WO2025259830A1 - Copolymeric compositions and methods of use thereof - Google Patents

Abstract

Compositions and methods for the delivery of a therapeutic agent are provided.

Description

Copolymeric Compositions and Methods of Use Thereof

By David Oupicky Sudipta Panj a

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63/659,061, filed on June 12, 2024. The foregoing application is incorporated by reference herein.

This invention was made with government support under Grant No. R01 DK 124095 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the delivery of therapeutic agents to a patient, particularly for the treatment of cancer.

BACKGROUND OF THE INVENTION

The development of polymer analogs of small-molecule drugs is a proven strategy to enhance therapeutic efficacy. These so-called macromolecular (polymeric) drugs are pharmacologically active molecules with multiple binding sites, enabling strong, multivalent interactions with target receptors, leading to enhanced therapeutic efficacy (Gestwicki, et al. (2002) J. Am. Chem. Soc., 124(50): 14922-14933). Polymeric drugs offer extended circulation times, lower immune responses, increased stability against enzymatic degradation, and an opportunity to form prodrugs that can be activated by specific stimuli at the target site. Polymeric drugs often display enhanced receptor binding and longer receptor residence times through mechanisms such as clustering, chelation, and statistical rebinding consequently leading to improved therapeutic efficacy. Furthermore, polymeric macromolecules present an opportunity to fabricate sophisticated drug delivery systems by incorporating additional active components that synergistically improve the overall therapeutic efficacy (Khandare, et al. (2006) Prog. Polym. Sci., 31 (4) : 359-397). Due to these advantages, polymeric drugs have been extensively explored as anticancer, antiviral, and antimicrobial agents, which often surpass the therapeutic efficacy of their small-molecule counterparts (Li, et al. (2015) J. Contr. Release 219:369-382).

Hydroxychloroquine (HCQ) is a promising adjuvant anti -cancer drug that acts through several mechanisms such as inhibition of cancer metastasis and autophagy, normalization of tumor vessels, and modulation of the tumor microenvironment (TME) (Yu, et al. (2019) J. Polym. Sci. Polym. Chem., 57(22):2235-2242; Yu, et al. (2016) ACS Macro Lett., 5(3):342-345; Xie, et al. (2018) Macromol. Biosci. 18(l):201700194). Inspired by the anti-cancer effects of HCQ and the advantages of polymeric macromolecular drugs, polymeric forms of HCQ have been synthesized. Compared to HCQ, PCQ demonstrated improved anti- metastatic efficacy in multiple cancer models (Yu, et al. (2016) J. Contr. Release 244(Pt B):347-356; Gestwicki, et al. (2002) J. Am. Chem. Soc., 124(50): 14922- 14933; Cairo, et al. (2002) J. Am. Chem. Soc., 124(8): 1615-1619). Improved polymeric HCQ (PCQ) with superior anti-metastatic activity than the existing macromolecules are needed.

SUMMARY OF THE INVENTION

In accordance with the instant invention, copolymers are provided. In certain embodiments, the copolymer is linear. In certain embodiments, the copolymer is a random copolymer. In certain embodiments, the copolymer is a polyacrylate or a polyacrylamide. In certain embodiments, the copolymer comprises a plurality of a first monomer, a plurality of a second monomer, and, optionally, a plurality of a third monomer. In certain embodiments, the copolymer backbone or the copolymer and plurality of third monomers are hydrophilic. In certain embodiments, the first monomer comprises an antimetastatic agent. In certain embodiments, the antimetastatic agent is chloroquine or hydroxychloroquine. In certain embodiments, the mole percent of the first monomer in the copolymer is about 1% to about 10%. In certain embodiments, the second monomer comprises a therapeutic agent. In certain embodiments, the second monomer comprises a cleavable linker which allows for the release of the therapeutic agent when cleaved. In certain embodiments, the cleavable linker comprises a disulfide bond. In certain embodiments, the therapeutic agent is a chemotherapeutic agent such as gemcitabine. In certain embodiments, the mole percent of the second monomer in the copolymer is at least about 1%. In certain embodiments, the third monomer is hydrophilic. Nanoparticles comprising a copolymer of the instant invention are also provided. Monomers (e.g., isolated) of the copolymers are also encompassed by the instant invention. Compositions comprising a copolymer and/or nanoparticle in a pharmaceutically acceptable carrier are also encompassed by the instant invention.

In accordance with another aspect of the instant invention, methods of treating, inhibiting, and/or preventing a disease or disorder in a subject in need thereof are provided. In certain embodiments, the method comprises administering a copolymer and/or nanoparticle of the instant invention to the subject. In certain embodiments, disease or disorder is cancer. In certain embodiments, the cancer is pancreatic cancer (e.g., PDAC).

BRIEF DESCRIPTIONS OF THE DRAWING

Figure 1 A provides the chemical structure and schematic representation of the PCQ polymers, PCQ(g)n and PCQ(r)n. The letter ‘r’ stands for random, and ‘g’ stands for gradient structure, and ‘n’ stands for the mol% of CQ content. Figure IB provides a graph of the copolymerization kinetics as evaluated by ¹ H NMR of PCQ(r)n synthesized by using DHPMA and CQMA monomers. Figure 1C provides a graph of the copolymerization kinetics as evaluated by 'H NMR of PCQ(g)n synthesized by using HPMAm and CQMA monomers. Figure ID provides a graph of the variation of molar composition of the reacting monomers in the growing chain of random PCQ(r)n and gradient PCQ(g)n copolymers as determined by ¹ H NMR. Figure IE provides a schematic of the synthesis of methacrylate derivative of gemcitabine monomer, GEM-SS-MA. Figure IF provides a chemical structure of CQMA. Figure 1G provides a chemical structure of PCQ(r)6. Figure 1H provides a chemical structure of PCQ(g)n. Figure II provides results of CellTiter-Blue® assay performed to evaluate the cell viability of random PCQ(r)n (upper row) and gradient PCQ(g)n (lower row) against KPC8060 (left column) and S2-013 (right column) cell lines. Data are shown as mean ± SD (n = 3). Figure 1 J provides results of a scratch wound healing assay with KPC8060 cells to evaluate the effect of inhibition of cell migration of different CQ containing PCQ(r)n and PCQ(g)n. KPC8060 cells were incubated with 25, 50, and 100 pM (equivalent to HCQ) concentration polymers over 48 hours. PCQ(r)6 shows a higher inhibition effect. Data are shown as mean ± SD (n = 3). Figure IK provides representative images of a scratched wound on KPC8060 cell at 0 hour and 30 hours after incubation with PCQ(r)6 and PCQ(g)7 at 100 pM concentration. Figure IK also provides a graph of the efficiency of inhibition of cell migration quantified by measuring the % of wound closed after 30 hours of incubation. Data are shown as mean ± SD (n = 3). Figure IL provides graphs of the Transwell® migration assay of KPC8060 (left) and S2-013 (right) cell lines using 10% FBS as a chemoattractant.

Figures 2A-2C show results from Transwell® migration assays to determine the dose-dependent anti-migration effect of PCQ. The assay was conducted with KPC8060 (Fig. 2 A) and S2-013 (Fig. 2B) cell lines using 10% FBS as a chemoattractant. Figure 2C provides representative images of migrated cells from various treatment groups, scale bar 200 pm. Data are shown as mean ± SD (n = 5).

Figure 3A provides the chemical structure of PCQ(r)6-SS-GEM12 and the ^JH NMR spectrum of PCQ(r)6-SS-GEM12 in DMSO- k The variables x, y, and z are independently an integer from 1 to 1,000, 1 to 500, 1 to 250, or 1 to 100. Figure 3B provides a graph of the hydrodynamic size of self-assembled PCQ(r)6-SS- GEM12 nanoparticles determined by DLS. Figure 3C provides representative TEM images demonstrating the bulk morphology of the nanoparticles. Figure 3D provides a graph of the glutathione (GSH, 10 and 2 mM) responsive GEM release kinetics, analyzed by HPLC.

Figures 4A-4C show the cellular uptake of polymers in 2D and 3D PDAC models, observed under confocal microscope and quantified by flow cytometry. Figure 4A provides representative confocal microscopy images of KPC8060 cells after 4 hours incubation with rhodamine-B dye-labeled polymers, nuclei stained with Hoechst and lysosomes stained with LysoTracker™, scale bar 20 pm. Figure 4B provides a graph of the mean fluorescence intensity (MFI) of the polymers at 4 hours post incubation in KPC8060 and S3-013 cells as assessed using flow cytometry. Results are shown as mean ± SD (n = 3). Figure 4C provides representative surface plots of KPC8060 multicellular 3D tumor spheroids after 12 hours of treatment with dye-labeled polymers. Figure 4D provides confocal microscopic images of S2-013 cells after 4 hours incubation with rhodamine-B dye- labeled polymers, nuclei stained with Hoechst and lysosomes stained with LysoTracker™, scale bar 20 pm. Figure 4E provides flow cytometric analysis of KPC8060 and S2-013 cells at 4 hours of post-incubation with dye-labeled polymers.

Figures 5A-5C show the dose-dependent cell viability of the polymers in 2D and 3D PDAC models. Figures 5A and 5B provide graphs of CellTiter-Blue® assays performed to evaluate the cell viability of polymers and polymer-GEM combinations after 48 hours of incubation with KPC8060 (Fig. 5A) and S2-013 (Fig. 5B) cell lines. PCQ(r)6 (•), GEM (■), HCQ + GEM (A), PCQ(r)6 + GEM (▼), PCQ(r)6-SS-GEM12 (♦). Figure 5C provides representative images of 3D-tumor spheroids captured under the fluorescence microscope after 48 hour incubation with respective treatments. Live cells were stained with Calcein AM, and dead cells were stained with EthD-III. Figure 5D provides a graph of dead cell population from the live/dead assay quantified by measuring the EthD-III intensity by using the microplate reader. Results are shown as mean ± SD (n = 3). Figure 5E provides representative images showing the structural integrity of 3D-tumor spheroid treated with polymers and polymer-drug combination; image (2D, bright-field) captured under EVOS xl microscope. Figure 5F provides a graph of in vivo colloidal stability of PCQ(r)6-SS-GEM12 nanoparticle (NP) in 10% mouse serum and 10% ascites. Figure 5G provides a graph of the change in surface zeta potential of NP after incubation with 10% mouse serum and 10% ascites. Figure 5H provides a graph of the IVIS® fluorescence intensity ratios of tumor/ liver. Figure 51 provides a graph of the IVIS® fluorescence intensity ratios of tumor/ kidney. Figure 5J provides HPLC chromatograms of NP incubated with 10% ascites and 10% serum supplemented with and without GSH. The chromatogram for ascites, serum and GEM with GSH represent control experiments. Figure 5K provides representative images of a-SMA positive stromal cells.

Figures 6A-6H show the biodistribution, treatment regimen, and antitumor effect of the polymeric prodrug nanoparticles. PCQ-based GEM prodrug nanoparticles showed significant inhibition of tumor growth and profound anti- metastatic effect with no toxic side effects. Figure 6A provides representative fluorescence images (ex vivo) of the tumor and major organs were obtained by using the IVIS®. Figure 6B provides a graph of the semiquantitative analysis of fluorescence intensity from the organs. Figure 6C provides representative confocal microscopic images of tumor tissue. Figure 6D provides a schematic diagram of the in vivo therapeutic study. The mice received IP injections of the nanoparticles (dose 10 mg/kg GEM equiv.). Figure 6E provides a graph of the weight of the tumor harvested at day 32. Figure 6F provides images of the tumor harvested at day 32. Figure 6G provides a graph of the change in tumor volume with time was measured by using an ultrasound imaging system. Figure 6H provides representative ultrasound images of the tumor on day 12 and day 31 of treatment. *P <0.05, **P <0.01, ***p <0.001 and ****p <0.0001, ns =no significance difference.

