herein are pharmaceutical compositions that include (a) a lipid nanoparticle comprising a protein kinase inhibitor (e.g., MEK inhibitor, PI3K inhibitor, or RAF inhibitor) and a chemotherapeutic agent; and (b) a hydrogel comprising a thermosensitive and biodegradable polymer. Also provided herein are methods of treating a cancer in a subject that include administering to the subject a therapeutically effective amount of any one of the pharmaceutical compositions described herein.

Description

[0001] USE OF A HYDROGEL-NANOPARTICLE SYSTEM FOR THE DELIVERY OF PROTEIN KINASE INHIBITORS AND CHEMOTHERAPEUTICS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U.S. Patent Application No. 63 / 671,448 filed on July 15, 2024, which is incorporated by reference herein in its entirety.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “44807-0473 WO 1_SL_ST26.XML.” The XML file, created on July 14, 2025, is 13,399 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] STATEMENT REGARDING FEDERAL FUNDING

[0007] This invention was made with government support under AG060903, AR081774, CA143868 and CA268083 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] The present disclosure relates to the field of biotechnology, and more specifically, to compositions and methods of delivering therapeutic agents.

[0010] BACKGROUND

[0011] Many human cancers are characterized by a mutation in a protein kinase, including a MEK kinase, PI3K kinase, and RAK tyrosine kinase. As one example, around 30% of human solid tumors are characterized by a mutation in the RAS gene leading to aberrant activation of the RAS-RAF-MEK-ERK pathway. MEK1 and MEK2 have crucial roles in turn ori genesis, cell proliferation and inhibition of apoptosis, making MEK inhibition an attractive therapeutic in numerous cancers. Several potent MEK1 / 2 inhibitors with high affinity for desired phosphosites have been developed and studied over the past decade. However, MAPK is a critical pathway in all cell types, so systemic delivery causes prohibitive toxicity in patients. Although an attractive candidate, MEK inhibitors have historically failed in the clinical setting due to narrow therapeutic index, lack of predictive biomarkers, and severe side effects.

[0012] SUMMARY

[0013] Provided herein are pharmaceutical compositions that include (a) a lipid nanoparticle comprising a protein kinase inhibitor and a chemotherapeutic agent; and (b) a hydrogel comprising a thermosensitive and biodegradable polymer.

[0014] In some embodiments, the lipid nanoparticle targets a501 integrin. In some embodiments, the lipid nanoparticle comprises a fibronectin-mimetic peptide (PR_b) on a surface of the lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a liposome. In some embodiments, the lipid nanoparticle comprises a micelle.

[0015] In some embodiments, the protein kinase inhibitor comprises a MEK inhibitor. In some embodiments, the MEK inhibitor comprises PD0325901, AZD6244, trametinib, cobimetinib, selumetinib, or binimetinib.

[0016] In some embodiments, the chemotherapeutic agent comprises vincristine, prednisone, dexamethasone, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, nivolumab, pembrolizumab, durvalumab, atezolizumab, radioisotopes, monomethyl auristatin E (MMAE; e.g., vedotin), calicheamicins, deruxtecan, DM1, and any combinations thereof. In some embodiments, the chemotherapeutic agent comprises doxorubicin.

[0017] In some embodiments, the thermosensitive and biodegradable polymer comprises a PVLA-PEG-PVLA triblock copolymer. In some embodiments, the lipid nanoparticle is encapsulated in the hydrogel.

[0018] Also provided herein are methods of treating a cancer in a subject that include administering to the subject a therapeutically effective amount of any one of the pharmaceutical compositions described herein. In some embodiments, a cancer cell from the cancer expresses 0.501 integrin on the cancer cell surface. In some embodiments, the cancer is a breast cancer, colon cancer, pancreatic cancer, lung cancer, prostate cancer, brain cancer, or melanoma. In some embodiments, the cancer is a triple negative breast cancer (TNBC). In some embodiments, the cancer is an E-cadherin positive cancer.

[0019] In some embodiments, the administration comprises intratumoral administration and / or local administration in the vicinity of the tumor. In some embodiments, the subject is a human.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0021] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0022] BRIEF DESCRIPTION OF DRAWINGS

[0023] FIGs. 1A-1G show preferential uptake of targeting liposomes in E-cad positive MDA-MB-231 cells and increased MEK inhibitor efficacy. FIG. 1A shows an exemplary schematic of the 3D two compartment organoid system composed of a Matrigel and cell inner core and collagen I outer layer with representative phase contrast image of the two-compartment organoid system, scale bar is 250 pm. FIG. IB shows a representative confocal laser scanning microscopy z-stack images from live-dead staining of E-cad+ and E-cad- MDA-MB-231 organoids at different cross sections. Live cells are shown in green and dead cells shown in red, scale bars are 200 pm. FIG. 1C shows maximum-intensity projection of a confocal microscopy image of Cy5 labeled liposomes (PR_b functionalized and non-targeting) after incubation with MDA-MB-231 organoids for 72 h at 37°C. Nuclei are shown in blue and liposomes shown in red, scale bars are 200 pm and inset scale bar 50 pm. FIG. ID shows an exemplary schematic of liposomes used in subsequent experiments with release profiles of iMEK and DOX loaded in liposomes at 37°C. FIG. IE shows cytotoxicity assessment of iMEK and DOX loaded liposomes (both PR_b and non-targeted) at different concentrations on E-cad+ and E-cad- MDA-MB-231 organoids after 72 h. Arrows indicate optimal concentrations used as a starting point for future experiments. Statistical significance determined via one-way ANOVA with Tukey’s HSD post-hoc analysis and showed that all PR b formulations were significant (P<0.01) when compared to their nontargeted pair. FIG. IF shows synergy calculations for the drug interaction between iMEK and DOX (loaded in PR_b liposomes), calculated by plotting cell killing observed in excess of the additive Bliss expectation between iMEK and DOX in E-cad+ and E-cad- MDA-MB-231 organoids. Green dotted box indicates combination with greatest synergy against E-cad induced hyper-proliferation in organoids. FIG. 1G shows cytotoxicity assessment of liposomes loaded with 1 pM iMEK + 2 pM DOX delivered with targeted or non-targeted liposomes to MDA-MB- 231 organoids after 72 h. Statistical significance was determined using a two-sided unpaired t test; *P<0.05, **P<0.01, ***P<0.001.

[0024] FIGs. 2A-2F shows hydrogel characterization and extended-release evaluation. FIG. 2A shows an exemplary schematic illustration of the PVLA-PEG-PVLA hydrogel with liposomes, demonstrating the polymer phase transition and entrapment of liposomes in the hydrogel. FIG. 2B shows PVLA-PEG-PVLA polymer as a liquid at 25°C and a gel at 37° C as confirmed by tube inversion. FIG. 2C shows Cryo-TEM images of PVLA-PEG-PVLA solution in water (0.25% w / v) at 25°C and 37°C, scale bar is 100 nm. FIG. 2D shows release profiles of iMEK and DOX loaded as a free drug entrapped in the hydrogel or FIG. 2E in liposomes in the hydrogel at 37°C. All results are reported as mean ± SEM (n = 3). FIG. 2F shows an exemplary schematic of a 12- day organoid growth inhibition experiment with MDA-MB-231 4-day old organoids (i.e., at t =0, organoids are at t_growth =4 days) exposed to different samples via a transwell insert. After each 3 -day treatment the same transwell inserts were employed onto new 4-day old organoids. Cell viability of MDA-MB-231 organoids compared to non-treated controls. Data are mean ± SEM (n = 3). Statistical significance determined via one-way ANOVA with Tukey’s HSD post-hoc analysis and p-values shown in comparison to the empty hydrogel control; *P<0.05, **P<0.01, ***p<0.001.

[0025] FIGs. 3A-3F show in vivo biodegradation of PVLA-PEG-PVLA hydrogel in the absence of nanoparticles. FIG. 3A shows an exemplary schematic of subcutaneous hydrogel injection in both NSG and BALB / c mice. FIG. 3B shows in vivo gel formation and retention analyzed after subcutaneous injection of PVLA-PEG-PVLA solution in the flank of BALB / c mice, scale bar is 1 cm. FIG. 3C shows degradation rate of hydrogel in NSG and BALB / c mice as measured by hydrogel volume via calipers. Data are shown as mean ± SEM (n = 4 per group). FIG. 3D shows body weight of NSG and BALB / c mice after polymer injection. Data are shown as mean ± SEM (n = 4 per group). FIG. 3E show representative images of H&E staining of injection area at different time points in BALB / c mice. Asterisk denotes injection site, vertical red arrows polymorphonuclear neutrophils and horizontal black arrows foamy macrophages. Scale bar is 500 pm and inset scale bar is 50 pm. FIG. 3F shows representative images of H&E staining of fdtration organs from BALB / c mice 90 days after polymer injection, scale bar is 100 pm. FIGs. A-4J show local and targeted delivery of iMEK and DOX in liposomes in TNBC xenograft mouse model. FIG. 4A shows an exemplary treatment schedule and survival curve for mice treated with orally delivered iMEK. E-cad+ MDA-MB-231 cells were implanted in the second mammary fat pad of NSG mice 9 days before treatment began on day 0. Oral iMEK was given every day for 5 days followed by 2 days of rest, and the cycle repeated 3 times. Statistical significance was determined using a two-sided log-rank (Mantel-Cox) test (n = 3). FIG. 4B shows an exemplary preparation and treatment schedule of mice. E-cad+ MDA-MB-231 cells were implanted in the second mammary fat pad of NSG mice day 0, 7 days before treatment began. IV nanoparticle injections were given every 7 days and hydrogel injections once every 14 days. Gels were administered via 3 separate injections every 14 days. FIG. 4C shows survival curves corresponding to different treatment groups (n = 7-10). Statistical significance was determined using a two-sided log-rank (Mantel-Cox) test. FIG. 4D shows body weight of mice in different groups during treatment. FIG. 4E shows tumor growth in different treatment groups over time. Data are mean ± SEM and statistical significance was determined via one-way ANOVA with Tukey’s HSD post-hoc analysis. FIG. 4F shows scaled pictures of the excised orthotopic tumors from all treatment groups. Scale bar is 10 mm. FIG. 4G shows representative images of H&E stained tumors and IHC staining for pERK and Ki67. Scale bar is 3 mm. FIG. 4H shows representative images of H&E stained hearts for various treatment groups, scale bar is 100 pm. FIG. 41 shows representative images of H&E stained lung sections. Scale bar is 3 mm and inset scale bar is 100 pm. FIG. 4 J shows relative expression level of HK2 in the lungs of treatment groups. Results are presented as mean ± SEM (n = 7-10) and statistical significance determined via one-way ANOVA with Tukey’s HSD post-hoc analysis. For all plots in this figure; *P<0.05, **P<0.01, ***P<0.001. FIGs. 5A-5N show local and targeted delivery of iMEK and DOX in liposomes in syngeneic mouse model. FIG. 5A shows an exemplary preparation and treatment schedule of mice. 4Tl-luc cells were implanted in the second mammary fat pad of BALB / c mice on day 0, 7 days before treatment began. Hydrogels were injected every 14 days. Gels were administered via 3 separate injections on each day of treatment. FIG. 5B shows survival curves corresponding to the different treatment groups (n = 7-9). Statistical significance was determined using a two-sided log-rank (Mantel-Cox) test. FIG. 5C shows body weight of mice in different groups during treatment. FIG. 5D shows representative bioluminescence images of mice at different time points. FIG. 5E shows quantification of tumor bioluminescence values in different treatment groups over time. Data are shown as means ± SEM. Statistical significance on day 17 was determined via one-way ANOVA with Tukey’s HSD post-hoc analysis. FIG. 5F shows bioluminescence images of excised orthotopic tumors upon animal death. FIG. 5G shows bioluminescence images of excised organs upon animal death. FIG. 5H shows quantification of excised tumor and organ bioluminescence values at the end of study. Values are shown as mean ± SEM. Statistical significance between organs of different groups was determined via one-way ANOVA with Tukey’s HSD post-hoc analysis. FIG. 51 shows representative images of H&E stained lung sections. Scale bars for full sections are 3 mm and high magnification insets are 100 pm. FIG. 5J shows representative images for IHC staining of apoptosis marker cleaved caspase 3, scale bar is 3 mm. FIG. 5K shows flow cytometric quantification of immune cells from 4T1 tumors, (FIG. 5K) all T cells (CD3+), (FIG. 5L) CD4+ T cells, (FIG. 5M) CD8+ T cells and (FIG. 5N) NK cells. Statistical significance was determined via one-way ANOVA with Tukey’s HSD post-hoc analysis. For all plots in this figure; *P<0.05, **P<0.01, ***P<0.001. For all unbracketed groups, P>0.05.

[0026] FIG. 6A shows a survival plot based on Metabric data set. Low E-cad expression is defined as 50 percentile or lower, while high E-cad expression is 50 percentile and above. N = 1902 patients. FIG. 6B shows maximum-intensity projections of confocal microscopy images of live- dead signal in E-cad+ and E-cad- organoids, different z heights are represented in FIGs. 1A-1G. Scale bar = 200 pm, inset = 50 pm. FIG. 6C shows cell proliferation of organoids assessed via PrestoBlue on days 1, 4, 7. Data are presented as mean ± SEM (n = 3 in quintuplicate). Statistical significance was determined using a two-sided unpaired t test. FIG. 6D shows a5 1 integrin expression on MDA-MB-231 organoids. Statistical significance was determined using a two- sided unpaired t test. FIG. 6E shows cCyo-TEM image of PR_b liposomes encapsulating iMEK+DOX. Scale bar = 200 nm. FIG. 6F shows MDA-MB-231 monolayer uptake experiment of PR b functionalized liposomes and non-targeting liposomes. Statistical significance was determined using a two-sided unpaired t test. FIG. 6G shows 184B5 healthy mammary cell uptake of liposomes. Statistical significance was determined using a two-sided unpaired t test. FIG. 6H shows IC50 of DOX and iMEK in both E-cad+ and E-cad- organoids. Data are presented as mean ± SEM (n = 3). For all plots in this figure; *P<0.05, **P<0.01, ***P<0.001. FIG. 7A shows cell viability of MDA-MB-231 E-cad+ and E-cad- organoids with PR_b functionalized liposomes and non-targeted liposomes loaded with various concentrations of iMEK and DOX. FIG. 7B shows E-cad+ organoids sustained release results for intermediate measurements (organoid 2 and 3) to complete data shown in FIGs. 2A-2H. FIG. 7C shows E- cad- organoid sustained release results for all organoid batches. For all plots in this figure data are presented as mean ± SEM (n = 3). Statistical significance determined via one-way ANOVA with Tukey’s HSD post-hoc analysis; *P<0.05, **P<0.01, ***P<0.001.

[0027] FIG. 8 shows liver, spleen, and kidney H&E from various time points throughout the hydrogel biodegradation study, scale bar is 250 pm.

[0028] FIG. 9A shows mouse weights during the iMEK oral delivery study, demonstrating early toxicity effects. FIG. 9B shows tumor weight of all groups after excision. Results are presented as mean ± SEM (n = 7-10) and statistical significance determined via one-way ANOVA with Tukey’s HSD post-hoc analysis; *P<0.05, **P<0.01, ***P<0.001. FIG. 9C shows H&E and IHC for pERK and Ki67 staining of primary tumors, scale bar is 3 mm. FIG. 9D shows RES organ assessment (spleen, liver, kidney) via H&E staining, scale bar is 200 pm. FIG. 9E shows H&E staining of lung sections from all groups. Scale bar is 3 mm and inset scale bar is 100 pm. All tissues were harvested upon animal termination criteria.

[0029] FIG. 10A shows tumor volume progression over time, measured with calipers in an orthotopic 4Tl-luc tumor model of TNBC. Data are presented as mean ± SEM (n = 7-9) and statistical significance determined via one-way ANOVA with Tukey’s HSD post-hoc analysis; *P<0.05, **P<0.01, ***P<0.001. FIG. 10B shows radiance images of tumors from Gel-NP(iMEK+DOX) group. FIG. 10C shows H&E staining of tumors from different treatment groups. Scale bar = 3 mm, inset = 100 pm. FIG. 10D shows RES organ assessment via H&E staining of kidney, liver, spleen, and heart for all groups, scale bar is 250 pm. FIG. 10E shows weight of spleens from different treatment groups. Statistical significance determined via one-way ANOVA with Tukey’s HSD post-hoc analysis; *P<0.05, **P<0.01, ***P<0.001. FIG. 10F shows IHC staining of tumors for Ki67. Scale bar = 3 mm. FIG. 10G shows IHC staining of tumors for cleaved caspase 3. Scale bar = 3 mm. All tissues were harvested upon animal termination criteria or end of the study.

[0030] FIG. 11 shows gating strategy for quantifying immune cells in 4T1 tumors.

[0031] FIGs. 12A-12F show flow cytometric quantification of (FIG. 12A) macrophages, (FIG. 12B) CD206+ macrophages, (FIG. 12C) CD86+ macrophages, (FIG. 12D) dendritic cells, (FIG. 12E) neutrophils and (FIG. 12F) B cells. Statistical significance was determined via one-way ANOVA with Tukey’s HSD post-hoc analysis. For all pairs in this figure, P>0.05.

[0032] DETAILED DESCRIPTION

[0033] The efficacy of protein kinase inhibitors (e.g., MEK inhibitors, PI3K inhibitors, or RAF inhibitors) can be improved by combining them with chemotherapeutics, due to the effectiveness of protein kinase inhibitors observed in pre-clinical experiments. However, the inclusion of protein kinase inhibitors in a chemotherapeutic regime for cancer (e.g., breast cancer) patients may not be feasible with systemic delivery.

[0034] As described herein, in some embodiments, a drug delivery system utilizing a thermosensitive and biodegradable hydrogel (e.g., PVLA-PEG-PVLA hydrogel) that encapsulates lipid nanoparticles (e.g., PR_b functionalized liposomes) can allow for the sustained release of a protein kinase inhibitor, (e.g., a MEK inhibitor, e.g., PD0325901 (iMEK)) and a chemotherapeutic agent (e.g., doxorubicin (DOX)) to cancer (e.g., triple-negative breast cancer (TNBC)) cells. In some embodiments, a hydrogel -nanoparticle system can be injected directly at the tumor site functioning as a local reservoir for the extended release of the protein kinase inhibitor, (e.g., a MEK inhibitor, e.g., PD0325901 (iMEK)) and the chemotherapeutic agent (e.g., DOX) to both inhibit the proliferative mechanism of the cancer and induce apoptosis.

[0035] This disclosure describes a design of a drug delivery system that allows for the local and extended release of therapeutics to proliferating cancer cells, as well as methods of using the same. Provided herein are pharmaceutical compositions that include (a) a lipid nanoparticle comprising a protein kinase inhibitor and a chemotherapeutic agent; and (b) a hydrogel comprising a thermosensitive and biodegradable polymer. Also provided herein are methods of treating a cancer in a subject that include administering to the subject a therapeutically effective amount of any one of the pharmaceutical compositions described herein.

[0036] Various non-limiting aspects of such methods and compositions are described herein and can be used in any combination without limitation.

[0037] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0038] As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0039] As used herein, a “cell” can refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.

[0040] As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder, or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more signs or symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered. In some embodiments, a subject is a human.

[0041] Pharmaceutical Compositions

[0042] Provided herein are pharmaceutical compositions that include (a) a lipid nanoparticle comprising a protein kinase inhibitor (e.g., MEK inhibitor, PI3K inhibitor, or RAF inhibitor) and a chemotherapeutic agent; and (b) a hydrogel comprising a thermosensitive and biodegradable polymer. In some embodiments, a pharmaceutical composition can include (a) a lipid nanoparticle comprising a MEK inhibitor and a chemotherapeutic agent; and (b) a hydrogel comprising a thermosensitive and biodegradable polymer.

[0043] As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, a pharmaceutical composition is suitable for administration to a human or animal subject, e.g., via a particular route of administration (e.g., intratumoral). In some embodiments, an active agent is present in a pharmaceutical composition in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0044] Lipid Nanoparticle including a Protein Kinase Inhibitor and Chemotherapeutic Agent

[0045] In some embodiments, a lipid nanoparticle can include a protein kinase inhibitor (e.g., MEK inhibitor, PI3K inhibitor, or RAF inhibitor) and a chemotherapeutic agent. As used herein, a “lipid nanoparticle” refers to a nanoparticle composed of lipids, wherein a lipid nanoparticle comprises a lipid membrane or a lipid outer layer and an aqueous core. Lipid nanoparticle (LNP) synthesis comprises (i) lipid component preparation, which involves the combination of different types of lipids to achieve the desired lipid composition; and (ii) aqueous phase preparation, which includes the hydrophilic substance to be encapsulated by the LNP. In some embodiments, a hydrophobic substance can be encapsulated in the lipid membrane. In some embodiments, a lipid nanoparticle can include a liposome, lipid nanoparticle, solid lipid nanoparticle, nanostructured lipid carrier, or cationic lipid-nucleic acid complex. In some embodiments, lipid nanoparticles can be used as a pharmaceutical drug delivery system.

[0046] In some embodiments, a lipid nanoparticle comprises a lipid. In some embodiments, the lipid nanoparticle comprises cholesterol, ALC-0315, ALC-0159, SM-102, D-Lin-MC3-DMA, D- Lin-DMA, D-Lin-D-DMA, D-Lin-KC2-DMA, DODMA, DOTAP, L319, MC3, amino alcohol lipids, DPPC, DSPC, DPPE, DSPE, DPPS, DSPS, DMG-PEG, DSG-PEG, DOPE-PEG, DSPE- PEG, DPPE -PEG, 1,2 DSPC, or PEG lipids where the PEG is modified to include any of the following end groups -COOH, -OH, -SH, -NHS, -mal, -amine, -azide, -alkyne, -DBCO, - aldehyde, -biotin, -vinylsulfone, or any combinations thereof. In some embodiments, a lipid nanoparticle comprises phosphatidyl serine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylinositol (PI), dimyristoyl phosphatidylglyerol (DMPG) and sphingomyelin (SM), l,2-dioleoyl-3 -trimethylammoniumpropane (DOTAP), l,2-dioleoyloxy-3- dimethylammoniumpropane (DODAP) and analogues, l,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phosphatidylcholine (PC) and dimyristoyl phosphatidylcholine (DMPC), 1,2-di-O-octadecenyl- 3-trimethylammoniumpropane (DOTMA), l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), dioctadecyldi-methylammonium (DODA(Br) / DDAB), dioctadecyldimethylammoniumchloride (DODAC), l,2-dimyristoyloxypropyl-l,3- dimethylhydroxy ethylammonium (DMRIE), 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA) analogues, cholesterol, sphingomyelin (SM), or any combinations thereof.