Figures 7A-7E show the antimetastatic effect and safety profile of polymeric prodrug nanoparticle. Figure 7A provides an immunohistochemical analysis of the tumor section including staining (apoptotic cells region surrounded by dotted lines and zoomed section highlighted by a square), cleaved caspase-3 (CC3), and CD8+ staining (scale bar 200 pm). Figure 7B provides a graph of thew quantification of cleaved caspase-3 (CC3) positive area from different treatment groups. Figure 7C provides a graph of the quantification of CD8+ positive area from different treatment groups. Figure 7D provides a graph of the macroscopically evaluated metastasis fraction for different treatment groups at day 32. Figure 7E provides a graph of the change in mice body weight during the treatment. *P < 0.05, **P < 0.01 and ****p < 0.0001.

Figures 8A-8G show RNA-sequencing data analysis to understand the responsible pathways of the anti-tumor effect. Figures 8A-8C provide volcano plots depicting the distribution of differentially expressed genes in PCQ(r)6-SS-GEM12 (Fig. 8A), (PCQ(r)6 + GEM) (Fig. 8B), and GEM (Fig. 8C). The abscissa in the figure is log2Fold change, and the ordinate is -loglOpadj values, the line indicates the threshold line for differential gene screening criteria. Figure 8D provides a Venn diagram illustrates the differential genes in the comparison of GEM, (PCQ(r)6 + GEM) and PCQ(r)6-SS-GEM12 treated groups. Figures 8E-8G provide Reactome pathway enrichment analysis of downregulated differentially expressed genes in PCQ(r)6-SSGEM12 (Fig. 8E), (PCQ(r)6 + GEM) (Fig. 8F), and GEM (Fig. 8G) treated groups. The most significant 20 Reactome pathways are selected for display. In the enrichment analysis, the abscissa represents the ratio of number of downregulated genes with the total number of genes corresponding to the same pathway. The size of the point represents the number of genes annotated to a specific Reactome pathway, and the scale represents the significant size of the enrichment.

DETAILED DESCRIPTION OF THE INVENTION

Combination therapy is a major therapeutic strategy employed to treat many cancers, including pancreatic ductal adenocarcinoma (PDAC) (Zhang, et al. (2021) ACS Nano 15(1): 1186-1198). PDAC is the third most common cause of cancer- related deaths and it is anticipated that PDAC could be the second leading cause of death by the year 2030 (Crowley, et al. (2021) Cancer Metastasis Rev., 40(3) : 891 - 908; Park, et al. (2021) JAMA 326(9):851-862). PDAC is asymptomatic at an early stage, and in most cases, is diagnosed post-metastasis. In such circumstances, the patient becomes ineligible for curative surgical resection leaving chemotherapy as one of the remaining viable treatment options (Tang, et al. (2021) Mol. Pharm., 18(12):4448-4458; Hang, et al. (2021) J. Contr. Release 333: 139-150). The standard chemotherapy regimens for PDAC at advanced stages include FOLFIRINOX (a combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin) and the combination of gemcitabine with nab-paclitaxel (Wang, et al. (2021) Biomaterials 278: 121176). Recently, NALIRIFOX, a regimen combining nanoliposomal irinotecan, 5-fluorouracil, leucovorin, and oxaliplatin, was approved based on findings from the NAPOLI 3 trial, which demonstrated improved overall survival compared to earlier doublet therapies (Nevala-Plagemann, et al. (2024) Nat. Rev. Clin. Oncol., 21(8):567-568). NALIRIFOX presents a potentially more effective and less toxic option by substituting traditional irinotecan with nanoliposomal irinotecan and reducing the oxaliplatin dose. The drugs in the combination often have diverse physicochemical properties resulting in varied half-lives, stabilities, and uneven exposure to tumor cells, ultimately reducing therapeutic efficacy (Haze, et al. (2024) Surgeries 5(l):49-62; Tang, et al. (2023) Biomater. Adv., 145:213236). Moreover, a major challenge in PDAC therapy remains the dense fibrotic stroma surrounding the tumor, which limits drug penetration and contributes to its aggressive growth. Current approaches aim to address this issue by using stromal- modifying agents such as PEGPH-20 or incorporating TME-penetrating moieties in the nanoformulation to improve drug delivery and enhance treatment effectiveness (Wang, et al. (2021) Biomaterials 278: 121176; Pramanik, et al. (2024) J. Contr. Release, 366:231-260). Therefore, the development of TME-penetrating polymer- based multiple-drug-containing systems could facilitate efficient tumor penetration and aid in improving the therapeutic outcomes in PDAC.

Gemcitabine (GEM) is an FDA-approved chemotherapeutic administered solely or in combination with other drugs to treat PDAC (Paroha, et al. (2021) Int. J. Pharm., 592: 120043). However, the systemic administration of GEM faces a serious hurdle due to its rapid deamination to an inactive metabolite, 2',2'-difluorouridine, by cytidine deaminase resulting in a short half-life (~15 minutes) (Gestwicki, et al. (2002) J. Am. Chem. Soc., 124(50): 14922-14933). A prodrug-based delivery strategy, utilizing a stimuli-responsive linker for the conjugation of GEM to a carrier, could enhance the efficacy and overcome the inherent limitations of GEM therapy. In a typical prodrug approach, the 4-(7V)-amino functional group of GEM is conjugated with various organic long-chain acids, for example, lauric acid, valeric acid, stearic acid, squalenic acid, poly(lactic acid) (Zhang, et al. (2021) ACS Nano 15(1): 1186-1198; Lee, et al. (2013) ACS Nano 7(3):2078-2089; Tam, et al. (2018) ACS Nano 12(7):7406-7414; Singh, A. (2021) Mater. Today Proc., 37:3301-3304; Gaudin, et al. (2016) Biomaterials 105: 136-144; Wang, et al. (2014) Mol. Pharm., 11(4): 1140-1150). Even though these modifications increase the GEM half-life, they make GEM more lipophilic which results in a reduced aqueous solubility. Alternative strategy relies on the fact that cancer cells generally exhibit elevated levels of glutathione (GSH) (up to 10 mM) compared to healthy cells. This higher GSH level enables reductive cleavage of disulfide bonds, selectively releasing free drugs in cancer cells (Liu, et al. (2019) Adv. Sci., 6(7): 1801987; Shetty, et al. (2020) Mol. Pharm., 17( 10): 3979-3989). Taken together, the conjugation of GEM through a disulfide linker to structurally well-defined PCQ could be used to achieve a smart anti-metastatic polymeric drug-based reduction-responsive GEM delivery system. Unlike conventional, pharmacologically inactive drug delivery systems, the PCQ- based GEM delivery system would be a pharmacologically active, macromolecular combination of drugs to improve treatment outcomes in PDAC.

Therefore, an anti-metastatic polymeric drug-based, reduction-responsive GEM delivery system as a combination therapy is provided herein to reduce or eliminate cancer metastasis and inhibit tumor growth. Random and gradient PCQ copolymers with different chloroquine (CQ) content were synthesized. Migration assays with PDAC cells were performed to select PCQ with superior anti -migratory activity. Selected PCQ was further improved by copolymerizing with the GEM monomer (MA-SS-GEM) to achieve the reduction -responsive PCQ-based GEM prodrug, PCQ(r)6-SS-GEM12. Physicochemical properties and GSH-responsive release of GEM from PCQ(r)6-SS-GEM12 were assessed to prove its feasibility. In vitro cellular uptake and cancer cell killing efficiency were examined in monolayer cancer cells and in a 3D-tumor spheroid model. In vivo biodistribution and therapeutic efficacy were evaluated in a murine orthotopic KPC8060 PDAC model. RNA sequencing was performed to understand the underlying mechanism of action. Overall, the data indicates the efficacy of the combination therapy using PCQ(r)6- SS-GEM12 in treating PDAC.

The present invention describes novel chemical compositions and methods of treating a disorder, disease, condition and/or an indication in a subject. In certain embodiments, the present invention encompasses pharmacologically active systems. In certain embodiments, the present invention encompasses synthetic carriers, nanocarriers, delivery systems, prodrugs or other like functions. In certain embodiments, the present invention comprises a structurally unique macromolecule which can self-assemble into nanoparticles or other like compositions. In certain embodiments, the copolymer of the present invention comprises, but is not limited to, a hydrophilic polymer carrier backbone, one or more chloroquine moieties, one or more disulfide linkers, one or more therapeutic agents, and/or other components. The one or more therapeutic agents may be used for treating one or more indications, be used as a prophylactic agent to prevent the development or recurrence of one or more indications, or other like uses.

As explained herein, a leading cause of cancer-related deaths is the result of cancer metastasis in patients. One therapeutic approach to slow or prevent metastasis is through targeting cellular migration factors. Current small molecular inhibitors used for inhibiting cellular migration in various indications often require higher doses to exhibit the desired outcomes. However, such high doses often result in ill-desired side effects. A means to improve the pharmacokinetic profile and overall activity of these agents is with polymeric drugs. Polymeric drugs are macromolecules with multiple available binding sites that can cooperatively bind to multiple subsites at a time. Furthermore, these macromolecules can exhibit several binding mechanisms such as subsite binding, receptor clustering, chelation, steric stabilization, and statistical rebinding. Polymeric drugs can adopt an energyefficient mechanism to bind with macromolecular receptors which are even not physically or sufficiently proximal to one another. Such macromolecules have been shown to increase the half-life, alter lipophilicity, and decrease aqueous solubility of the therapeutics conjugated to form the polymeric drug. A macromolecular polymeric drug like polymeric chloroquine (PCQ) can affect their interactions with cells as a copolymer. U.S. Patent Application Publication No. 2021/0161825 (incorporated herein by reference) provides examples of polymeric chloroquine. A copolymer drug may exhibit higher potency and require less doses to have the same therapeutic effect as compared to small molecular drugs alone. PCQ enables for structurally distinct polymeric drug with anti-metastatic effect but when combined with different densities of small molecular drugs within the polymer, it can significantly influence the therapeutic effect.

Herein, novel chemical compositions comprising pro-drug nanoparticles of polymeric chloroquine conjugated to a therapeutic agent via a sensitive or cleavable linker (e.g., a disulfide linker) are provided. The disulfide linker allows for release of the therapeutic in the reducing environment of tumors. The compositions were designed for treating pancreatic ductal adenocarcinoma but can be used for other cancers. The PCQ-based multiple-drug-containing nanoparticles can facilitate deep therapeutic penetration, aid in efficient beneficial therapeutic effects in various indications, as well as serve as bioactive carriers for therapeutic delivery.

The compositions of the present invention may serve to respond to stimuli. The response to stimuli may trigger the release of one or more therapeutic agents. The release of one or more therapeutic agents may be the result of the reduction and cleavage of thiol groups. The present invention may comprise one or more disulfide linkers. The one or more disulfide linkers of the present invention may serve to conjugate one or more therapeutic agents. The one or more disulfide linkers of the present invention may comprise a thiol group. The thiol group of the disulfide linker may be reduced or cleaved. The stimuli that may trigger the release of one or more therapeutic agents from the present invention may be a reductive environment. The tissues of the subject may be targeted by the present invention. The tissues of the subject may be that of an indication. The targeted tissue of the indication may be cancerous tissue. The tissue of the subject may comprise a reductive environment. The reductive environment of the tissue in the subject may be the stimuli that triggers the release of one or more therapeutic agents. For example, reductive environment may be a tumor microenvironment, thereby resulting in the release of the therapeutic agent in and/or near the tumor.

In accordance with the instant invention, copolymers are provided. Copolymers are polymers formed from two or more different monomers. The copolymer may be linear or branched. In certain embodiments, the copolymer is biocompatible. In certain embodiments, the copolymer is linear. In certain embodiments, the copolymer is a gradient or random copolymer. In certain embodiments, the copolymer is a random copolymer. In certain embodiments, the probability of finding a given monomer residue at a particular point in the copolymer is equal to the mole fraction of that monomer residue in the copolymer. In certain embodiments, the copolymer is not a block copolymer. In certain embodiments, the copolymer is not a gradient copolymer. In certain embodiments, the copolymer is a statistical copolymer. In certain embodiments, the copolymer is a copolymer synthesized by free radical polymerization. In certain embodiments, the copolymer is a copolymer synthesized by reversible addition-fragmentation chaintransfer polymerization (RAFT).

In certain embodiments, the copolymer comprises a polymer backbone, particularly a hydrophilic polymer backbone. In certain embodiments, the hydrophilic polymer is biocompatible. Examples of hydrophilic polymers include, without limitation, poly{y-2-[2-(2-methoxyethoxy)ethoxy] ethoxy-s-caprolactone}, poly etherglycols, dextran, gelatin, albumin, polyethylene oxide), methoxy- poly(ethylene glycol), polysaccharides, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyltriazole, N-oxide of polyvinylpyridine, N-(2-hydroxypropyl) methacrylamide (HPMA), polyortho esters, polyglycerols, polyacrylamide, polyoxazolines (e.g., methyl or ethyl poly(2-oxazolines)), polyacroylmorpholine, and copolymers or derivatives thereof. In certain embodiments, the hydrophilic polymer is a polyacrylamide. In certain embodiments, the hydrophilic polymer comprises poly(diethylene glycol methyl ether methacrylate), poly(triethylene glycol methyl ether methacrylate), poly(N-isopropylacrylamide), and/or poly(2- (dimethylamino)ethyl methacrylate). In certain embodiments, the polymer backbone is a polyacrylamide (e.g., polymethacrylamide) or polyacrylate (e.g., poly(methyl acrylate)) or a mixture thereof.

In certain embodiments, the copolymer comprises a plurality of a first monomer and a plurality of a second monomer. In certain embodiments, the copolymer comprises a plurality of a first monomer, a plurality of a second monomer, and a plurality of a third monomer. In certain embodiments, the first monomer comprises an antimetastatic agent. In certain embodiments, the second monomer comprises a therapeutic agent. In certain embodiments, the first monomer and second monomer are randomly located throughout the copolymer. In certain embodiments, the distribution of the first monomer and second monomer throughout the copolymer is controlled. In certain embodiments, the copolymer has a molecular weight of about 10 kDa to about 100 kDa, particularly about 1 kDa to about 50 kDa. In certain embodiments, the copolymer further comprises a third monomer. In certain embodiments, the third monomer is a monomer of the polymer backbone. In certain embodiments, the third monomer is hydrophilic. In certain embodiments, the third monomer is an acrylate, acrylamide, or a combination thereof. In certain embodiments, the third monomer is a hydrophilic acrylate, acrylamide, or a combination thereof. The presence of the third monomer is used to control aqueous solubility (e.g., hydrophilicity of copolymer) and/or the density, concentration, and/or distribution of the first and second monomers. In certain embodiments, the third monomer is selected from the group consisting of N-(2 -hydroxypropyl) methacrylamide (HPMAm), 2,3-dihydroxypropyl methacrylate (DHPMA), di(ethylene glycol) methyl ether methacrylate, triethylene glycol methyl ether methacrylate, N-isopropyl acrylamide), poly(2-(dimethylamino)ethyl methacrylate), and combinations thereof (or the residue after copolymerization). In certain embodiments, the third monomer is N-(2-hydroxypropyl) methacrylamide (HPMAm) (or the residue of HPMAm after copolymerization). In certain embodiments, the third monomer is 2,3-dihydroxypropyl methacrylate (DHPMA) (or the residue of DHPMA after copolymerization). In certain embodiments, the third monomer includes 2,3-dihydroxypropyl methacrylate (DHPMA) and N-(2- hydroxypropyl) methacrylamide (HPMAm) (or the residues after copolymerization).

In certain embodiments, the third monomer comprises wherein m is 0 to 10, particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, the third monomer comprises wherein m is 0 to 10, particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, m is 0 or 1. In certain embodiments, m is 0. The groups are attached to a hydrophilic group, particularly a hydrophilic alkyl such as a C1-C5 hydrophilic alkyl.

In certain embodiments, the third monomer has the structure: wherein m is 0 to 10, particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, m is 0 or 1. In certain embodiments, m is 0.

In certain embodiments, the third monomer has the structure:

As stated hereinabove, the first monomer comprises an antimetastatic agent. Antimetastatic agents are compounds that inhibit, reduce or decrease metastasis of cancer cells. In certain embodiments, the antimetastatic agent is a small molecule. Examples of antimetastatic agents include, without limitation: chloroquine, hydroxychloroquine, a chloroquine analog or derivative, BMS262084, cilengitide, dasatinib, saracatinib, curcumin, lapatinib, and tucatinib. In certain embodiments, the first monomer comprises chloroquine, hydroxychloroquine, or a chloroquine analog or derivative. In certain embodiments, the chloroquine analog or derivative is selected from the group consisting of methacryloyl chloroquine, quinacrine, 8- hydroxyquinoline, primaquine, sontoquine, azidoquine, and methacryloyl triazole chloroquine.

In certain embodiments, the antimetastatic agent (e.g., chloroquine, hydroxychloroquine, or a chloroquine analog or derivative) is attached to a monomer of the polymer backbone, either directly or via a linker. In certain embodiments, the antimetastatic agent (e.g., chloroquine, hydroxychloroquine, or a chloroquine analog or derivative) is attached to an acrylate or acrylamide, either directly or via a linker.

In certain embodiments, the first monomer has the formula: wherein Ri is an alkene, acrylate (e.g., methacrylate), or an acrylamide (e.g., methacrylamide). In certain embodiments, Ri is acrylate (e.g., methacrylate) or an acrylamide (e.g., methacrylamide). In certain embodiments, Ri is selected from the wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, Ri is selected from the group consisting , wherein m is 0 to 10, particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, m is 0 or 1. In certain embodiments, m is 0. In certain embodiments, the first monomer has the structure: is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, m is 0 or 1. In certain embodiments, m is 0. In certain embodiments, the first monomer has the structure:

In certain embodiments, the mole percent (mol%) of the first monomer in the copolymer is about 0.1% to about 10%, about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 8%, or about 5% to about 7%. In certain embodiments, the mole percent (mol%) of the first monomer in the copolymer is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the mole percent (mol%) of the first monomer in the copolymer is less than about 10%, less than about 9%, less than about 8%, less than about 7%, or less than about 6%.

As stated hereinabove, the second monomer comprises a therapeutic agent. In certain embodiments, the therapeutic agent is a small molecule. In certain embodiments, the therapeutic agent is an immune boosting agent. In certain embodiments, the therapeutic agent is an endocrine agent. In certain embodiments, the therapeutic agent is a chemotherapeutic agent. Chemotherapeutic agents are compounds that exhibit anticancer activity and/or are detrimental to a cell (e.g., a toxin or cytotoxic). Suitable chemotherapeutic agents include, but are not limited to: receptor tyrosine kinase inhibitors, toxins (e.g., saporin, ricin, abrin, ethidium bromide, diptheria toxin, Pseudomonas exotoxin, and others listed above; thereby generating an immunotoxin when conjugated or fused to an antibody); alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes such as cisplatin and carboplatin; bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase I inhibitor (e.g., topotecan, camptothecin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate); pyrimidine antagonists (analogs) such as fluorouracil (5 -fluorouracil), gemcitabine, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists (analogs) such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase; ribonucleotide reductase inhibitors (such as hydroxyurea); tubulin interactive agents (e.g., vincristine, vinblastine, docetaxel, and paclitaxel (Taxol®)); hormonal agents (e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbesterol; chlortrianisen; idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone); adrenal corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone, and prednisolone); leutinizing hormone releasing agents or gonadotropin-releasing hormone antagonists (e.g., leuprolide acetate and goserelin acetate); immunomodulator (e.g., levamisole); and antihormonal antigens (e.g., tamoxifen, antiandrogen agents such as flutamide; and antiadrenal agents such as mitotane and aminoglutethimide). In certain embodiments, the chemotherapeutic agent is gemcitabine, camptothecin, doxorubicin, paclitaxel, docetaxel, cyclophosphamide, or 5 -fluorouracil. In certain embodiments, the chemotherapeutic agent is a nucleoside analog. In certain embodiments, the chemotherapeutic agent is a pyrimidine analog or purine analog. In certain embodiments, the chemotherapeutic agent is a pyrimidine analog. In certain embodiments, the chemotherapeutic agent is gemcitabine.

In certain embodiments, the therapeutic agent is attached to a monomer of the polymer backbone, either directly or via a linker. In certain embodiments, the therapeutic agent is attached to an acrylate or acrylamide, either directly or via a linker. In certain embodiments, the therapeutic agent is attached via a cleavable linker (e.g., such that cleavage releases the therapeutic agent). In certain embodiments, the cleavable linker is cleaved in the tumor microenvironment. In certain embodiments, the cleavable linker is cleaved by the reductive conditions of the tumor microenvironment. In certain embodiments, the cleavable linker is cleaved by GSH. In certain embodiments, the cleavable linker is cleaved by a thiol exchange reaction. In certain embodiments, the linker comprises at least one disulfide bond.

In certain embodiments, then second monomer has the formula: wherein X is a therapeutic agent, wherein each n is independently 0 to 10, particularly wherein n is 0 to 5, 0 to 3, 0 to 2, or 0 to 1, and wherein Ri is an alkene, acrylate (e.g., methacrylate), or an acrylamide (e.g., methacrylamide). In certain embodiments, n is 0 or 1. In certain embodiments, n is 1. In certain embodiments, Ri is acrylate (e.g., methacrylate) or an acrylamide (e.g., methacrylamide). In wherein m is 0 to 10, particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, Ri is selected from the group consisting wherein m is 0 to 10, particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1. In certain embodiments, m is 0 or 1.

In certain embodiments, the second monomer has the structure

wherein X is a therapeutic agent, wherein each n is independently 0 to 10 (particularly wherein n is 0 to 5, 0 to 3, 0 to 2, or 0 to 1), and wherein m is 0 to 10 (particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1). In certain embodiments, m is 0 or 1. In certain embodiments, n is 0 or 1.

In certain embodiments, the second monomer has the structure:

wherein each n is independently 0 to 10 (particularly wherein n is 0 to 5, 0 to 3, 0 to

2, or 0 to 1), and wherein m is 0 to 10 (particularly wherein m is 0 to 5, 0 to 3, 0 to 2, or 0 to 1). In certain embodiments, m is 0 or 1. In certain embodiments, n is 0 or

1.

In certain embodiments, the second monomer has the structure:

In certain embodiments, the mole percent (mol%) of the second monomer in the copolymer is about 0.1% to about 95%, about 0.1% to about 50%, about 1% to about 25%, about 2% to about 25%, about 5% to about 20%, about 7% to about 17%, or about 10% to about 15%. In certain embodiments, the mole percent (mol%) of the second monomer in the copolymer is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%. In certain embodiments, the mole percent (mol%) of the second monomer in the copolymer is at least about 1%, at least about 5%, at least about 8%, at least about 10%, or at least about 12%. In certain embodiments, the mole percent (mol%) of the second monomer in the copolymer is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 15%.