[0047] In some embodiments, a lipid nanoparticle can include a mixture of lipids suitable to form lipid nanoparticles and / or liposomes for encapsulation of a therapeutic agent (e.g., a chemotherapeutic agent or inhibitor). In some embodiments, a suitable lipid solution is chloroform based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure chloroform (i.e., 100% chloroform). In some embodiments, a suitable lipid solution is ethanol based. In some embodiments, a suitable lipid solution is isopropyl alcohol based. In some embodiments, a suitable lipid solution is dimethylsulfoxide-based. In some embodiments, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, chloroform, ethanol, isopropyl alcohol and dimethylsulfoxide.

[0048] In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of about 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 6.0 mg / ml, 7.0 mg / ml, 8.0 mg / ml, 9.0 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or 100 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from about 0.1-100 mg / ml, 0.5-90 mg / ml, 1.0-80 mg / ml, 1.0-70 mg / ml, 1.0-60 mg / ml, 1.0-50 mg / ml, 1.0-40 mg / ml, 1.0-30 mg / ml, 1.0-20 mg / ml, 1.0-15 mg / ml, 1.0-10 mg / ml, 1.0-9 mg / ml, 1.0-8 mg / ml, 1.0-7 mg / ml, 1.0-6 mg / ml, or 1.0-5 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration up to about 100 mg / ml, 90 mg / ml, 80 mg / ml, 70 mg / ml, 60 mg / ml, 50 mg / ml, 40 mg / ml, 30 mg / ml, 20 mg / ml, or 10 mg / ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a molar ratio, wherein the mixture of desired lipids comprises DPPC:cholesterol:DPPE-PEG750 in chloroform at a molar ratio of 64:35: 1. In some embodiments, any desired lipids may be mixed at any ratios suitable for encapsulating therapeutic agents (e.g., chemotherapeutic agents or inhibitors).

[0049] In some embodiments, the lipid nanoparticle targets asPi integrin. In some embodiments, a lipid nanoparticle includes a fibronectin mimetic peptide, wherein the fibronectin mimetic peptide specifically binds to ocsP i integrin. In some embodiments, a lipid nanoparticle comprises a fibronectin-mimetic peptide on a surface of the lipid nanoparticle, wherein the fibronectinmimetic peptide is a PR_b peptide. In some embodiments, a lipid nanoparticle comprises a liposome. In some embodiments, a lipid nanoparticle comprises a micelle.

[0050] In some embodiments, a lipid nanoparticle can include a protein kinase inhibitor (e.g., MEK inhibitor, PI3K inhibitor, and RAF inhibitor) and a chemotherapeutic agent (e.g., doxorubicin). In some embodiments, a lipid nanoparticle can include a MEK inhibitor and a chemotherapeutic agent. In some embodiments, a lipid nanoparticle can include a PI3K inhibitor and a chemotherapeutic agent. In some embodiments, a lipid nanoparticle can include a RAF inhibitor and a chemotherapeutic agent.

[0051] As used herein, a “protein kinase inhibitor” refers to a molecule that blocks a protein kinase, wherein a protein kinase is an enzyme that modifies other proteins by chemically adding the terminal y-phosphate group of adenosine triphosphate (ATP) to serine, threonine or tyrosine residues which is also known as phosphorylation. Protein kinases are known to help control important functions, such as cell signaling, metabolism, division, and survival. In some embodiments, certain kinases are more active in some types of cancer cells and blocking them may help keep the cancer cells from growing. In some embodiments, a protein kinase inhibitor may also block the growth of new blood vessels that tumors need to grow. In some embodiments, a protein kinase inhibitor can be used to treat cancer, wherein the kinase inhibitor is efficacious for the treatment of cancer by targeting specific mutations that drive tumorigenesis. In some embodiments, a protein kinase inhibitor includes a MEK inhibitor. In some embodiments, a MEK inhibitor includes PD0325901, AZD6244, trametinib, cobimetinib, selumetinib, or binimetinib. In some embodiments, a protein kinase inhibitor includes a PI3K inhibitor. In some embodiments, a PI3K inhibitor includes copanlisib, buparlisib, alpelisib, duvelisib, umbralisib, or idelalisib. In some embodiments, a protein kinase inhibitor includes a RAF inhibitor. In some embodiments, a RAF inhibitor includes vemurafenib or dabrafenib. In some embodiments, a protein kinase inhibitor includes a cyclin dependent kinase 4 / 6 inhibitor. In some embodiments, a cyclin dependent kinase 4 / 6 inhibitor includes abemaciclib, ribociclib, or trilaciblib. In some embodiments, a protein kinase inhibitor includes a Bruton kinase inhibitor. In some embodiments, a Bruton kinase inhibitor includes acalabrutinib. In some embodiments, a protein kinase inhibitor includes a KRAS inhibitor. In some embodiments, a KRAS inhibitor includes adagrasib or sotorasib. In some embodiments, a protein kinase inhibitor includes an EGFR inhibitor. In some embodiments, an EGFR inhibitor includes afatinib, erlotinib, gefitinib, mobocertinib, or osimertinib. In some embodiments, a protein kinase inhibitor includes a HER1 inhibitor. In some embodiments, a HERl inhibitor includes erlotinib. In some embodiments, a protein kinase inhibitor includes a HER2 inhibitor. In some embodiments, a HER2 inhibitor includes afatinib, neratinib, palbociclib, or tucatinib. In some embodiments, a protein kinase inhibitor includes a HER1,2,3 inhibitor. In some embodiments, a HER1,2,3 inhibitor includes dacomitinib. In some embodiments, a protein kinase inhibitor includes a ALK inhibitor. In some embodiments, an ALK inhibitor includes alectinib, brigatinib, ceritinib, or crizotinib. In some embodiments, a protein kinase inhibitor includes an ABL1 Myristoyl inhibitor. In some embodiments, an ABL1 Myristoyl inhibitor includes asciminib. In some embodiments, a protein kinase inhibitor includes a PDGFRA inhibitor. In some embodiments, a PDGFRA inhibitor includes avapritinib, ripretinib, or nintedanib. In some embodiments, a protein kinase inhibitor includes a PDGFRB inhibitor. In some embodiments, a PDGFRB inhibitor includes tivozanib, or nintedanib. In some embodiments, a protein kinase inhibitor includes a PDGF inhibitor. In some embodiments, a PDGF inhibitor includes regorafenib, or sunitinib. In some embodiments, a protein kinase inhibitor includes a KIT inhibitor. In some embodiments, a KIT inhibitor includes avapritinib, ripretinib, sunitinib, or tivozanib. In some embodiments, a protein kinase inhibitor includes a VEGFR 1-3 inhibitor. In some embodiments, a VEGFR 1-3 inhibitor includes axitinib, pazopanib, regorafenib, sorafenib, tivozanib, nintedanib, or pegaptanib. In some embodiments, a protein kinase inhibitor includes a VEGFR-2 inhibitor. In some embodiments, a VEGFR-2 inhibitor includes cabozantinib, or vandetanib. In some embodiments, a protein kinase inhibitor includes a BRAF inhibitor. In some embodiments, a BRAF inhibitor includes binimetinib, dabrafenib, encorafenib, or vemurafenib. In some embodiments, a protein kinase inhibitor includes a proteasome inhibitor. In some embodiments, a proteasome inhibitor includes bortezomib, or carfilzomib. In some embodiments, a protein kinase inhibitor includes a BCR- ABL inhibitor. In some embodiments, a BCR-ABL inhibitor includes bosutinib, dasatinib, nilotinib, or ponatinib. In some embodiments, a protein kinase inhibitor includes a MET inhibitor. In some embodiments, a MET inhibitor includes cabozantinib, capmatinib, or tepotinib. In some embodiments, a protein kinase inhibitor includes a mutant IDH-1 inhibitor. In some embodiments, a mutant IDH-1 inhibitor includes olutasidenib. In some embodiments, a protein kinase inhibitor includes a mutant IDH-2 inhibitor. In some embodiments, a mutant IDH- 2 inhibitor includes enasidenib. In some embodiments, a protein kinase inhibitor includes a NTRK inhibitor. In some embodiments, a NTRK inhibitor includes entrectinib. In some embodiments, a protein kinase inhibitor includes a ROS1 inhibitor. In some embodiments, a RO SI inhibitor includes entrectinib. In some embodiments, a protein kinase inhibitor includes a FGFR inhibitor. In some embodiments, a FGFR inhibitor includes erdafitinib, futibatinib, pemigatinib, or nintedanib. In some embodiments, a protein kinase inhibitor includes a JAK-2 inhibitor. In some embodiments, a JAK-2 inhibitor includes fedratinib, or pacritinib. In some embodiments, a protein kinase inhibitor includes a JAK-1 / 2 inhibitor. In some embodiments, a JAK-1 / 2 inhibitor includes momelotinib, or ruxolitinib. In some embodiments, a protein kinase inhibitor includes a FLT3 inhibitor. In some embodiments, a FLT3 inhibitor includes midostaurin, pacritinib, or pexidartinib. In some embodiments, a protein kinase inhibitor includes a PARP inhibitor. In some embodiments, a PARP inhibitor includes niraparib, olaparib, rucaparib, or talazoparib. In some embodiments, a protein kinase inhibitor includes a CSF1 inhibitor. In some embodiments, a CSF1 inhibitor includes pexidartinib. In some embodiments, a protein kinase inhibitor includes a RET inhibitor. In some embodiments, a RET inhibitor includes pralsetinib, or selpercatinib. In some embodiments, a protein kinase inhibitor includes a Hedgehog inhibitor. In some embodiments, a Hedgehog inhibitor includes sonidegib, or vismodegib. In some embodiments, a protein kinase inhibitor includes a BTK inhibitor. In some embodiments, a BTK inhibitor includes zanubrutinib. In some embodiments, a protein kinase inhibitor includes a Janus kinase inhibitor. In some embodiments, a Janus kinase inhibitor includes abrocitinib, baricitinib, ritlecitinib, tofacitinib, or upadacitinib. In some embodiments, a protein kinase inhibitor includes a Tyrosine Kinase inhibitor. In some embodiments, a Tyrosine Kinase inhibitor includes deucravacitinib, or fostamatinib.

[0052] As used herein, a “chemotherapeutic agent” can refer to one or more pro-apoptotic, cytostatic and / or cytotoxic agents, for example specifically including agents utilized and / or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In some embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g. microtubule targeting agents such as taxanes, maytansine and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some embodiments, a chemotherapeutic agent comprises vincristine, prednisone, dexamethasone, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, nivolumab, pembrolizumab, durvalumab, atezolizumab, radioisotopes, monomethyl auristatin E (MMAE; e.g., vedotin), calicheamicins, deruxtecan, DM1, and any combinations thereof. In some embodiments, a chemotherapeutic agent can include doxorubicin.