In certain embodiments, the copolymer comprises terminal groups. In certain embodiments, the terminal groups are from chosen for the method of synthesis (e.g., RAFT polymerization). In certain embodiments, the terminal groups are the residual of a chain transfer agent. Examples of terminal groups include, without limitation phenyl-carbonothioylthio, 4-cyano-pentanoic acid, ethylsulfanylthiocarbonyl-sulfanyl, (dodecyl sulfanylthiocarbonyl)sulfanyl, (2- cyano-propyl, and dodecyl trithiocarb onate.

The copolymer of the instant invention may, optionally, be linked (conjugated) to one or more targeting moieties, which may be used to direct the nanoparticle to a specific tissue or cell type (e.g., cancer cell). To limit potential toxic side effects, it is desirable to confine the copolymer specifically to the target tissue or cell type (e.g., cancer cell or tumor). The term “targeting moiety” or ligand refers to any molecular structure, which preferentially binds a particular tissue or cell type over other tissues or cell types (e.g., via binding of cell surface marker (e.g., protein), particularly one preferentially expressed on the targeted tissue or cell). For example, lipids, peptides, antibodies, antibody fragments, lectins, ligands, sugars, steroids, hormones, carbohydrates, small molecules, and proteins may serve as targeting moieties. The targeting moiety may be an antibody or fragment thereof immunologically specific for a cell surface marker (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type (e.g., cancer cell or tumor). The targeting moiety may be a ligand of a cell surface marker or receptor (e.g., protein or carbohydrate) preferentially or exclusively expressed on the targeted tissue or cell type (e.g., cancer cell or tumor). In a particular embodiment, the targeting moiety preferentially binds cancer cells.

The targeting moiety may be conjugated to the polymer directly (e.g., a bond) or via a linker. In a particular embodiment, the targeting moiety is linked to a terminal group of the copolymer. The linker may be non-degradable or degradable under physiological conditions. In a particular embodiment, the targeting moiety is conjugated via a non-degradable linker.

The present invention also encompasses nanoparticles. The copolymers of the instant invention can self-assemble into nanoparticles. In certain embodiments, the nanoparticles are spherical. In certain embodiments, the nanoparticle of the instant invention is up to about 1 pm in diameter (e.g., z-average diameter). In certain embodiment, the diameter or longest dimension of the nanoparticle is about 5 to about 500 nm, about 5 nm to about 250 nm, about 5 nm to about 200 nm, about 5 nm to about 150 nm, or about 5 nm to about 100 nm. In certain embodiments, the nanoparticles have a zeta potential of about 5 to about 40 mV, about 5 to about 30 mV, about 10 to about 25 mV, about 10 to about 20 mV, about 12 mV to about 16 mv, or about 14 mV.

The nanoparticles of the instant invention may comprise a mixture of the copolymers described herein or may comprise a single type of copolymer. In other words, the nanoparticles need not be made of a single homogenous copolymer. The amount of any of the above copolymers can be from about 0.001 to 100% (by weight).

The nanoparticles of the instant invention may further comprise at least one therapeutic agent. In certain embodiments, the therapeutic agent is an anticancer drug (chemotherapeutic agent). The therapeutic agent (e.g., anticancer drug) may be encapsulated within the nanoparticle.

Compositions comprising a copolymer and a carrier are also encompassed by the instant invention. The present invention also encompasses compositions comprising a nanoparticle and a carrier. In certain embodiments, the carrier is a pharmaceutically acceptable carrier. The compositions of the instant invention may further comprise other agents such as therapeutic agents (e.g., chemotherapeutic agents).

The present invention also encompasses methods for preventing, inhibiting, and/or treating a medical condition (e.g., a disease or disorder) in a subject. The copolymers and/or nanoparticles (or compositions comprising the same) of the instant invention can be administered to an animal, in particular a mammal, more particularly a human, in order to treat/inhibit/prevent the medical condition. In a particular embodiment, the medical condition is cancer (e.g., pancreatic cancer), including metastases. Examples of cancers that can be treated include, without limitation: leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia), lymphoma (e.g., Hodgkin lymphoma, Non-Hodgkin lymphoma), multiple myeloma, breast cancer, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer, liver cancer, neuroblastoma, brain cancers (e.g., gliomas, meningiomas, pituitary adenomas, etc.), lung cancer, prostate cancer, ovarian cancer, stomach cancer, skin cancer (e.g., melanoma), cervical cancer, testicular cancer, kidney cancer, carcinoid tumors, and bone cancer. In certain embodiments, the cancer is pancreatic cancer, breast cancer, glioblastoma, colorectal cancer, lung cancer, or prostate cancer. In certain embodiments, the cancer is pancreatic ductal adenocarcinoma (PDAC).

Additional therapeutic agents (e.g., chemotherapeutic agents) may be administered with the copolymers and/or nanoparticles of the instant invention. Therapeutic agents include without limitation: anti-cancer agent, anti-migration agent, anti-metastatic agent, anti-inflammatory agent, immune-modulatory agent, anti-stromal agents, gene silencing agents or other like agents. The additional therapeutic agent may be administered in the same or in separate composition from the copolymers and/or nanoparticles of the instant invention. The compositions may be administered at the same time (e.g., simultaneously) and/or at different times (e.g., sequentially).

The copolymers and/or nanoparticles described herein will generally be administered to a patient as a pharmaceutical preparation. The term “patient” as used herein refers to human or animal subjects. These copolymers and/or nanoparticles may be employed therapeutically, under the guidance of a physician or other healthcare professional.

The pharmaceutical preparation comprising the copolymers and/or nanoparticles of the invention may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of copolymers and/or nanoparticles in the chosen medium will depend on the hydrophobic or hydrophilic nature of the medium, as well as the size, drug activity, and other properties of the nanoparticles. Solubility limits may be easily determined by one skilled in the art. The pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in pill or dried powder form (e.g., lyophilized).

In yet another embodiment, the pharmaceutical compositions of the present invention can be delivered in a controlled release system, such as using an intravenous infusion, a pump (e.g., implantable osmotic pump), a transdermal patch, liposomes, or other modes of administration. In a particular embodiment, particularly for the treatment/inhibition of inflammation, the copolymers and/or nanoparticles may be delivered in an implantable biomaterial.

As used herein, “pharmaceutically acceptable medium” or “carrier” includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding discussion. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the copolymers and/or nanoparticles to be administered, its use in the pharmaceutical preparation is contemplated.

The dose and dosage regimen of a nanoparticle according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition for which the copolymers and/or nanoparticles are being administered and the severity thereof. The physician may also take into account the route of administration of the copolymers and/or nanoparticles, the pharmaceutical carrier with which the copolymers and/or nanoparticles are to combined, and the copolymer’s and/or nanoparticle’s biological activity.

Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the copolymers and/or nanoparticles of the invention may be administered by direct injection into a desired area or intravenously. In these instances, the pharmaceutical preparation comprises the nanoparticles dispersed in a medium that is compatible with the site of injection.

Copolymers and/or nanoparticles may be administered by any method such as intravenous injection or intracarotid infusion into the blood stream, oral administration, or by subcutaneous, intramuscular, intrathecal injection, or intraperitoneal injection. In certain embodiments, the copolymers and/or nanoparticles are administered intra-tumorally or directly to the tumor and/or the tumor microenvironment. The methods of administration may include but are not limited to parenterally, subcutaneously, orally, topically, pulmonarily, rectally, vaginally, intravenously, intraperitoneally, intrathecally, intracerebrally, epidurally, intramuscularly, intradermally, or intracarotidly. Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the copolymers and/or nanoparticles, steps should be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.

Pharmaceutical compositions containing a copolymers and/or nanoparticles of the present invention as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. Injectable suspensions may be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. Additionally, the copolymers and/or nanoparticles of the instant invention may be administered in a slow-release matrix.

A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient.

Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unit for the administration of copolymers and/or nanoparticles may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of nanoparticle pharmaceutical preparations may be administered to mice, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the copolymers and/or nanoparticles treatment in combination with other standard drugs. The dosage units of nanoparticles may be determined individually or in combination with each treatment according to the effect detected.

The pharmaceutical preparation comprising the copolymers and/or nanoparticles may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient.

Definitions

The following definitions are provided to facilitate an understanding of the present invention:

The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind.

As used herein, the term “lipophilic” refers to the ability to dissolve in lipids. As used herein, the term “hydrophilic” means the ability to dissolve in water. The term “isolated” may refer to protein, nucleic acid, compound, or cell that has been sufficiently separated from the environment with which it would naturally be associated, so as to exist in “substantially pure” form. “Isolated” does not necessarily mean the exclusion of artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification.

“Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., cancer) resulting in a decrease in the probability that the subject will develop the condition. A “therapeutically effective amount" of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular disorder or disease and/or the symptoms thereof.

As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

“Linker”, “linker domain”, and “linkage” refer to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attach at least two compounds, for example, a therapeutic agent to a polymer. The linker can be linked to any synthetically feasible position of the compounds, but preferably in such a manner as to avoid blocking the compound’s desired activity. Linkers are generally known in the art. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic alkyl group or an optionally substituted aryl group. In a particular embodiment, the linker may contain from 0 (i.e., a bond) to about 500 atoms, about 1 to about 100 atoms, or about 1 to about 50 atoms. The linker may also be a polypeptide (e.g., from about 1 to about 20 amino acids). The linker may be biodegradable under physiological environments or conditions. The linker may also be non-degradable and can be a covalent bond or any other chemical structure which cannot be cleaved under physiological environments or conditions.

As used herein, the term “biodegradable” or “biodegradation” is defined as the conversion of materials into less complex intermediates or end products by solubilization hydrolysis under physiological conditions, or by the action of biologically formed entities which can be enzymes or other products of the organism. The term “non-degradable” refers to a chemical structure that cannot be cleaved under physiological condition, even with any external intervention.

As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 4,000, less than 2,000, particularly less than 1 kDa or 800 Da). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids, though they may be amino acids or dipeptides.

An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof (e.g., scFv), that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule.

As used herein, the term “immunologically specific” refers to proteins/polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

The following example provides illustrative methods of practicing the instant invention and is not intended to limit the scope of the invention in any way.

EXAMPLE

The intractable and devastating nature of pancreatic ductal adenocarcinoma (PDAC) necessitates an urgent need for novel therapies. This study presents the development of a novel polymer prodrug system for the combination treatment of PDAC, based on an optimized pharmacologically active anti-metastatic macromolecular carrier, PCQ, conjugated with gemcitabine (GEM). Structureactivity relationship evaluations showed that random PCQ copolymers exhibited superior anti-migratory activity compared to the gradient PCQ analogs. GEM was incorporated into the random PCQ copolymers using disulfide linker to prepare a reduction-responsive prodrug, PCQ(r)6-SS-GEM12. The resultant therapeutic system presents a pharmacologically active delivery strategy that targets both the proliferative and the metastatic phenotype in PDAC. The PCQ(r)6-SS-GEM12 prodrug demonstrated a selective release of GEM under the reductive tumor environment leading to a significant inhibition of tumor growth with pronounced anti-metastatic effect. Collectively, the data show that the combination of antimetastatic PCQ and cytotoxic GEM-based reduction-responsive prodrug polymer provides an innovative strategy to treat PDAC.