[0053] Hydrogel including a Thermosensitive and Biodegradable Pol mer

[0054] In some embodiments, any of the pharmaceutical compositions described herein can include a hydrogel comprising a thermosensitive and biodegradable polymer. As used herein, a “hydrogel” refers to a 3D network of amphiphilic polymer chains. A hydrogel includes a polymer that will self-assemble to form a three-dimensional (3D) hydrogel network. In some embodiments, a hydrogel polymer can include any convenient hydrogel polymers, such as, but not limited to, acrylamide and derivatives thereof (e.g., alkyl acrylamide), bis-acrylamide and derivatives thereof (e.g., N,N'-alkylene bis-acrylamide, such as N,N' -methylenebisacrylamide), acrylate and derivatives thereof (e.g., sodium acrylate or alkyl acrylate), methacrylate and derivatives thereof (e.g., alkyl methacrylate or methacryloyl), bis-acrylate and derivatives thereof, polyacrylamide and derivatives thereof, poly(ethylene glycol) (PEG) and derivatives thereof (e g., PEG-acrylate (PEG-DA), PEG methacrylate (PEGMA), PEG-RGD), polyethylene oxide (PEG) and derivatives thereof, gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols (PVA) and derivatives thereof, polycaprolactone (PCL) and derivatives thereof, polypropylene fumarate) (PPF) and derivatives thereof, poly(lactic-co-glycolic) acid (PLGA or PLG) and derivatives thereof, poly(lactic acid) (PLA, PDLA, PDLLA, PLLA) and derivatives thereof, poly(valerolactone) (PVL) and derivatives thereof, poly(aldehyde guluronate) and derivatives thereof, polyanhydrides and derivatives thereof, polypropylene glycol and derivatives thereof, polytetramethylene oxide, polyvinyl pyrrolidone, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), cellulose, collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, heparin, fibrin, alginate, glutaraldehyde, protein polymers, methylcellulose, and the like, or combinations thereof. In some embodiments, a hydrogel polymer can include poly(N-alkyl) substituted acrylamides (e.g., Poly(N-isopropylacrylamide) (PNIPAAm), Poly(N-iso-propylmethacrylamide) (PNIPMAM), Poly(N-cyclopropylacrylamide) (PNCPAL), Poly(N, N-di ethylacrylamide) (PDEA), PolyN-(2, 2-Dimethyl-l, 3-dioxan-5-yl)Methacrylamide (PNDMM), PolyN-(2, 2-Dimethyl-l, 3-dioxan-5- yl)Acrylamide (PNDMA)), poly(N-vinylalkylamides) (e.g., Poly(N-vinylcaprolactam) (PVCL)), poly ethers (e.g., Poly(Polyethers methyl vinyl ether) (PMVE), Poly(ethylene oxide)- Poly(propylene oxide) (PEO-PPO)), poloxamer (e.g., Poly(ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) (PEO-PPO-PEO), PLGA-PEG-PLGA, or PEG-PLLA-PEG.

[0055] For any compounds described herein (e.g., monomers, catalysts, etc.), salt forms may be employed (e.g., sodium salts, hydrochloride salts, and the like), and ionic forms may be employed (e.g., anionic or cationic forms). In some embodiments, a thermosensitive and biodegradable hydrogel polymer comprises a poly(6-valerolactone-co-lactide)-£>-poly(ethylene- glycol)-Z>-poly(8-valerolactone-co-lactide) PVLA-PEG-PVLA triblock copolymer. In some embodiments, a hydrogel is a thermosensitive and biodegradable hydrogel. As used herein, a “thermosensitive hydrogel” refers to a hydrogel that responds to changes in temperature and usually undergoes a sol-gel phase transition when the temperature changes from room to physiological temperature. In some embodiments, a thermosensitive hydrogel comprises triblock copolymers made up from poly(ethylene glycol) (PEG) linked to hydrophobic polymer blocks. As used herein, a “biodegradable hydrogel” refers to a hydrogel that has the capability to be degraded into biocompatible, nontoxic, and less complex materials that can be easily eliminated from a subject’s body. In some embodiments, a biodegradable hydrogel comprises a biodegradable polymer derived from various natural, semi synthetic, and synthetic sources. In some embodiments, a biodegradable hydrogel comprises a hydrogel polymer that can include, but not limited to, collagen / gelatin, chitosan, hyaluronic acid, chondroitin sulfate, alginate, agar / agarose, fibrin, PEG / PEO, PVA, PPF / OPF, PNIPAAm, PEO-PPO-PEO, PLGA-PEG- PLGA, PEG-PLLA-PEG, poly(aldehyde guluronate), or polyanhydrides.

[0056] Examples of hydrogels suitable for use in any one of the pharmaceutical compositions described herein are described in U.S. Patent Application Serial No. 63 / 180,995, U.S. Patent Application Serial No. 63 / 214,056, Vidyasagar et al., ACS Macro Lett. 2017 Oct 17;6(10): 1134- 1139, and Shabana et al., Int J Pharm. 2021 Jan 25:593: 120139, the entire contents of each of which are incorporate herein by reference.

[0057] In some embodiments, a lipid nanoparticle comprises a protein kinase inhibitor (e.g., MEK inhibitor, PI3K inhibitor, or RAF inhibitor) and a chemotherapeutic agent, wherein the lipid nanoparticle is encapsulated in a hydrogel. In some embodiments, a lipid nanoparticle comprises a protein kinase inhibitor (e.g., MEK inhibitor, PI3K inhibitor, or RAF inhibitor) and a chemotherapeutic agent, wherein the protein kinase inhibitor and / or the chemotherapeutic agent is conjugated to a hydrogel polymer.

[0058] Methods of Treatment

[0059] Provided herein are method of treating cancer in a subject that includes administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions described herein. In some embodiments, a therapeutically effective amount of any of the pharmaceutical compositions described herein is administered to a subject, thereby treating a cancer in the subject. As used herein, the terms “cancer” and “tumor”, refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. Exemplary cancers that may be treated with a composition or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and Non-Hodgkin's Lymphomas. Exemplary cancers that may be treated with a composition or method provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, breast cancer, triple negative breast cancer, invasive ductal carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, metastatic brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a cancer cell from the cancer expresses oc5(31 integrin on the cancer cell surface. In some embodiments, a cancer is a breast cancer, colon cancer, pancreatic cancer, lung cancer, prostate cancer, brain cancer, or melanoma. In some embodiments, a cancer is a triple negative breast cancer (TNBC). In some embodiments, a cancer is an E-cadherin positive cancer.

[0060] As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be parenteral, intra-arterial, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), etc. In some embodiments, administration can be intratumoral. In some embodiments, administration can include local administration in the vicinity of a tumor. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0061] As used herein, an “effective amount” or a “therapeutically effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease. A “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder. For example, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0062] As used herein, the term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, stabilizes one or more characteristics of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. For example, in some embodiments, term “therapeutically effective amount”, refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse a cancer-supportive process occurring in said individual, or will enhance or increase a cancer-suppressive process in said individual.

[0063] In the context of cancer treatment, a “therapeutically effective amount” is an amount which, when administered to an individual diagnosed with a cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual. A particularly preferred “therapeutically effective amount” of a composition described herein reverses (in a therapeutic treatment) the development of a malignancy or helps achieve or prolong remission of a malignancy. A pharmaceutically effective amount administered to an individual to treat a cancer in that individual may be the same or different from a pharmaceutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a “cure” for cancer; rather the methods of treatment are directed to the use of the described compositions to “treat” a cancer, i.e., to affect a desirable or beneficial change in the health of an individual who has cancer. Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, a stabilization of patient condition, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise), improved feeling of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as the result of treatments described herein) may also be assessed by taking samples of cancer cells from the site of a tumor (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to monitor the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype. In some embodiments, a pharmaceutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a pharmaceutically effective amount may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0064] For any pharmaceutical composition described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art. In some embodiments, as is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0065] In some embodiments, the methods provided herein can be used to reduce or eliminate the number of cancer cells present within a subject having a cancer. For example, any one of the methods described herein can be used to reduce the number of cancer cells present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the methods described herein can be used to reduce the size (e.g., volume) of one or more tumors present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some embodiments, the number of cancer cells present within a subject being treated can be monitored. Any appropriate method can be used to determine whether or not the number of cancer cells present within a subject is reduced. For example, imaging techniques can be used to assess the number of cancer cells present within a subject.

[0066] In some embodiments, any one of the methods provided herein can be used to improve survival of a subject having a cancer. For example, any one of the methods described herein can be used to improve the survival of a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the methods described herein can be used to improve the survival of a subject having cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more). An effective amount (e.g., effective dose) of a pharmaceutical composition can vary depending on the severity of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and / or the judgment of the treating physician.

[0067] An effective amount of a pharmaceutical composition can be any amount that can treat a subject having a cancer without producing significant toxicity to the subject. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., a triple negative breast cancer) may require an increase or decrease in the actual effective amount administered.

[0068] The frequency of administration of a pharmaceutical composition can be any frequency that can treat a subject having a cancer without producing intolerable toxicity to the subject. For example, the frequency of administration can be once a day, once a week, once every 2 weeks, or once every 4 weeks. In some embodiments, an administration can include a continuous infusion of a pharmaceutical composition. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a pharmaceutical composition can include rest periods. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., severity of cancer) may require an increase or decrease in administration frequency.

[0069] An effective duration for administering a pharmaceutical composition can be any duration that treat a subject having a cancer without producing significant toxicity to the subject. For example, the effective duration can vary from several days to several weeks, months, or years. In some embodiments, the effective duration for the treatment of a subject can range in duration from about one month to about 10 years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition (e g., a breast cancer) being treated. In some embodiments, any of the pharmaceutical compositions described herein can be used as the sole active agent to treat a subject having a cancer. In some embodiments, one or more (e.g., one, two, three, four, five or more) additional treatments (e.g., therapeutic interventions) that are effective to treat cancers can be used. For example, a subject in need thereof (e.g., a subject having a cancer) can be administered the pharmaceutical composition in combination with one or more therapeutic interventions. Examples of therapeutic interventions that can be used as described herein to treat a cancer include, without limitation, cancer surgeries, radiation therapies, chemotherapies, and any combinations thereof. In some embodiments, the one or more additional treatments that are effective to treat cancers can be performed at the same time as the administration of the pharmaceutical composition. In some embodiments, the one or more additional treatments that are effective to treat cancers can be performed before and / or after the administration of the pharmaceutical composition.

[0070] EXAMPLES

[0071] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0072] Materials

[0073] Polyethylene glycol 1500 (PEG1500) was purchased from Millipore Sigma, D,L-lactide was purchased from Acros Organics, 8- valerolactone was purchased from Alfa Aesar, stannous octoate, sephadex G-50 and calcein were purchased from Sigma- Aldrich.

[0074] Dipalmitoylphosphatidylcholine (DPPC), cholesterol and l ,2-dipalmitoyl-.w-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (DPPE-PEG750) (ammonium salt) were purchased from Avanti Polar Lipids. PrestoBlue assay was purchased from Thermo Fisher Scientific. All organic solvents (HPLC grade) were purchased from Sigma-Aldrich and Fisher Scientific. All other chemicals and materials were purchased from Sigma-Aldrich.

[0075] Synthesis of PVLA-PEG-PVLA tri-block copolymer

[0076] The PVLA-PEG-PVLA triblock copolymer was synthesized by utilizing bulk ringopening polymerization of 8-valerolactone and D,L-lactide, with PEG as a macro-initiator, and stannous octoate as a catalyst as previously published, with full material characterization. Briefly, PEG was purified by drying at 50°C overnight in a vacuum oven, 5-valerolactone was vacuum distilled to remove polymerized monomer, and D, L-lactide was recrystallized in ethyl acetate three times. For the polymer synthesis 10.94 g of PEG were first transferred to a reactor, then 5.10 g of 5-valerolactone and 20.41 g of D, L-lactide were added along with stannous octoate (0.02 g / mL). The reaction was carried out at 130°C for 14 h under argon atmosphere. The reaction mixture was cooled to room temperature then dissolved in dichloromethane and precipitated in ice cold ethyl ether. The precipitate was dried in a vacuum oven with no heat for 3 days and stored at -20°C until further use.