Methods

Synthesis of methacrylate derivative ofHCQ (CQMA)

HCQ sulfate was reacted with NH4OH to obtain HCQ-free base, which was extracted with dichloromethane (DCM), and dried in a rotavapor to get solid HCQ. CQMA was synthesized by l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) coupling reaction. In brief, 0.77 g (5.96 mmol) of MA was taken with 2 mL of dry DCM in a 50 mL flask under N2 atmosphere followed by addition of 0.8 mL of ERN on ice. A solution of EDC (2.28 g; 11.92 mmol) in 5 mL of DCM with a catalytic amount of 4-dimethylaminopyridine (DMAP) was slowly added to the reaction mixture under stirring for 1 hour. Thereafter, 2 g (5.96 mmol) of HCQ dissolved in 5 mL of DCM was added to the flask and the reaction was continued overnight at room temperature under N2 atmosphere. The reaction mixture was diluted with DCM and washed thrice with a saturated solution of NaHCCL. The crude product was purified by silica column chromatography using a 20: 1 DCM/MeOH mixture as an eluent. Purified CQMA was characterized by ^JH NMR and mass spectrometry.

'H NMR of CQMA: (400 MHz, CDCI3, 25° C) 3 8.48 (-CH-N), 7.93 (-CH- CC1), 7.79 (-CH), 7.33 (-CH-CC1), 6.40 (-CH-CNH), 6.06 (H-C=C, cis), 5.51 (H- C=C, trans), 5.13 (-NH-CHCH3), 4.20 (-OCH2-), 3.69 (-NH-CHCH3), 2.73-2.38 (N-CH2), 1.90 (CH₃-C=C), 1.78-1.43 (-CH2-), 1.30 (-NH-CHCH3) and 1.00 (CH3CH2N-). MALDI: [M+H]⁺ calculated for [C₂2H3iClN3O₂]⁺, 404.21; found, 404.21.

Synthesis of methacrylate GEM monomer (GEM-SS-MA)

To synthesize GEM-SS-MA, MA was first reacted with excess HEDS by EDC coupling. In brief, 1 g (11.61 mmol) of MA was dissolved in 2 mL of dry DCM in a 50 mL flask under N2 atmosphere followed by the addition of 3.4 mL of EtsN on ice. A 5 mL DCM solution of EDC (2.88 g; 15.09 mmol) with a catalytic amount of DMAP was slowly added to the reaction mixture and stirring was continued for 1 hour. Thereafter, 3.92 g (25.51 mmol) of HEDS dissolved in 5 mL of DCM was added to the reaction flask and the reaction was continued overnight at room temperature under N2 atmosphere. The reaction mixture was washed with saturated NaHCCL solution, followed by purification by silica column chromatography using 4: 1 hexane/ethyl acetate mixture as an eluent. The purified intermediate HO-SS-MA (Fig. IE) was characterized by *HNMR spectroscopy.

'H NMR of HO-SS-MA: (400 MHz, CDCI3, 25°C) 3 6.09 (H-C=C, cis), 5.56 (H-C=C, trans), 4.37 (-OCH2-), 3.83 (-CH2-OH), 2.93 (-CH2CH2-S-), 2.84 (- SCH2-CH2OH) and 1.90 (CH₃-C=C). In the next step, the primary hydroxyl of HO-SS-MA was activated by CDI by dissolving 0.6 g (3.48 mmol) of HO-SS-MA in 10 mL of DCM along with 0.85 g (5.22 mmol) of CDI in a reaction flask under N2 atmosphere. The solution was stirred for 12 hours at room temperature. The reaction mixture was then diluted with 10 mL of DCM and washed with water thrice. The synthesized CDI-activated intermediate; CIO-SS-MA (Fig. IE) was characterized by 'HNMR spectroscopy.

'H NMR of CIO-SS-MA: (400 MHz, CDCI3, 25 °C) 3 8.23 (N=CH-N), 7.48 and 7.13(=CH-N), 6.16 (H-C=C, cis), 5.56 (H-C=C, trans), 4.72 (-OCH2-), 4.46 (- CH₂O-C=O), 3.10 (-CH2CH2-S-), 3.03 (-SCH2- CH₂O-C=O) and 1.98 (CH₃- C=C).

To synthesize GEM-SS-MA, CDI-activated intermediate, CIO-SS-MA was reacted with GEM. In brief, 0.5 g (1.58 mmol) of CIO-SS-MA and 0.54 g (2.05 mmol) of GEM were dissolved in 10 mL dry DMF and kept at 45°C for 3 days. The reaction mixture was diluted with 20 mL of ethyl acetate, washed with saturated NaCl solution, and the organic layer was collected and concentrated in rotavapor. The crude product was then purified by silica column chromatography using a 2:8 hexane:ethyl acetate mixture to yield GEM-SS-MA (Fig. IE). Synthesized GEM- SS-MA was characterized by ¹ H NMR and mass spectrometry.

'H NMR of GEM-SS-MA: (400 MHz, d⁴ MD3OD and DMSO-t/6, 25°C) 3 8.19 (=CH-N), 7.90 (NHCO-), 7.05 (N-CHCF2)) 6.25 (=CH-C =), 6.14 (-OCH- CH2OH), 6.00 (H-C=C, cis), 5.63 (H-C=C, trans), 5.24 (CF2-CH-OH), 4.32 (- CH2-OCO), 4.13 (CF2CH-OH), 3.85 (-CH2OH), 3.75-3.62 (-CH2OH), 3.00 (- SCH₂) and 1.83 (CH₃-C=C). HRMS (m/z): [M+H]⁺ calculated for [CI₈H₂₄ F₂N3O₈S2]⁺, 512.09; found, 512.09.

Synthesis ofDHPMA

To synthesize DHPMA, glycidyl methacrylate was reacted with water at 80°C in the pressure vessel for 12 hours. Thereafter, water was removed under reduced pressure to yield the viscous liquid. The viscous liquid was dissolved in DCM, passed through aluminum oxide (basic) column, concentrated under reduced pressure, and directly used for polymerization.

Copolymer synthesis and characterization RAFT copolymerization was used to synthesize PCQ copolymers by a slight modification of a reported procedure (Yu, et al. (2019) J. Polym. Sci. Polym. Chem., 57(22):2235-2242). In a typical copolymerization, a mixture of monomers, 4-cyano- 4-(phenylcarbonothioylthio)pentanoic acid, and 2,2'-azobisisobutyronitrile with the molar ratio of 20: 1 :0.25 were dissolved in 1 : 1 DMSO: 1,4-di oxane at the concentration of 100 mg/mL and placed inside the Schlenk tube. The tube was sealed with septum with a magnetic bar inside. The tube was purged with N2 for 30 minutes, immersed in a preheated oil bath at 65 °C, and kept under stirring for 24 hours. The reaction mixture was removed from the oil bath and immersed in a benchtop liquid N2 container to quench the reaction. Once the reaction mixture was solidified, it was taken out of the liquid N2 and allowed to come to room temperature. The reaction mixture was then diluted with an equal volume of 0.1 M acetic acid and dialyzed (3.5 kDa cut off) against water for 4 days. The dialyzed solution was lyophilized to obtain the copolymers as white solids.

The chemical structure of the copolymers was characterized by ^JH NMR (400 MHz Bruker Advance NMR spectrometer, Bruker Biospin, Rheinstetten, Germany) and molecular weights were determined using SEC Agilent 1260 Infinity LC system equipped with Wyatt’s miniDAWN TREOS and Optilab T-rEX detectors, and TSKgel G5000PWXL-CP column. Eluent, sodium acetate buffer (0.1 M, Ph 5) was used with a flow rate of 0.5 mL/minute. The particle size and surface zeta potential of the self-assembled nanoparticles were characterized by DLS (Malvern Zetasizer Nano Series Nano-ZS, Westborough, MA) and the morphology of the particles was visualized under TEM (Tecnai G2 Spirit Bio-twin, FEI, Eindhoven, Netherlands).

Fluorescently labeled polymers were synthesized by adding a supplementary monomer, RhBMA (molar equivalent to CTA) during the polymerization. The unreacted dye and other monomers were removed by dialysis against water. The concentration of RhBMA in the copolymers was determined by UV-visible spectroscopy using an absorbance vs. concentration calibration curve.

‘H NMR of PCQ(r)n (Fig. 1G): (400 MHz, DMSO-t/6, 25°C) 3 8.40 (- CH=N ), 7.80 (-CH=CH-CC1), 7.47 (-CH=C-C1), 7.08 (-CH-C-C1), 6.58 (- CH=CNH), 5.22-4.48 (-CH2-O), 4.20-3.49 (-CH(OH)-CH₂OH) and 2.22-0.50 (- CH₂- and -CH₃). 'H NMR of PCQ(g)n (Fig. 1H): (400 MHz, DMSO-t/6, 25°C) 3 8.40 (- CH=N ), 7.80 (-CH=CH-CC1), 7.47 (-CH=C-C1), 7.17 (-CH-C-C1), 6.62 (-CH=C- NH-), 4.71 (-CH(CH₃)0H), 3.68(-CH(CH₃)OH), 2.91 (-CH2-NHCO-) and 2.09- 0.50 (-CH2- and -CH₃).

‘H NMR of PCQ(r)n-SS-GEM (Fig. 3 A): (400 MHz, DMSO-t/6, 25°C) 3 8.40 (-CH=N-), 7.79 (-CH=CH-CC1), 7.68 (-CH=C-C1), 7.46 (-CH=N-), 7.02 (- CH-C-C1), 6.54 (-CH=CNH-), 6.25 (-OCH-CH2OH), 5.84 (CF₂-CH(OH)-), 5.47- 4.28 (-CH2O-), 4.27-3.48 (-CH(CH₃)NH, -CH (OH), -CH₂(OH)), 2.24-1.40 (-CH₂- CH(CH₃)-) and 1.39-0.5 (-CH₃).

Orthotopic PDAC cancer model

The orthotopic cancer model was generated by following the protocol approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. In brief, KPC8060 cells were suspended in a cooled Matrigel/PBS mixture (1/1 v/v) at a concentration of 2.5 ^Z I O⁴ cells/40 pL. Thereafter, 7-week-old C57BL/6 mice (procured from Charles River Laboratories) were anesthetized by using isoflurane. Incision was made in the abdomen and 40 pL of the cell mixture was injected into the tail of the pancreas. Two layers of 5-0 chromic catgut sutures and soft staples were used to close the incision. On day 10 post-surgery, the soft staples were removed.

Therapeutic study

On day 12 of post KPC8060 cells implantation, mice were randomly divided into six groups and given IP injection of PBS, PCQ(r)6, GEM, (HCQ + GEM), (PCQ(r)6 + GEM) and PCQ(r)6-SS-GEM12 (10 mg/kg GEM) on day 12, 15, 18, 21, 24, 27, and 30 (Fig. 6D). Throughout the treatment, the animals were weighed, and tumor growth was monitored by using Vevo® 3100 ultrasound imaging system (Fujifilm, VisualSonics, Toronto, ON) equipped with MX550D transducer. The Vevo Lab software was used to analyze the shape and volume of the tumors. On day 32, blood samples were collected, and the mice were sacrificed. Macroscopic metastases in the major organs were identified and primary tumor weight was recorded. The isolated organs and tumors were fixed with 4% formaldehyde and 70% ethanol and embedded in paraffin. The paraffin-embedded organs and tumors were sectioned and subjected to immunohistochemical staining. Materials l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4- dimethylaminopyridine (DMAP), l,r-carbonyldiimidazole, GSH, 2,2'- azobisisobutyronitrile (AIBN), glycidyl methacrylate and 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid (CTA) were purchased from Sigma Aldrich, St. Louis, MO. HCQ-sulfate, methacrylic acid and triethylamine were from Acros Organics (Fisher Scientific, Pittsburgh, PA). 2-Hydroxyethyl disulfide (HEDS) was from TCI America, Portland, OR. GEM was from LC Laboratories, Woburn, MA. N-(2-Hydroxypropyl) methacrylamide, and methacryloxyethyl thiocarbamoyl rhodamine B (RhBMA) were from Poly sciences (Warrington, PA). Phosphate-buffered saline PBS, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum FBS, trypsin, penicillin, and streptomycin were purchased from Thermo Scientific (Waltham, MA).