[0077] Cryo-TEM studies

[0078] The morphologies of self-assembled polymer nanoparticles were observed with cryo- TEM at various temperatures. To prepare samples, polymer solution in water was prepared at 20 w / v% and then further diluted to 0.25 w / v% followed by heating to 25°C and 37°C for 1 h using a dry bath. Samples were deposited onto carbon copper grids pretreated with glow discharge and vitrified in liquid ethane by Vitrobot using the following parameters: 3 s blot time, 0 offset, 3 s wait time, 0 s drain time, 0 blot force, 100% relative humidity. Prepared sample grids were stored under liquid nitrogen until they were transferred to a F200C Talos TEM operated with an acceleration voltage of 200 kV (Integrated Imaging Center, Institute for NanoBioTechnology) and images were acquired using a Ceta camera. iMEK+DOX-loaded liposomes were also examined with cryo-TEM. Approximately 5 pL of aqueous suspension of liposomes at 9 mM lipids were deposited onto carbon copper grids and vitrified in liquid ethane by Vitrobot using the following parameters: 2 s blot time, 0 offset, 0 s wait time, 0 s drain time, 0 blot force, 100% relative humidity. The prepared samples were then stored and imaged.

[0079] Preparation and characterization of liposomes

[0080] Liposomes were prepared using the dry lipid film technique. Stock solutions of DPPC, cholesterol and DPPE-PEG750 in chloroform were mixed with the molar ratio of 64:35: 1. The lipid mixture was placed on a rotary evaporator at 50°C to remove organic solvent and then dried under vacuum overnight. For cellular uptake studies, the lipid film was rehydrated in 300 pM of sulfo-Cy5 dye in PBS (pH 7.4) for 1 h at 65°C. The vesicles were subjected to extrusion through 100 nm polycarbonate membranes for 21 cycles at 65°C. The unencapsulated dye was removed by gel permeation chromatography using Sephadex G-50 prepacked column.

[0081] PD0325901 (iMEK) (Selleckchem, S1036) loaded liposomes were prepared in a similar manner, where iMEK solution in chloroform was added to the lipid mixture at varying concentrations. The unencapsulated iMEK was removed by filtering the liposome suspension through 0.45 pm poly vinylidene fluoride (PVDF) membrane filter. Doxorubicin HC1 (DOX) (Selleckchem, S1208) loaded liposomes at different concentrations were prepared using an active loading procedure. The lipid film was rehydrated with 1 mL of 250 mM ammonium sulfate solution in Milli-Q water for 1 h at 65°C. The generated liposomes were subjected to extrusion as described above and the unencapsulated ammonium sulfate was removed through dialysis at 4°C for 1.5 h. The liposomal suspension was then mixed with stock solution of DOX in HEPES (Quality Biological, 118-089-721) for 3 h at 65°C. Unencapsulated DOX was removed through dialysis at 4°C overnight.

[0082] To assess encapsulation efficiency of iMEK and DOX the drug loaded liposomes were ruptured with ethanol to make a 50:50 water ethanol solution and centrifuged to pellet down undissolved lipids and obtain a clear supernatant. The supernatant was analyzed using UV-Vis carried out on a Spectramax M3 plate reader. iMEK was detected at 275 nm and DOX at 485 nm and concentrations were determined using a calibration curve.

[0083] PR_b functionalized liposomes were prepared using a PR_b peptide-amphiphile synthesized as previously described18and added to the lipid mixture at 5 mol% initial concentration. Lipid concentration was determined using a Stewart Assay, and final concentration of the peptide on the liposome surface was determined by the BCA protein assay (Thermo Fisher Scientific, 23225) following the manufacturer’s protocol. The actual peptide concentration on the surface of the PR_b functionalized liposomes was 3.4 ± 0.5 mol%. The particle size and zeta potential of liposomes were determined by a Zetasizer (Malvern Panalytical).

[0084] Preparation of hydro el encapsulating liposomes

[0085] A polymer solution of P VLA-PEG-P VL A in 1 mM HEPES was prepared at a concentration of 29 w / v% at 4°C. HEPES buffer was added, and the pH of the solution was titrated to 7.4 with 1 M NaOH resulting in a final gel concentration of 26.4 w / v%. Liposomes loaded with DOX / iMEK (2: 1 mol / mol) were mixed with the polymer solution at a 1 :5 v / v ratio by stirring at 4°C for 2 h. The resulting mixtures had a final polymer concentration of 22 w / v% and liposomes at 3 mM of lipids for in vitro and 9mM lipids in vivo. Solutions were placed in an incubator at 37°C to form the hydrogel-nanoparticle system. Gelation was observed in ~ 1 min and gels were allowed to form for 1 h before use.

[0086] Drug release studies

[0087] The release of drugs from hydrogels encapsulating iMEK / DOX-loaded liposomes was carried out as previously described. Briefly, the hydrogel-nanoparticle system was prepared as discussed above. 2.5 mL of the mixture was placed in a dialysis tube and allowed to gel at 37°C for 1 h. The dialysis tubes were placed in 20 mL of release media (PBS, with 0.1% v / v tween 80) and placed on a rotary shaker at 50 RPM. At different time points the release media was collected and replaced with prewarmed fresh media. The collected samples were analyzed using UV-Vis. For the release of free drug from the hydrogel 22 w / v% polymer was combined with DOX dissolved in PBS and iMEK dissolved in PBS with 1% v / v tween 80 for a final volume of 2.5 mL. The dialysis tubes were placed in 20 mL of release media and the experiment was performed as described above for the nanoparticle-hydrogel system. For the release of drugs from free liposomes in solution, the same protocol was followed with 2 mL of liposome solution placed in a dialysis tube in 20 mL of release media.

[0088] Cell culture

[0089] Human breast carcinoma cells MDA-MB-231, (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning, 10-013-CV) supplemented with 10% (v / v) fetal bovine serum (FBS, Corning, 35-010-CV) and 1% penicillin-streptomycin (Gibco, 15140-122). Human breast healthy epithelial cells 184B5 (ATCC), were cultured in Mammary Epithelial Cell Growth Medium (MEGM, Lonza CC-3150). Mouse breast carcinoma cells 4Tl-luc (ATCC) were cultured in RPML1640 ATCC modification (ThermoFisher, A1049101) supplemented with 10% (v / v) FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C and 5% CO2 in a humidified incubator during cell culture. To generate MDA-MB-231 E-cad knock-in cells, briefly, the lentiviral vector of E-cadherin-EGFP was generated from EGFP;pCS-CG (Addgene, 12154), via cloning full-length E-cadherin upstream of EGFP between the Nhe 1 and Age 1 sites of pCS-CG to generate an EGFP- fused protein. A scramble sequence was also inserted (Addgene, 162011) and the scramble control line was used in all experiments for the E-cad- cell line. Genetically modified cells were kept in puromycin selection during maintenance only.

[0090] Multi-compartment tumor organoids

[0091] Organoid cores were generated with high-concentration Matrigel (Corning, 354248) at a density of 10,000 cells / pL. 1 pL droplets of Matrigel / cell solution were made utilizing oil-in- water droplet technology as described previously. Cores were then wrapped in a 2 mg / mL collagen I gel. Briefly, collagen I (Coming, 354249) was mixed with reconstitution buffer and titrated to pH 7.4. 10 pL of collagen I matrix and a single Matrigel core were pipetted to generate the multi-compartment organoid. The PrestoBlue assay was used at different timepoints to determine the fold change in cell number over time.

[0092] Expression ofintegrin asfii

[0093] The expression of 0(501 integrin receptors on MDA-MD-231 organoids was investigated using flow cytometry. Organoids were allowed to grow for 4 days before cells were harvested and analyzed. Briefly the organoids were dissociated by first incubating in 20 pg / mL collagenase (Gibco, 17018029) for 30 min at 37°C. Organoids were then washed twice with PBS and incubated in Cell Recovery Solution (Corning, 354253) for 30 min at 4°C. Cells were pelleted and washed twice in PBS before being resuspended in FACS buffer (PBS, pH 7.4, 5% FBS, ImM sodium azide). Cells were then incubated with phycoerythrin (PE) conjugated anti-human (1 01 (BD Biosciences, 555617), or mouse IgG isotype control (BD Biosciences, 555749) at 1 : 100 dilution for 30 min. Cells were pelleted by centrifugation, washed twice with cold FACS buffer, and flow cytometric analysis was performed immediately using a BD FACSCanto (Integrated Imaging Center, Institute for NanoBioTechnology).

[0094] Liposome internalization in organoids via confocal microscopy

[0095] To study liposome internalization in 2D, 5,000 cell / well of MDA-MB-231 cells were seeded in a glass bottom 96-well culture plate. After cells were allowed to adhere overnight, cells were treated with 150 pM Cy5-loaded PR_b liposomes or non-targeted liposomes in media for 24 h at 37°C. Following treatment cells were washed with PBS three times and fixed with 4% paraformaldehyde solution for 15 min. Cell nuclei were then stained with Hoechst 33342 at 1 : 1000 dilution for 10 min and imaged.

[0096] To study internalization in 3D MDA-MB-231 cells were prepared in multi-compartment organoids as described above. Four-day old organoids were then treated with 300 pM Cy5- loaded PR_b liposomes or non-targeted liposomes in media for 72 h at 37°C. Following treatment cells were washed with PBS three times and fixed using 4% paraformaldehyde solution overnight. Cell nuclei were then stained with Hoechst 33342 at 1 :250 dilution for 1 hr. Organoids were then washed and placed on a glass Petri dish for imaging. Cells were imaged on a Nikon Al confocal microscope and multiple z-scans were collected for every field of view.

[0097] Confocal microscopy of organoids live / dead assay

[0098] Four-day old organoids were treated with a live / dead assay that was preformed according to manufacturer’s protocol. Briefly, cells were exposed to 2 pM calcein-AM and 3 pM propidium iodide for 4 h (Sigma-Aldrich, R37601). Following incubation, organoids were imaged live with a Nikon Al confocal microscope.

[0099] Cytotoxicity studies

[0100] The IC50 values of free drugs was determined by treating organoids with different concentrations of iMEK or DOX added directly to the culture media for 72 h. Following treatment, the cytotoxicity was measured using the PrestoBlue assay as compared to an untreated control. The log of drug concentration was then plotted against cell viability, the data fit using the Hill equation in Prism, and the IC50 value extracted.

[0101] The cytotoxicity of hydrogels encapsulating different therapeutics was carried out in a 96 well transwell plate. Hydrogels containing free drugs, targeted or non-targeted liposomes were loaded into the upper inserts and maintained at 37°C for 1 h. After incubating the organoids with hydrogels at 37°C for 72 h, the media for all treatment groups was exchanged for fresh media. After 24 h cytotoxicity was measured using the PrestoBlue assay. The same transwell inserts were immediately placed on top of new 4-day old organoids and the experiment was repeated on a total of four batches of organoids over 12 days. Cytotoxicity was calculated as the percent compared to non-treated controls. Bliss expectation was calculated as (A + B) - (A x B), where A and B are the fractional growth inhibitions induced by agents A and B at a given dose. Delta Bliss is calculated as the difference between the Bliss expectation and actual observed inhibition of the combination of A and B at a given dose.

[0102] Hydrogel safety and biodegradation

[0103] To determine the degradation profile and safety of the hydrogel, 5-week-old female BALB / c and NSG mice (Jackson Laboratories) were injected with the polymer solution and tracked over time. The hair was removed from the left hind flank of the mice, 200 pL of 22 w / v% hydrogel solution in PBS was injected subcutaneously, and the volume was measured via calipers over time. At specific time points animals were sacrificed and the injection site was excised for macroscopic observation. Samples were also fixed in 10% formalin and sectioned for H&E assessment. Liver, spleen, and kidneys were also harvested for cytotoxic assessment. The tissue scans were evaluated by a Doctor of Veterinary Medicine (DVM) and an American College of Veterinary Pathologists (ACVP) board-certified pathologist at the Phenotyping Core for pathology support at Johns Hopkins University.

[0104] Animal studies

[0105] All mouse experiments were carried out according to protocols approved by the Johns Hopkins University Animal Care and Use Committee in accordance with the NTH Guide for the Care and Use of Laboratory Animals. To prepare the human xenograft tumor model, 1 x 106MDA-MB-231 Ecad+ cells in a 1 : 1 mix of PBS:Matrigel were injected into the second mammary fat pad of 5-week-old female NSG mice (Jackson Laboratories). After the tumor reached -200 mm3mice were randomly assigned to 6 groups. Mice receiving hydrogel treatments were injected once intratumorally with 50 pL of hydrogel-nanoparticle solution, and two 125 pL peritumoral injections (300 pL total) containing an aqueous solution of 22 w / v% polymer and 9 mM lipids. Controls were injected in the same manner with PBS or empty hydrogel. Hydrogel injections contained liposomes with 1.5 mg / kg iMEK and 3.6 mg / kg DOX per 300 pL and were given every 14 days.

[0106] For intravenous administration of the nanoparticles a total of 0.75 mg / kg iMEK and 1.8 mg / kg DOX was given via once-weekly injection for five weeks. The tumor volume was calculated from x,y dimensions measured every three days with calipers and mouse weights were recorded. To mimic standard of care, iMEK was orally administered via peanut butter pellets daily at a dosage of 20 mg / kg for 5 days followed by a 2-day rest period. The treatment cycle was then repeated three times. Tumors were categorized as spheres or ellipses by calculating the difference between x and y. If x-y < 1 mm, the tumor volume was calculated using the volume formula for a sphere. If x-y > 1mm, the tumor volume was calculated using the volume formula for an ellipse. Tumor bearing mice were sacrificed when tumor volumes exceeded 1500 mm3or when body -weight loss exceeded 20%. Upon sacrifice, mouse organs were harvested and fixed in formalin and sent for sectioning and staining to the Johns Hopkins Oncology Tissue Services core.

[0107] For the syngeneic mouse model, 1 x 1054Tl-luc cells in a 1 : 1 mix of PBS:Matrigel were injected into the second mammary fat pad of 5-week old female BALB / c mice. After the tumor reached -200 mm3mice were randomly assigned to 3 groups and injected as in the previous mouse model. Mice receiving the hydrogel treatment were injected once intratumorally with 50 pL of hydrogel-nanoparticle solution, and two 125 pL peritumoral injections (300 pL total) containing an aqueous solution of 22 w / v% polymer and 9 mM lipids. Controls were injected in the same manner with PBS or empty hydrogel. Hydrogel injections contained liposomes with 1.5 mg / kg iMEK and 3.6 mg / kg DOX per 300 pL and were given every 14 days. Tumor burden was observed using an IVIS Spectrum imaging system as described below. Tumor bearing mice were sacrificed when tumor volumes exceeded 1500 mm3or when body -weight loss exceeded 20%. Upon sacrifice mouse organs were harvested and fixed in formalin and sent for sectioning and staining to the Johns Hopkins Oncology Tissue Services core. qPCR of harvested mouse tissue

[0108] Mouse lung tissue was broken down with a tissue homogenizer and DNA was extracted from tissue using PureLink Genomic DNA mini kit (ThermoFisher, KI 82002). qPCR was conducted with iTaq-SYBR Green (Bio-Rad, 1725121) using the Bio-Rad CFX Touch Real-Time PCR detection system. Primers were obtained from Integrated DNA Technologies.

[0109] In vivo bioluminescence and imaging

[0110] Mice were monitored by bioluminescence imaging for local tumor growth at predetermined time points. 10 min after intraperitoneal (i.p.) injection of D-luciferin (GOLDBIO, LUCNA-100) at a dosage of 150 mg / kg, mice were anesthetized with 2% isoflurane and imaged using an IVIS Spectrum imaging system (Perkin Elmer, Molecular Imaging Service Center). Ex vivo images of organs were acquired by sacrificing animals 10 min after i.p. injection. Organs were harvested and submerged in a 300 pg / mL luciferin bath for 5 min before imaging. Bioluminescence images were analyzed using Living Image software and fluorescence intensity was quantified as the average radiance (photons s'1cm’2sr’1).

[0111] Immunohistochemistry and H&E staining o f tissue sections

[0112] Immediately after excision, a portion of the tumors were fixed in formalin and then sent for paraffin embedding and staining to the Oncology Tissue Service core at JHMI. Tumor slices were stained for proliferation marker Ki67 and apoptotic marker cleaved caspase 3, in addition to H&E for visualization of the tumor microenvironment.

[0113] Tumor dissociation and myeloid and T-cell subset analyses

[0114] 4T1 tumors from mice were minced and incubated in RPMI medium containing collagenase / hyaluronidase and DNAse I (Stemcell Technologies) for 30 min at 37°C. The dissociated tumor was then passed through a 70 pm strainer, pelleted, and resuspended in ammonium chloride solution for 5 min at room temperature. Cells were then washed, counted, and resuspended at 2 x 106cells in 100 pL of BD Horizon Brilliant Stain Buffer (BD Biosciences) containing lx Tandem stabilizer (Biolegend). FcR on the cells were blocked with TruStain FcX (anti-mouse CD16 / CD32) Antibody (Biolegend). Cells were then stained with antibodies listed below:

[0115] Samples were analyzed using an LSRFortessa Flow Cytometer (BD Biosciences). Gating was done on FlowJo and subsequently immune cell subsets were quantified.

[0116] Example 1 - E-cad positive breast tumors preferentially uptake otsPi targeting liposomes and respond to MEK inhibition

[0117] To overcome the narrow therapeutic index limitation of MEK inhibitors given orally, a fibronectin mimetic peptide was employed, PR_b, to enhance the delivery of liposomes loaded with iMEK. It has been have demonstrated that PR_b functionalized liposomes can selectively bind to human TNBC MDA-MB-231 cells and effectively improve the delivery of chemotherapeutics. PR_b decorated liposomes have been shown to target the asPi integrin receptor, and have been shown to enhance cellular uptake in pancreatic cancer cells upon release from the PVLA-PEG-PVLA thermosensitive and biodegradable hydrogel in vitro. Clinically, E-cad expression correlates with a worse overall survival in breast cancer patients, further confirming that its classification as a tumor suppressor gene is no longer accurate (FIG. 6A). E-cad+ and E-cad- TNBC cells growth progression was studied in a novel multi-compartment tumor organoid system (FIG. 1A). Live-dead staining of cells was conducted in the organoids (FIG. IB, FIG. 6B), and a necrotic core was observed in both E-cad- and E- cad+ organoids on day 7. It was determined that the optimal intervention for nanoparticle delivery would be on day 4 of culture, when the proliferation rates (i.e., fold change in PrestoBlue measurements) between the E-cad+ and E-cad- organoids were not significantly different, thus, ensuring cell cycle changes did not impact uptake (FIG. 6C). With organoid growth behavior and optimal intervention point determined, it was next studied how E-cad manipulation impacted asPi integrin expression in the organoid system by conducting flow cytometry on cells cultured in the organoids (FIG. 6D). More than 2-fold increase in the expression of asPi integrin on the surface of MDA-MB-231 E-cad+ organoids was observed compared to MDA-MB-231 E-cad- organoids (FIG. 6D). MDA-MB-231 cells do not endogenously express E-cad, so the increase in otsPi integrin can be correlated with the gain of E- cad expression.

[0118] Targeted liposomes were generated with 3.4 ± 0.5 mol% PR_b on the surface, with a size of 126 ± 2.3 nm and a zeta potential of 12.2 ± 0.8 mV. This positive zeta potential developed with the inclusion of the positively charged PR_b peptide-amphiphile, as the non-targeted liposomes are 118 ± 1.8 nm with a zeta potential of -14.1 ± 1.1 mV (Table 1). Liposomes were visualized with cryogenic transmission electron microscopy (cryo-TEM) that showed unilamellar vesicles with the presence of a few multivesicular vesicles (FIG. 6E). To functionally demonstrate the preferential binding of PR_b liposomes to E-cad+ organoids, an uptake experiment was conducted with Cy5.5 loaded liposomes (FIG. 1C). The increase in ccsPi integrin expression on the surface of E-cad± organoids led to increased uptake of the PR_b liposomes when compared to E-cad- organoids (FIG. 1C), as anticipated based on the integrin expression results (FIG. 6D). Also, the benefit of adding a targeting peptide to the liposomes was demonstrated, as organoids showed minimal uptake of the non-targeting liposomes after 72 h (FIG. 1C). Furthermore, cell association of nanoparticles was compared with flow cytometry and it was found that TNBC cell monolayers showed 15 times the nanoparticle signal when compared to monolayers of non-cancerous, healthy breast cell line 184B5 (FIGs. 6F-6G), confirming the preferential uptake of PR_b functionalized liposomes by cancerous cells. With the confirmation that asP i integrin is an effective target for E-cad+ breast cancer organoids and an established treatment timeline, the design of the nanoparticles was validated for the proposed drug delivery system.

[0119] Table 1. Size and zeta potential of liposomes in HEPES buffer as determined by a Zetasizer

[0120] From a clinical perspective, the addition of a toxic inhibitor therapeutic to the standard- of-care chemotherapies offered to patients with IDC breast cancer is not feasible, as chemotherapeutics employed for IDC, specifically the TNBC subset, cause significant side effects without the added burden of a MEK inhibitor. When designing the proposed drug delivery system, it was desired to not only lower the off-target toxicity of iMEK PD0325901, but also to demonstrate the feasibility of co-delivering the iMEK with a traditional chemotherapeutic, i.e., DOX. To co-deliver these two small molecules, hydrophilic DOX (DOX- HC1) was encapsulated inside the liposome, with hydrophobic iMEK contained inside the lipid membrane, achieving 74% encapsulation efficiency (EE) of iMEK and 97% EE of DOX (FIG. ID, Table 2). Then, the release of iMEK / DOX from the liposomes was investigated and a burstrelease was observed in the first 12 h followed by a slower release with 34 ± 1% of iMEK and 90 ± 1% of DOX releasing in 14 days (FIG. ID). Previous studies have reported that the codelivery of a MEK inhibitor with cisplatin can decrease cell viability in melanoma and colon cancer, but these studies were conducted in 2D models, lacking physiological relevance. To demonstrate the importance of the PR_b peptide, targeted and non-targeted liposomes were delivered to E-cad+ and E-cad- organoids for 72 h after which organoid viability was assessed. As anticipated, based on the nanoparticle uptake data, the incorporation of PR_b significantly improved the efficacy of both iMEK and DOX loaded liposomes (FIG. IE). While DOX loaded liposomes reduced viability similarly between E-cad+ and Ecad- organoids, iMEK loaded liposomes were much more effective against the E-cad+ organoids, as expected from the observed IC50 of 0.38 pM for E-cad+ cells and E7 pM for E-cad- cells (FIG. 6H).