Polymerization kinetic

To study the kinetics of PCQ copolymerization, an aliquot was taken out from the reaction mixture after a specific interval of reaction time, and ^JH NMR was performed to analyze the monomer conversion. Based on the ^JH NMR analysis, % of monomer conversion vs reaction time and the variation of the monomer composition in the copolymer vs reaction time were evaluated.

In vitro GEM release

In vitro GEM release was performed by dissolving 20 mg of PCQ(r)6-SS- GEM12 in 2 mL of PBS and placing it into a dialysis tube (cut off MW 3.5 kDa). The dialysis tube was then immersed in 18 mL of PBS with and without 10 mM GSH. The increasing concentration of released GEM with time was determined by taking out the aliquot from the released medium and analyzing it on HPLC equipped with a C18 column (5 pm, 4.6x 150 mm), water-acetonitrile mixture (80:20 v/v) as mobile phase at a flow rate of 0.5 mL/minute and the detection wavelength of 270 nm was used to determine the GEM concentration.

Cell culture KPC8060 mouse pancreatic cancer cell line has been established from the genetically engineered PDAC mouse model (KrasLSL-G12D/+, Trp53LSL-R172H/+, and Pdx-l-Cre). The S2-013 human pancreatic tumor cell line has been isolated from liver metastasis. The cell lines were cultured in a high- glucose DMEM medium containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 pg/mL) at 37°C with 5% CO2 in a humidified chamber.

Wound healing assay

KPC8060 cells were seeded in a 96-well plate at a density of 2* 10⁴ cells/well and cultured for 18 hours. WoundMaker (Sartorius, Goettingen, Germany) was used to make precise and reproducible scratches. The media was then aspirated, cells were washed with fresh media, and incubated with 100 pL of media containing different treatments. The 96-well plates were then placed in the IncuCyte® S3 live-cell analysis system (Sartorius, Goettingen, Germany). Built-in scan type “scratch wound” was selected and images automatically taken at lOx magnification every 4 hours for 48 hours. Images were processed to calculate the wound width. The average wound width for each set of experiments was compared with the untreated group and the results represented as % of wound closure against time.

Transwell® migration assay

KPC8060 cells were suspended in a serum-free DMEM medium containing PCQ or HCQ. The cell suspension (6*10⁴ cells per insert in 300 pL of media) was placed in each insert (8.0 pm pores, Fisher Scientific, Pittston, PA). The inserts were immersed into a 24-well plate containing DMEM medium with and without 10% FBS. After 16 hours of incubation, cells on the bottom of the inserts were fixed with 100% MeOH and stained with Crystal Violet solution (0.2%). The cotton swab was used to remove the non-migrated cells from the top of the chamber. The migrated cells were imaged under EVOS™ XL microscope at 20x magnification. The results were calculated as the percent of migrated cells relative to PBS-treated cells/imaging field ± SD (n = 5).

Cellular uptake KPC8060 and S2-013 cells were seeded (15>< 10³ cells per well) in 8-well glass chamber and cultured for 24 hours. Thereafter, the medium was aspirated, and cells were further incubated with serum-free medium containing RhB-labeled polymers for 4 hours. After the allotted time, cells were washed with PBS and stained with Hoechst 33342 (blue) followed by LysoTracker™ (green) for live-cell imaging. The images were captured using a confocal microscope (LSM 800 Zeiss, Jena, Germany). To quantify the uptake, cells were seeded in a 12-well plate and incubated with RhB-labeled polymers for 4 hours. Cells were then washed with PBS and trypsinized for flow cytometry analysis (BD® LSRII, BD Biosciences, San Jose, CA).

3D-tumor spheroid penetration

3D-tumor spheroids were grown by seeding KPC8060 cells (1 x 10⁴ cells per well) in an ultralow-attachment 96-well plates (Corning, NY) for 7 days with media being changed every other day. Once the spheroid size reached approximately 300 pm, they were incubated with RhB-labeled polymers for 12 hours, washed with PBS, and imaged with confocal microscope using Z-stack mode with a step size 10 pm. Captured images were transformed into 3D surface plots using ImageJ software.

Cell viability assay

The cell viability of polymers and free drugs against KPC8060 and S2-013 cell lines was evaluated by CTB assay. Four thousand cells were seeded per well in a 96-well plate and incubated overnight. Thereafter, the cells were treated with 100 pL of media containing different treatment concentrations and incubated for 48 hours, when the treatment was replaced with a 100 pL fresh media and CTB mixture (100/20 v/v) and incubated for 2 hours. A microplate reader (SpectraMax® iD3, Molecular Devices, CA) was used to measure the fluorescence intensity (FI) (Ex/Em = 560/590 nm). Cell viability (%) was determined as ([FI]treated ^x 100)/ [FI]_Untreated). The half-maximal inhibitory concentration, IC50 was calculated using nonlinear fit in GraphPad Prism. The combination index (CI) was also evaluated from the cell viability date using CompuSyn software.

Live/dead assay Live and dead cell populations in the 3D-tumor spheroids were evaluated by a live/dead assay kit (Molecular Devices). Spheroids were incubated with copolymers, free drug, and a mixture of copolymer and drug over 48 hours. Thereafter, live/dead staining solution was added and incubated for another 1 hour. Confocal microscope was used to capture the live (green; calcein-AM dye) and dead (red; EthD-III dye) cells. Microplate reader was used to measure the intensity of EthD-III to quantify the dead cell population in each treatment group.

Stability in ascites and serum

The GEM prodrug nanoparticles were incubated in 10% ascites and 10% mice serum solution at 37°C for 48 hours and changes in size and zeta potential were monitored by Zetasizer Nano ZS. To evaluate the degradation stability, nanoparticles were incubated with 10% serum and 10% ascites, with and without 10 mM GSH at 37°C for 48 hours. The incubated mixture was filtered by passing through spin column (cut off MW 3.5 kDa) at 10,000 rpm for 15 minutes. Free GEM in the filtrate was then analyzed by HPLC as described above.

Biodistribution study

The biodistribution of RhB-labeled samples, PDHPMA-RhB, PCQ(r)-RhB and PCQ(r)-SS-GEM-RhB was determined in the orthotopic pancreatic cancer model. The samples were administered by IP injection. After 24 hours of administration, the mice were sacrificed, and the tumors and major organs were harvested for ex vivo fluorescence imaging under the Xenogen IVIS® 200 optical imaging system. The Living Image software was used to quantify the fluorescence intensity in the tissues. The tumors were also embedded in OCT compound and cut into frozen sections (10 pm), followed by staining with DAPI. To visualize the intra-tumoral distribution and the depth of polymer penetration, the tissue sections were imaged under confocal microscope.

RNA sequencing and analysis

RNA isolation was performed using RNeasy® Plus Mini Kit following manufacture’s protocol. The RNA sequencing library was prepared (by Novogene Corporation Inc., CA), and sequenced by using Illumina NovaSeq™ 6000 platform with paired-end reads according to manufacturer’s protocol. The sequencing generated an average of 50 million paired-end reads for each sample. The data obtained from the sequencing platform was converted into sequence data in the FASTQ format (reads) using CASAVA base recognition. High-quality data was obtained by filtering out raw reads containing adapter sequences, reads where uncertain nucleotides(N) constitute more than 10% of either read or reads with low- quality nucleotides constituting more than 50% of the read. The average Q30 (an error probability of 0.001) of the sample after filtering raw reads was above 94%. An average of 96% reads mapped to the mouse genome with an average of 93% reads mapping to the exonic region suggesting the good quality of sequencing data. Afterward, the reads were aligned to the mouse reference genome Mus Musculus (GRCm38/mml0) using Hisat2 (2.0.5) (Kim, et al. (2019) Nat. Biotechnol., 37:907- 915). To quantify the gene expression, the number of reads mapping to each gene was calculated using feature Counts (1.5.0-p3) (Liao, et al. (2014) Bioinformatics 30:923-930). The counts were used as input for differential gene expression analysis using DESeq2 (1.20.0) (Love, et al. (2014) Genome Biol., 15:550). Iog2-fold Change > 0 and padj <= 0.05 were set as the threshold for significantly differential gene expression. Volcano Plot analysis was applied to visualize the distribution of differentially expressed genes in GEM, (PCQ(r)6 + GEM) and PCQ(r)6-SS-GEM12 groups. Reactome is an open-source relational database of signaling and metabolic molecules and their relations organized into biological pathways and processes (Croft, et al. (2014) Nucleic Acids Res., 42:D472-477). Reactome pathway enrichment analysis was carried out using cluster profiler (3.8.1) (Yu, et al. (2012) OMICS 16:284-287). Reactome pathway enrichment analysis was carried out with padj < 0.05 as the threshold for significant enrichment and the most significant 20 Reactome pathways were selected for display.

Statistical analysis

Results are represented as means ±SD. One-way analysis of variance was done to evaluate the statistical significance between different treatment groups. P <0.05 (*) was set as a cut-off value to indicate a significant difference. Fisher’s exact test was used to compare the proportion of metastases at each of the sites separately between the groups. The Kruskal-Wallis test was used to compare the number of metastases per mouse between groups. Pairwise comparisons were tested with Wilcoxon rank sum test and Bonferroni method adjusted for multiple comparisons. SAS software version JMP Pro 16.1.0 was used for analysis (SAS Institute Inc., Cary, NC).

Results

Despite advancements in cancer therapy, metastasis remains the leading cause of cancer-related death, accounting for over 90% of cases (Ganesh, et al. (2021) Nat. Med., 27(l):34-44). Metastasis is a complex process in which a tumor cell migrates to a distant site and establishes a new tumor (Zhu, et al. (2016) Adv. Sci., 3(11): 1600229). The metastatic cascade involves five key steps: invasion, intravasation, survival in blood circulation, extravasation, and colonization in distant organs (Steeg, P.S. (2016) Nat. Rev. Cancer 16(4):201-218; Steeg, P.S. (2006) Nat. Med., 12(8):895-904). In principle, inhibiting any step in the metastatic cascade can aid in preventing metastasis. Inhibiting cell migration is a strategy extensively employed to prevent metastasis. Several potential anti-metastatic agents, including maraviroc, SCH 527123, reparixin, BX 471, UCB 35625, SB 65693, DF 2156A, SCH 479833, AMG 487, AMD 3100, and HCQ have been used to target the cell migration (Allegretti, et al. (2012) Immunol. Lett., 145(l-2):68-78; Kim, et al. (2012) PLoS One 7(2):e31004; Li, et al. (2015) J. Contr. Release 219:369-382). The development of polymeric analogs of these small-molecule drugs is a promising strategy to improve their pharmacokinetic profile and overall therapeutic efficacy.