[0121] Table 2. Encapsulation efficiency (EE) of iMEK and DOX loaded in liposomes

[0122] Additionally, the potential synergy of the co-delivery treatment between iMEK and DOX was investigated using targeted liposomes, as measured by the amount of synergistic killing in excess of the additive Bliss expectation. The Bliss model of synergy was chosen because of the independent mechanisms of action by iMEK and DOX. The drug combination was synergistic in E-cad+ organoids when iMEK concentration was at or above 1 pM for different DOX concentrations, while the combination did not demonstrate synergy in E-cad- organoids (FIG. IF). This result agrees with previous findings, as without the hyper-active MEK / ERK cascade, iMEK has little impact on cell viability and therefore cannot be expected to produce a synergistic killing effect with a chemotherapeutic. The combination of 1 pM iMEK with 2 pM DOX dramatically reduced cell viability in the E-cad+ organoids by more than 75% (FIG. 1G), than either drug alone (FIG. IE). This drug combination was not very effective at reducing viability in E-cad- organoids, as anticipated. Furthermore, PR_b liposomes were still better at killing organoids than non-targeted liposomes when loaded with iMEK / DOX, demonstrating the need of targeting. The 1 pM iMEK with 2 pM DOX was selected for further in vitro studies, as it decreased cell viability to similar levels when compared with higher drug concentrations (FIG. 7A). Example 2 - PVLA-PEG-PVLA hydrogel is thermosensitive and provides sustained release of targeted nanoparticles encapsulating iMEK and DOX

[0123] The PVLA-PEG-PVLA triblock copolymer was synthesized, and the number average molecular weight was confirmed by 'H NMR to be 1570-1500-1570. The triblock copolymer undergoes a liquid-gel transition via a spherical-to-wormlike micelle transformation (FIG. 2A). The PVLA-PEG-PVLA triblock copolymer forms a liquid at room temperature and an opaque hydrogel upon heating to physiological temperature (FIG. 2B). Cryo-TEM was used in the dilute limit (0.25 w / v%) to confirm the transition from spherical to cylindrical micelles, whose highly clustered nature suggests some amount of triblock copolymer bridging (FIG. 2C). This result agrees with previous studies that showed the spherical-to-wormlike transition with cryo-TEM as well as small angle neutron scattering (SANS) measurements at higher concentrations (20 w / v%). This phase transition is uncommon and has been shown before in triblock copolymers, such as certain pluronics, at high temperatures (T > 60°C) or in the presence of high salt concentrations.

[0124] With the polymer system characterized, it was sought to optimize the iMEK and DOX loaded, PR_b functionalized liposomes by testing them with the organoid model and confirming the ability of the hydrogel-nanoparticle system to provide an extended-release platform. To characterize the proposed drug delivery system, fundamental in vitro release studies was performed analyzing the release of drugs into PBS. Based on previous work, in which the thermosensitive hydrogel was used to deliver standard of care chemotherapeutics to pancreatic cancer spheroids in vitro the platform should provide extended release. The ability of the hydrogel was previously confirmed to entrap targeted liposomes, and also that the inclusion of liposomes did not impact the degradation or stiffness of the hydrogel. The free drugs in the hydrogel released 50 ± 2% iMEK and 96 ± 2% DOX after only 7 days (FIG. 2D). In contrast, the encapsulation of the liposomes in the hydrogel resulted in sustained release with 21 ± 4% of iMEK and 57 ± 3% of DOX released after 30 days (FIG. 2E). Once the nanoparticles are internalized by cells the liposomes will release the entire drug payload allowing for the synergistic cytotoxic effect of iMEK and DOX. Thus, encapsulating the liposomes in the hydrogel extends the release timeline of the drugs, providing an optimal platform for sustained release after local delivery. To functionally test the sustained delivery of the liposomes from the thermosensitive hydrogel, multiple batches of organoids were subjected to the same empty hydrogel, hydrogel containing free drugs, or liposomes.

[0125] Over a period of 12 days, four separate batches of organoids were treated on day 4 of organoid culture with the same formulation / DDS (FIG. 2F, FIGs. 7B-7C). The proposed system was tested: PR_b liposomes encapsulating both drugs, entrapped in the thermosensitive hydrogel, against various controls: (1) free non-targeted liposomes encapsulating both drugs, (2) free PR_b liposomes encapsulating both drugs, (3) empty hydrogel, (4) hydrogel containing free drugs, and (5) non-targeted liposomes encapsulating both drugs, entrapped in the hydrogel. As the multi-compartment organoids utilized in these experiments are truly 3D (i.e., floating in medium during culture), a transwell insert was used to treat the organoids with the various treatment groups, allowing for diffusion of the nanoparticles or drugs into the culture medium. Four-day old organoids were exposed to the different formulations through the transwell inserts for 3 days. At the end of treatment, the organoids were transferred to fresh control medium and the transwell inserts were immediately transferred to a new batch of untreated 4-day old organoids. The treated organoids were cultured for 1 more day before the final PrestoBlue viability measurement.

[0126] In the first batch of organoids, both free PR_b liposomes loaded with the two drugs (iMEK / DOX) and hydrogels encapsulating free drugs resulted in reduced organoid viability (FIG. 2F). This strong initial effect is markedly different from the organoid viability results of the fourth batch of organoids, in which the PR_b liposomes in the hydrogel have the greatest impact, reducing viability to 38%, compared to the free PR_b liposomes, or free drugs in hydrogel which did not lower cell viability in a significant way. The free drugs in the hydrogel had minimal impact on cell viability of the fourth organoid as 96% of the DOX and 50% of iMEK had been released by day 7 (FIG. 2D), leaving low amounts of drug to affect the final organoid. Similarly, the free liposomes had little impact on cell viability since 80% of the DOX and 30% of the iMEK were already released by day 14 (FIG. ID) from the transwell insert prior to being moved to the fourth organoid. The PR_b liposomes in the hydrogel reduced viability more than the non-targeted liposomes in the hydrogel demonstrating the benefit of including a cell targeting ligand and enhancing cell uptake of liposomes. The results of this study highlight the benefit of using targeted liposomes entrapped in a thermosensitive hydrogel as a delivery system. Example 3 - PVLA-PEG-PVLA hydrogel is thermosensitive and biocompatible in vivo

[0127] To further characterize this unique thermosensitive polymer, the in vivo biocompatibility and biodegradation rate of the hydrogel was determined. To quantify the rate of biodegradation, in vivo studies were conducted in both NSG and BABL / c mice, as these are the two species of mice used in later orthotropic mouse models. The polymer was subcutaneously injected into the flank of the animal (FIG. 3A) and calipers were used to measure x, y dimensions, allowing calculations of the volume of the implant. Immediately after injection, a dome was observed under the skin that could be palpated and no gel leaked from the injection site, confirming the rapid formation of a hydrogel upon reaching physiological temperature. One day after injection, gross macroscopic examination verified gel formation, which was observed as a slightly opaque hydrogel upon dissection (FIG. 3B). The implant volume degradation was also tracked over the course of 25 days (FIG. 3C), at which point the implant was too small to accurately measure (< 150 mm3). The biodegradation in vivo study was first terminated on day 35, when no implant was palpable, indicating complete degradation. Absence of hydrogel was confirmed by macroscopic observation during dissections (FIG. 3B). Throughout the study, mice weights were recorded when gel implant was measured as a first indication of potential toxicity from the hydrogel formation. Weight loss was not observed in the animals for the duration of the study (FIG. 3D), observing steady weight gain as anticipated in healthy animals.

[0128] To capture the various stages of degradation, animals were sacrificed at multiple timepoints, harvested the gel / site of implantation as well as the liver, kidneys, and spleen. All tissues were fixed and stained with hematoxylin and eosin (H&E) allowing for pathological assessment of biocompatibility One day after polymer injections, acute inflammation was observed that began resolving by day 14, characterized by the presence of polymorphonuclear neutrophils and foamy macrophages (FIG. 3E). On day 35, the inflammation was continually resolving into the formation of fibrotic tissue with few foamy macrophages remaining. Then, it was desired to verify the long term impact of the hydrogel and a 90 day study was conducted to ensure no permanent changes to the tissue. After three months, the inflammation completely resolved with few immune cells present and the remaining fibrotic tissue shrinking. Over the course of 90-days, histological analysis of tissues revealed no significant changes in the kidney, liver, or spleen indicating no systemic toxicity from the hydrogel (FIG. 3F). These results indicate the biodegradable nature of the hydrogel with no collagenous fibrous capsule forming, chronic inflammation resolving, and no pathological anomalies observed in the reticuloendothelial system (RES) organs when compared to animals that did not receive a polymer injection (FIG. 8), demonstrating the gel is biocompatible. Taken together these results demonstrate that the hydrogel is biocompatible and suitable for use as a carrier for liposomes in vivo.

[0129] Example 4 - Sustained, local, and targeted delivery of iMEK and DOX effectively slows E- cad+ tumor progression in a xenograft model

[0130] With the confirmation that the polymer-nanoparticle system provides sustained codelivery of iMEK and DOX encapsulated in targeted liposomes, it was sought to test the translatability of the system to animal models. A pre-clinical model of breast cancer would allow the determination of whether local, targeted delivery can increase the therapeutic index of iMEK and provide an effective method to translate iMEK combination therapy to the clinical setting. To evaluate and compare the drug delivery system to other methods of delivery, an orthotopic xenograft study of MDA-MB-231 E-cad+ breast cancer was designed in NSG mice. To compare to the clinically standard administration route of iMEK, a pilot study was first run in which iMEK was delivered orally following a dosage schedule that mirrors a clinical trial: animals were orally administered iMEK for 5 days, followed by 2 days of rest and then repeated the cycle (FIG. 4A) Animal weights were tracked throughout the study and significant weight loss was seen in the oral delivery cohort (FIG. 9A). The systemic delivery of iMEK via oral administration resulted in many off-target and toxic effects, resulting in deaths of animals before they succumbed to the tumor (FIG. 4A), further highlighting the need for local targeted delivery to overcome this toxicity limitation.

[0131] To deliver the hydrogel -nanoparticle system to mice bearing orthotopic tumors, the thermosensitive polymer solution was mixed with PR_b liposomes which contained iMEK and DOX. The polymer-nanoparticle solution was injected directly into the tumor, followed by peripheral tumor injections, a scheme developed based on past work demonstrating local hydrogel injections can successfully treat breast tumors (FIG. 4B). Furthermore, any nanoparticles that drain from the tumor site will still be able to target the tumor from systemic circulation due to the targeting peptide. The novel drug delivery system (Gel-NP(iMEK+DOX)) was compared to the following controls: (1) phosphate buffer saline injection (PBS), (2) empty hydrogel (Gel), (3) intravenous injection of targeted nanoparticles (IV-NP(iMEK+DOX)), and (4,5) single agent targeted nanoparticles in the hydrogel (Gel-NP(iMEK) and Gel-NP(DOX)). Compared to the controls (PBS and Gel), the Gel-NP(iMEK+DOX) system doubled both the lifetime of the mice from 24 days to 50 days and the median survival time from 22 days to 40 days (FIG. 4C). Mice weights were tracked for the duration of the study to capture any potential toxic effects of the treatments. No significant weight loss was observed in this study, indicating off-target effects were mitigated (FIG. 4D). When assessing the impact of the Gel-NP- (iMEK+DOX) on tumor volume progression over time, a significant slowing, and in some cases halting, of primary tumor growth was observed compared to all other groups (FIGs. 4E-4F).