Synthesis ofPCQ copolymers

It was hypothesized that chemical composition and CQ spacing in the PCQ copolymers could affect the interactions with cells and thus influence the overall biological activity of the polymeric drug (Gestwicki, et al. (2002) J. Am. Chem. Soc., 124(50): 14922-14933; Cairo, et al. (2002) J. Am. Chem. Soc., 124(8): 1615- 1619). Therefore, two series of distinct PCQs were synthesized from a methacrylate derivative of HCQ (CQMA) and a hydrophilic comonomer to control aqueous solubility and the density and distribution of the CQ moieties. The hydrophilic comonomers, 2,3-dihydroxypropyl methacrylate (DHPMA) and N-(2- hydroxypropyl) methacrylamide (HPMAm) were chosen due to their distinct copolymerization reactivities, enabling the synthesis of both, random and gradient PCQ copolymers. CQMA monomer was synthesized and characterized (Fig. IF) (Yu, et al. (2016) J. Contr. Release 244(Pt B):347-356). CQMA was copolymerized with DHPMA and HPMAm using reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare random PCQ(r)n and gradient PCQ(g)// copolymers, respectively (‘n’ denotes the mol% of CQMA in the copolymers). The CQMA content was determined by ^JH NMR (Figs. 1G and 1H) using the ratio of integration area between the quinoline protons of CQMA (d 8.40, 7.80, 7.47, 7.08, and 6.56 ppm) and methylene protons of DHPMA and HPMAm (d 4.92-4.50 ppm). The molecular weights, determined by size exclusion chromatography (SEC), ranged from 11.5 to 30.8 kDa with a narrow dispersity D (1.05-1.20) (Table 1).

The polymerization kinetic study was performed to evaluate changes in the composition of the synthesized copolymers with reaction conversion (Fig. 1). The ratio of the comonomers in the growing chain was determined at various time points using ¹ H NMR. The results revealed similar reactivities of DHPMA and CQMA as indicated by the unchanged ratio of DHPMA to CQMA in the growing polymer chain throughout the reaction (Fig. IB and D).

In contrast, the rate of copolymerization of CQMA was higher than that of HPMAm (Fig. 1C) as indicated by the increasing ratio of HPMAm to CQMA in the PCQ(g)n copolymers with increasing conversion (Fig. ID), thus resulting in the formation of gradient copolymers. For the subsequent studies, three gradients and three random copolymers each with distinct CQMA content were synthesized: PCQ(r)6, 10, and 23 and PCQ(g)7, 13, and 23 (Table 1). PHPMAm and PDHPMA homopolymers were also synthesized and used as controls (Table 2). The copolymers self-assemble into nanoparticles in an aqueous environment at pH 7.4 due to their amphiphilic nature attributable to the hydrophobic backbone and nonionized CQ moieties. The observed hydrodynamic size of the formed nanoparticles followed an increasing trend with increasing CQ content (Table 1). As expected, the nanoparticles displayed a positive zeta potential due to the protonated secondary (pKa 8.3) and tertiary amines (pKa 9.7) in CQMA units (Table 1) (Derendorf, et al. (2020) Int. J. Antimicrob. Agents 55(6): 106007).

Table 1: Characterization of PCQ copolymers, (r): random, (g): gradient. HCQ and GEM mol% were calculated from the ¹ H NMR peak integration areas. Particle size and zeta were determined by DLS. Mn and D determined by SEC using acetate buffer (pH 5) as mobile phase.

Table 2: Summary of physiochemical properties of homopolymers. Mn and D determined by SEC-MALS with acetate buffer (pH 5) as the mobile phase. Zeta potential was determined using DLS with HEPES buffer as a dispersant.

Anti-migration activity of PCQ

Cancer metastasis is a dynamic process that relies on the ability of cancer cells to migrate to distant sites (Chen, et al. (2019) Int. J. Pharm., 560:57-64). The ability of PCQ to inhibit the migration of PDAC cells was assessed by a scratch wound healing assay (Jonkman, et al. (2014) Cell Adhes. Migrat., 8(5):440-451). Prior to conducting the assay, the cytotoxicity of the copolymers and HCQ was examined in a mouse PDAC cell line, KPC8060, and in a human PDAC cell line, S2-013. The results revealed no significant toxicity of any tested samples at concentrations as high as 400 pM HCQ equivalent (Fig. II). Based on these results, 100 pM HCQ equivalent was chosen as a safe dose for the wound healing assay. The untreated control group showed complete wound healing after 30 hours in KPC8060 cells (Figs. 1 J, IK, and IL). All the tested PCQ copolymers inhibited cell migration. Copolymers with higher CQ content, PCQ(r)10, PCQ(r)23, PCQ(g)13 and PCQ(g)23 showed inhibition of the wound healing by 45, 7.8, 5.8 and 11 %, respectively (Fig. 1 J). In comparison, copolymers with lower CQ content PCQ(r)6 and PCQ(g)7 demonstrated better inhibition of wound healing (70 and 53 %, respectively, Fig. 1 J), indicating that copolymers with low CQMA content have a higher inhibitory effect than copolymers with high CQ content at constant HCQ equivalent concentrations. Additionally, random copolymers demonstrated better inhibition of wound healing than gradient copolymers. Without being bound by theory, the likely reasons for the difference include better aqueous solubility which leads to improved presentation of the CQ moieties. Additionally, when the concentration of HCQ is kept constant, the number of effective PCQ molecules is lower in the polymer with a higher CQ content compared to the one with a lower CQ content. To better assess the inhibitory effect of PCQ(r)6 and PCQ(g)7 on cancer cell migration, a Transwell® migration assay was conducted. PCQ inhibits cell migration in a relatively broad manner, with the role of CXCR4 chemokine pathway implicated (Yu, et al. (2016) J. Contr. Release 244(Pt B):347-356). To simulate the broad range of signaling pathways involved in cell migration, fetal bovine serum (FBS) was used as a chemoattractant. As expected, no inhibitory effect was observed for PHPMAm and PDHPMA (Fig. IL). In both the KPC8060 and S2-013 cells, PCQ treatment proved to be more efficacious than treatment with control parent HCQ (Fig. 2). Both PCQ(r)6 and PCQ(g)7 demonstrated a dose-dependent inhibitory effect between 0.625 and 10 pM (Fig. IL). Interestingly, 10 pM PCQ(r)6 completely inhibited KPC8060 cell migration while PCQ(g)7 showed 22% cell migration (Figs. 2A and C), indicating that the random PCQ(r)6 was more effective in inhibiting KPC8060 cell migration than the gradient analog. Similarly, random PCQ(r)6 was more effective in the inhibition of S2-013 cell migration than the gradient PCQ(g)7 (Fig. 2B). The representative image of the migrated KPC8060 and S2-013 cells are shown in Fig. 2C. Although the seeding density of these two cell types (KPC8060 and S2-013) was the same, there was a clear difference in the number of migrated cells in PBS. This indicates that the migration rates of the two cell types were different.

Synthesis and characterization of gemcitabine-containing PCQ prodrug PCQ(r) 6- SS-GEM12

To combine the anti-migratory activity of PCQ with anti-cancer activity, the best performing PCQ(r)6 was used for the synthesis of novel terpolymer prodrugs to simultaneously deliver GEM and PCQ. To facilitate the intracellular release, a GEM monomer, GEM-SS-MA, was synthesized with a reduction-responsive disulfide linker in a three-step reaction (Fig. IE). First, MA was reacted with an excess molar ratio of 2-hydroxy ethyl disulfide (HEDS) to synthesize monosubstituted HEDS, HO-SS-MA. In the second step, the hydroxyl group in HO-SS- MA was activated with l,l'-carbonyldiimidazole (CDI) and subsequently reacted with the amine group of GEM to obtain the GEM-SS-MA. The ^JH NMR spectrum of GEM-SS-MA displayed signals at <5 8.19, 7.90, 7.05, 6.25 ppm that were assigned to GEM and signals at 3 4.32 and 3.00 ppm that were assigned to the methylene protons of HEDS. Signals at 3 6.00, 5.63 and 1.83 ppm were assigned to the methacrylate protons. The observed mass spectrum peak at mlz 512.09 [M+H]⁺ (calculated 512.09) validated the successful synthesis of GEM-SS-MA.

The PCQ(r)6-SS-GEM12 was synthesized by RAFT polymerization of DHPMA, CQMA, and GEM-SS-MA. The 'H NMR spectrum of PCQ(r)6- SS- GEM12 displayed signals at 3 8.40, 7.79, 7.86, 7.02, and 6.54 ppm assigned to the quinoline protons of CQMA (Fig. 3A). The signals at 3 7.46, 6.25, and 5.84 ppm were assigned to the GEM protons, respectively, in Fig. 3 A. The signals between 5.47-4.28 and 4.27-3.48 ppm were assigned to the DHPMA protons, which merged with CQMA and GEM-SS-MA protons. The molecular weight of the PCQ(r)6-SS- GEM12 was determined as 41.97 kDa (by SEC) with D =1.33 (Table 1). PCQ(r)6- SS-GEM12 contained 5.6 mol% of CQ and 12.2 mol% of GEM which was calculated from the ¹ H NMR integration areas corresponding to CQ protons (at 3 8.40, 7.68 and 6.54 ppm), GEM protons (at 3 5.84 ppm) and DHPMA protons (between 3 A'l- . ppm). PCQ(r)6-SS-GEM12 is a high molecular weight polymer with a complex structure that can undergo folding or twisting. As a result, variations in the proton environments of the repeating units likely contribute to unequal peak integration areas (Senthil Kumar, et al. (2024) Magn. Reson. Chem., 62(8):619-628). This led the use of the average of multiple peak integration areas when analyzing the mol % of CQ and GEM.

PCQ(r)6-SS-GEM12 demonstrated self-assembly into nanoparticles with a hydrodynamic size of 92 nm (Fig. 3B and Table 1). The formed nanoparticles had a spherical shape as assessed by transmission electron microscopy (TEM) (Fig. 3C). The particle size obtained from TEM was smaller (~26 nm) than that obtained from DLS due to sample drying during TEM (Panja, et al. (2016) ACS Appl. Mater. Interfaces 8(19): 12063-12074; Panja, et al. (2015) Polymer 61 :75-86). The selfassembled nanoparticles showed a zeta potential of 14.1 mV indicating exposure of protonated CQMA moieties on the particle surface, an important prerequisite for their interactions with cancer cells.

The cellular uptake along with the intracellular drug release are two key factors that significantly influence the therapeutic efficacy of drug delivery systems. Cancer cells, including those in PDAC, are characterized by an elevated level of GSH which can cleave the disulfide linker via a thiol exchange reaction and release the GEM (Shetty, et al. (2020) Mol. Pharm. 17(10):3979-3989.; Xin, et al. (2020) Sci. Adv., 6(46): eabd6764). An in vitro release study was performed in the presence and absence of GSH to analyze the GEM release kinetics (Fig. 3D). PCQ(r)6-SS-GEM12 showed a maximum of 83% of GEM release after 24 hours of incubation with 10 mM GSH. In contrast, only 9% GEM release was observed on incubation with 2 mM GSH for 48 hours. Additionally, no significant release was observed upon incubation in saline and FBS. Taken together, these results confirmed the stability of PCQ(r)6-SS-GEM12 in the physiological medium and their reduction-responsive GEM release in the cancer cell environment.