[0132] At the conclusion of the study, tumors were excised and preserved for further analysis as well as organs of interest (i.e., lungs, liver, kidneys, spleen, pancreas, and heart). Final tumor weight confirmed the effectiveness of the treatments on tumor proliferation and growth. When comparing the Gel-NP(iMEK+DOX) final tumor weights to all other groups, a significant reduction in tumor weight of 56% compared to the PBS control was observed (FIG. 9B). Also, H&E and immunohistochemistry (IHC) analysis was performed on the excised tumors and a lowering of ERK phosphorylation (pERK) and subsequently Ki67 was observed, as expected given the proposed mechanism of iMEK inhibition (FIG. 4G, FIG. 9C). Histological examination of heart tissues, the most common place for side effects from DOX therapy, showed no abnormal tissue changes in any group, except a slight decrease in muscle cell size in the IV group (FIG. 4H). Similarly, there were no significant findings in the liver, spleen, or kidneys of any group (FIG. 9D), indicating all treatments were effective in limiting systemic toxicity associated with delivery of free DOX, such as nephrotoxicity or hepatotoxicity.

[0133] It was further investigated if local delivery of Gel-NP(iMEK+DOX) had an impact on tumor metastasis. A significant reduction was observed in lung metastases, the most common site of metastasis in TNBC, was evident from H&E sections of lungs that were inflated with agarose and then fixed in formalin after excision (FIG. 41, FIG. 9E). Quantitative polymerase chain reaction (qPCR) analysis was also performed on lungs excised from the mice and the expression of human genomic material marker HK2 was measured. The Gel-NP(iMEK+DOX) group resulted in significant reduction of metastatic burden when compared to all other experimental groups (FIG. 4J). These results show that the locally delivered hydrogel- nanoparticle system can reduce tumor burden at the primary site as well as metastatic burden.

[0134] Example 5 - Sustained, local delivery of iMEK and DOX effectively slows tumor progression and metastasis in a syngeneic mouse model of TNBC

[0135] To further demonstrate the translation potential, the hydrogel-nanoparticle system was tested in a syngeneic mouse model of breast cancer, which also allowed a study on how the immune system responds to the treatment. Murine breast cancer cell line 4T1 was injected in the mammary fat pad and grown for 1 week to initiate orthotopic breast tumors. Next, the polymer- nanoparticle solution was directly injected into the tumor (site 1, FIG. 5A) and the tumoral periphery (sites 2 and 3, FIG. 5A). A luciferase-tagged 4T1 cell line (4Tl-luc) was utilized, allowing the study to monitor tumor volume as a function of radiance via an IVIS imaging system as well as via caliper measurements. The luciferase tag also allowed assessment of metastatic spread at the conclusion of the study.

[0136] The novel hydrogel-nanoparticle drug delivery system Gel-NP(iMEK+DOX) was compared to PBS and empty gel controls. The Gel-NP(iMEK+DOX) treatment increased median survival by a factor of approximately 2, similarly to how it performed in the NSG mouse model. No difference was observed between the control groups (PBS and empty hydrogel injections) either, aligning with observations from the NSG study (FIG. 5B). Furthermore, the Gel- NP(iMEK+DOX) resulted in long term survival due to a halt in tumor progression for 4 out of 9 mice. Importantly, mouse weight was tracked for the duration of the study to capture any potential toxic effect of the treatments, and no significant animal weight loss was observed in this study (FIG. 5C). This was supported by analysis of H&E sections of the liver, spleen, heart, and kidneys that were collected upon termination of the study, which showed no pathological abnormalities in response to the treatment (FIG. 10D). The Gel-NP(iMEK+DOX) treatment resulted in a nearly complete loss of radiance in the tumor mass by day 22 of the study, suggesting eradication of the tumor cells (FIGs. 5D-5E). A significant decrease was also observed in tumor volumetric progression in the treatment group, as calculated from x, y dimension caliper measurements (FIG. 10A), confirming what was observed in the NSG survival study. Images of excised tumors further confirmed that the hydrogel-nanoparticle system killed most of the tumor cells, as treated tumors displayed only a small area of luciferase positive cells at the conclusion of the study (FIG. 5F, FIG. 10B). H&E staining of tumor sections revealed that by the end of the study, treated tumors consisted of collagen and other extracellular matrix components with much fewer cancer cells present compared to the PBS and empty hydrogel controls (FIG. IOC). Together, these data indicate that the proposed hydrogel- nanoparticle system, designed to deliver iMEK+ DOX locally and slowly, can slow down and even halt tumor progression.

[0137] To assess the impact of the treatment on metastasis, IVIS imaging and analysis of H&E sections of excised livers, lungs, spleens, and kidneys were performed at the end of the study. The Gel-NP(iMEK+DOX) treatment group had significantly decreased luciferase signal in the lungs when compared to control groups (FIGs. 5G-5H). There were no visible areas of luciferase signal in the livers of the Gel-NP(iMEK+DOX) group (FIG. 5G), but the difference was not significant when compared to both control groups (FIG. 5H). No major metastasis was present in H&E sections of the liver (FIG. 10D). No group showed any positive signal in the spleen or kidneys (FIGs. 5G-5H, FIG. 10D). The absence of cancer cells in the lungs from the GEL-NP(iMEK+DOX) group was further confirmed via H&E staining of lung sections. There were macro-metastases in both control groups (PBS and empty gel), but not in the Gel- NP(iMEK+DOX) group showing that the treatment minimized metastasis (FIG. 51). H&E sections from kidneys, livers, hearts, and spleens of the PBS control group, empty gel (Gel) and treatment group (Gel-NP(iMEK+DOX)) did not contain any cancer cells (FIG. 10D). The low levels of metastasis observed in the treatment group are likely the result of cancer cells that disseminated from the primary tumor in the first week of growth, prior to the injection of the hydrogel. 4T1 cells are particularly aggressive and previous studies show 4T1 cells quickly metastasize, and once the cancer cells infiltrate the lung tissue, treatment has little to no impact on preventing metastatic growth. Despite similar levels of spleen white pulp between all groups (FIG. 10D), the Gel-NP(iMEK+DOX) system suppressed the enlargement of the spleens by approximately 50%, as measured by weight (FIG. 10E). Splenomegaly is a sign of myeloid cell expansion due to inflammation caused by cancer, which can lead to major issues in blood cell count and health.

[0138] To further explore the tumor microenvironment, H&E and IHC was performed on tumor sections. H&E staining revealed that the dual therapy hydrogel -nanoparticle system reduced the density of cancer cells in the tumor microenvironment, marked by the lack of nuclei in the tumor mass that was excised (FIG. IOC). Staining was also performed for apoptotic marker cleaved caspase 3 and proliferation marker Ki67. An increase in cleaved caspase 3 signal was observed in the Gel-NP(iMEK+DOX) group when compared to both PBS and empty gel controls (FIG. 5J, FIG. 10G). All tumors showed a necrotic core with a population of apoptotic cells and lack of cells in the core as evident in H&E and IHC staining (FIGs. IOC and 10F), confirming the observations in the organoid model (FIG. IB). A decrease in Ki67 was also observed in the dual therapy hydrogel-nanoparticle group (FIG. 10F). A decrease in proliferative cells is expected given the cytotoxic nature of the therapies that is delivered to the tumor.

[0139] To assess the interplay of the immune response and the Gel-NP(iMEK+DOX) system and empty gel (Gel), flow cytometry was performed on tumors from all groups. It was determined to quantify the following immune cell populations: macrophages, neutrophils, dendritic cells, T cells, B cells, and natural killer (NK) cells (FIG. 11), in an attempt to understand how the co-delivery of DOX and iMEK resulted in long-term survivors. A significant increase in the CD4+ T cell population was observed in the Gel-NP(iMEK+DOX) case compared to both PBS and empty gel (FIG. 5L), while there was no significant difference in the total T cells between groups (FIG. 5K). This increase in T-helper cell (CD4+) population without an increase in total T cells is quite intriguing, indicating a shift in phenotype without an increase in overall production or requirement of T cells to the tumor microenvironment. Conversely, the CD8+ population was lower in the combination treatment compared to the controls (FIG. 5M). An increase in the NK cell population was also observed in the Gel- NP(iMEK+DOX) group when compared to the empty gel group (FIG. 5N). All other immune cells populations analyzed did not exhibit a statistically significant difference between the different groups (FIGs. 12A-12F). In summary, the novel hydrogel-nanoparticle system can be utilized to both slow tumor progression and reduce metastatic burden, with an overall positive impact on survival rate.

Claims

1. WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising:(a) a lipid nanoparticle comprising a protein kinase inhibitor and a chemotherapeutic agent; and(b) a hydrogel comprising a thermosensitive and biodegradable polymer.

2. The pharmaceutical composition of claim 1, wherein the lipid nanoparticle targets ocsPi integrin.

3. The pharmaceutical composition of claim 1 or 2, wherein the lipid nanoparticle comprises a fibronectin-mimetic peptide (PR_b) on a surface of the lipid nanoparticle.

4. The pharmaceutical composition of any one of claims 1-3, wherein the lipid nanoparticle comprises a liposome.

5. The pharmaceutical composition of any one of claims 1-3, wherein the lipid nanoparticle comprises a micelle.

6. The pharmaceutical composition of any one of claims 1-5, wherein the protein kinase inhibitor comprises a MEK inhibitor.

7. The pharmaceutical composition of claim 6, wherein the MEK inhibitor comprises PD0325901, AZD6244, trametinib, cobimetinib, selumetinib, or binimetinib.

8. The pharmaceutical composition of any one of claims 1-7, wherein the chemotherapeutic agent comprises vincristine, prednisone, dexamethasone, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, nivolumab,pembrolizumab, durvalumab, atezolizumab, radioisotopes, monomethyl auristatin E (MMAE; e.g., vedotin), calicheamicins, deruxtecan, DM1, and any combinations thereof.

9. The pharmaceutical composition of any one of claims 1-8, wherein the chemotherapeutic agent comprises doxorubicin.

10. The pharmaceutical composition of any one of claims 1-9, wherein the thermosensitive and biodegradable polymer comprises a PVLA-PEG-PVLA triblock copolymer.

11. The pharmaceutical composition of any one of claims 1-10, wherein the lipid nanoparticle is encapsulated in the hydrogel.

12. A method of treating a cancer in a subject, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 1-11.

13. The method of claim 12, wherein a cancer cell from the cancer expresses otsfJi integrin on the cancer cell surface.

14. The method of claim 12 or 13, wherein the cancer is a breast cancer, colon cancer, pancreatic cancer, lung cancer, prostate cancer, brain cancer, or melanoma.

15. The method of claim 14, wherein the cancer is a triple negative breast cancer (TNBC).

16. The method of claim 15, wherein the cancer is an E-cadherin positive cancer.

17. The method of any one of claims 12-16, wherein the administration comprises intratumoral administration and / or local administration in the vicinity of the tumor.

18. The method of any one of claims 12-17, wherein the subject is a human.

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