Cellular uptake of prodrug nanoparticles

Cell uptake of rhodamine-B (RhB, red) labeled PDHPMA, PCQ(r)6 and PCQ(r)6-SS-GEM12 was determined in KPC8060 and S2-013 PDAC cells using live-cell confocal microscopy at 4 hours post-incubation. Prior to the imaging, nuclei and lysosomes of the cells were stained with Hoechst 33342 (blue) and Lysotracker™ (green), respectively. PDHPMA had a very low cellular uptake (Fig. 4A and 4D) due to the large number of hydrated hydroxyl groups that limited interaction with the cells. As expected, the positively charged PCQ(r)6 and PCQ(r)6-SS-GEM12 exhibited high cell uptake (Fig. 4A and 4D). The red fluorescence of the nanoparticles was mostly distributed throughout the cytoplasm. The low colocalization of the nanoparticle fluorescence with the lysosomal marker’s fluorescence indicates either that the nanoparticles efficiently escaped from the lysosomes due to the pH-dependent membrane activity of CQMA moieties in PCQ, or that they translocated to the cytoplasm through an alternative pathway (Yu, et al. (2016) J. Contr. Release 244(Pt B):347-356). The cellular uptake of polymers was quantified by using flow cytometry. The highest cellular uptake was found for PCQ(r)6-SS-GEM12 with the mean fluorescence intensity (MFI) -67 times higher than PDHPMA and ~2.8 times higher than PCQ(r)6 in KPC8060 cells (Fig. 4B and 4E). Similarly, in S2-013 cells, the uptake of PCQ(r)6-SS- GEM12 was ~47 and ~3.2 times higher than PDHPMA and PCQ(r)6, respectively (Fig. 4B and 4E) (Tan, et al. (2010) Chem. Mater., 22(7):2239-2247). To better simulate the complexity of the solid tumors, cellular uptake was assessed in 3D tumor spheroids. The spheroids were prepared by seeding KPC8060 cells in an ultra-low attachment 96-well plate. After 1 week of culture, when the size of the spheroids reached -400 pm, they were incubated with PDHPMA-RhB, PCQ(r)6-RhB and PCQ(r)6-SS-GEM12-RhB for 12 hours. The z- stack mode of the confocal microscope was used to capture the uptake and penetration of polymer nanoparticles in the spheroids (Fig. 4C). The spheroid uptake of PDHPMA-RhB was insignificant as it was mostly located in the peripheral layer of cells of the spheroid. In contrast, PCQ(r) 6-SS-GEM12-RhB and PCQ(r)6- RhB exhibited high spheroid penetration. Moreover, in comparison with PCQ(r)6- RhB, PCQ(r) 6-SS-GEM12-RhB showed a deeper penetration (-70 pm) with partly homogeneous distribution throughout the tumor spheroid. Considering the larger size of the PCQ(r)6-SS-GEM12-RhB nanoparticles, the results indicate that a transcellular transport mechanism, as opposed to diffusion, was responsible for better uptake and penetration. The nanoparticles are first taken up by peripheral cells and are then transported to the inner layer of the tumor spheroid via exocytosis to achieve a deeper penetration (Tang, et al. (2021) Mol. Pharm., 18(12):4448- 4458).

Cancer cell killing efficiency of the prodrug nanoparticles

To investigate the feasibility of PCQ(r)6-SS-GEM12 for PDAC therapy, its PDAC cell-killing ability was evaluated using the CTB assay (Fig. 5A and B). The prodrug showed the best KPC8060 cell killing ability (IC50 =5 nM) even when compared with free control GEM (IC50 =37 nM) (Fig. 5A). A similar outcome was observed in the S2-013 cells (Fig. 5B), where the IC50 values for PCQ(r)6-SS- GEM12 and GEM were 32 nM and 60 nM, respectively.

To further understand this finding, the cell viability of a mixture of PCQ(r), GEM, HCQ, and GEM was examined. In comparison with GEM, both the (PCQ(r)6 + GEM) and (HCQ + GEM) showed a higher KPC8060 cell killing ability with IC50 5.9 nM and 14.1 nM, respectively (Fig. 5A). Similar results were also obtained for the S2-013 cell line (Fig. 5B). These data clearly show that PCQ played a prominent role in enhancing the cancer cell-killing ability of GEM, even though PCQ itself had no significant cell-killing ability. While PCQ alone does not kill cancer cells, it enhances the cell uptake of PCQ(r)6-SS-GEM12. In the redox environment of cancer cells, PCQ(r)6-SS-GEM12 releases GEM, allowing it to exert its cytotoxic effect. Moreover, the effect of PCQ on synergistic signaling pathway(s) is likely behind this observation. Indeed, analysis of the combination index (CI) showed a synergistic CI of 0.37 for the prodrug in KPC8060 cells (Table 3).

Table 3: A summary of combination index (CI), analyzed by using CompuSyn software and cell viability results. PCQ(r)6-SS-GEM copolymer with three different GEM contends were synthesized and their cancer cell killing ability was evaluated along with the equivalent doses of individual drugs, PCQ and GEM.

Cell-killing efficiency was then studied also in the multicellular KPC8060 spheroid model using live/dead staining with calcein-AM (green) and ethidium homodimer III (EthD-III, red) (Fig. 5C). In line with the monolayer cytotoxicity results, treatment with PCQ(r)6-SS-GEM12 led to the highest number of dead cells in comparison with all other treatment groups (Fig. 5C). Quantification of the dead cells (Fig. 5D) revealed that spheroids treated with PCQ(r)6-SS-GEM12 had ~1.8 times more dead cells than GEM-treated spheroids and -1.5 times higher than (PCQ(r)6 + GEM) treated spheroids. In addition, the spheroids started to disintegrate in the PCQ(r)6-SS-GEM12 group, whereas they remained intact in the other treatment groups (Figs. 5C and 5E). The higher cell killing efficiency of PCQ(r)6-SS-GEM12 was likely due to the PCQ-mediated deeper penetration in the spheroids, followed by the GSH-responsive release of GEM (Fig. 4C). Hence, the results from the monolayer and 3D-tumor model studies confirmed a strong PDAC cell-killing efficiency of the developed prodrug nanoparticles.

Stability of the prodrug nanoparticles

The in vivo stability of nanoparticles is a key parameter to achieve the desired therapeutic effect (Wang, et al. (2016) Nano Today 11(2): 133-144.). To test the stability of the prodrug nanoparticles, the particles were incubated in serum and in ascites obtained from mice with implanted pancreatic tumors. Incubation with mouse serum resulted in a slight increase of particle size from 92 to -153 nm (Fig. 5F) within the first 1 hour, with no further increase seen up to 24 hours, indicating good colloidal stability of the nanoparticles. The increase in particle size was attributed to serum protein adsorption. This was corroborated by the reversal of the nanoparticle surface charge from positive to negative (Fig. 5G). A similar result was also obtained when the nanoparticles were incubated with ascites (Figs. 5F and 5G). To evaluate the potential degradation of the prodrug and premature release of GEM, the nanoparticles were incubated with 10% ascites and 10% serum with and without GSH at 37°C for 48 hours. The HPLC chromatogram revealed no signature peaks for GEM degradation products, indicating the stability of the nanoparticles in ascites and serum (Fig. 5J). However, nanoparticles incubated with ascites or serum supplemented with GSH showed a signature peak for GEM, which confirmed the reduction-responsive GEM-releasing proficiency of the PCQ(r)6-SS-GEM12 nanoparticles.

Biodistribution and tumor accumulation of the prodrug nanoparticles

Encouraged by the favorable in vitro activity and stability of PCQ(r) 6-SS- GEM12, in vivo biodistribution studies were performed in an orthotopic PDAC mouse model established by implanting KPC8060 cells into the tail of the pancreas. Intraperitoneal (IP) administration has benefits for effective accumulation of nanoparticles in peritoneal tumors (Yuan, et al. (2016) Adv. Funct. Mater. 26(47):8631-8642; Xie, et al. (2020) ACS Nano 14(I):255-271).

RhB-labeled polymers were used and biodistribution was assessed 24 hours after IP injection in tumor-bearing mice. As shown in Fig. 6A and 6B, the prodrug nanoparticles showed 3.8- and 1.9-fold higher tumor accumulation than PCQ(r)6- RhB and PDHPMA-RhB, respectively. The phenomenon of higher tumor accumulation of positively charged nanoparticles is well established (Wang, et al. (2016) Nano Today 11(2): 133-144). Notably, hydrophobicity plays a significant role in tumor accumulation; for example, cholesterol or 2,3, 5,6-tetrafluoro-p-toluic acid derivative of cationic polymers demonstrated a higher tumor accumulation than simple polycations (Tang, et al. (2021) Mol. Pharm., 18(12):4448-4458; Hang, et al. (2021) J. Contr. Release 333: 139-150). Introducing hydrophobic MA-SS-GEM units likely contributed to the observed tumor accumulation of the prodrug nanoparticles. Organ distribution analysis revealed that after the tumor, the kidney was the next highest accumulation site, followed by the liver. A comparable tumor/liver distribution ratio was seen for PCQ(r)6-RhB and PCQ(r) 6-SS-GEM12- RhB (Fig. 5H). However, the tumor/kidney (T/K) ratios (Fig. 51) for PCQ(r)6-RhB and PCQ(r)6-SS-GEM12-RhB were 1.3 and 2.8, respectively indicating a higher tumor selectivity for PCQ(r) 6-SS-GEM12-RhB.

PDAC tumors, including the KPC8060 model used here, are commonly associated with a high density of pancreatic stellate cells (PSCs) and fibrosis, which creates a barrier to deep tumor penetration (Han, et al. (2020) J. Am. Chem. Soc., 142(10):4944-4954). The tumor sections confirmed a significant number of a- smooth muscle actin (a-SMA) positive PSCs (Fig. 5K), a drug penetration barrier. Therefore, to better understand the intratumoral distribution and depth of tumor penetration of the prodrug nanoparticles, frozen tumor sections were processed and counter-stained with DAPI. The confocal microscopy images revealed deep penetration of both the PCQ (r)6-SS-GEM12-RhB and PCQ(r)6-RhB, as shown by the homogeneous distribution of the red fluorescence throughout the tumor section (Fig. 6C). In contrast, PDHPMA-RhB was mostly retained on the tumor surface. In comparison with PDHPMA-RhB and PCQ(r)6-RhB, PCQ(r) 6-SS-GEM12-RhB treated group showed an intense red fluorescence which further complemented its highest tumor accumulation. The high tumor accumulation and deep penetration of PCQ(r)6-SS-GEM12-RhB, particularly in the presence of PSCs and fibrosis, were likely due to two factors: the strong interaction between the tumor cells and PCQ, and the hydrophobic nature of the GEM repeating units. Overall, the PCQ(r) 6-SS- GEM12 nanoparticles demonstrated good stability in both ascites and serum and showed the highest tumor accumulation and deep tumor penetration. Antitumor effect of PCQ(r)6-SS-GEM12

To evaluate the antitumor effect in vivo, mice with orthotopic KPC8060 tumors received 10 mg/kg equivalent GEM IP dose of various treatments following the schedule shown in Fig. 6D. The progression of tumor volume over the treatment period was determined by using an ultrasound imaging system (Fig. 6G). The representative ultrasound images of the tumor on day 12 and day 31 are shown in Fig. 6H. All treatment groups containing GEM inhibited tumor growth (Fig. 6E- 6G), with the PCQ(r)6-SS-GEM12 nanoparticles exhibiting the best efficacy, followed by the combination of HCQ + GEM and PCQ(r)6 + GEM groups, and GEM. The PCQ(r)6-SS-GEM12 treated group showed ~82 % inhibition of tumor growth, while the combination of HCQ +GEM and PCQ(r)6 +GEM showed tumor growth inhibitions of ~47 % and ~51 % respectively (Fig. 6G) and results were well corroborated with the tumor weight (Fig. 6E). The lowest inhibition of tumor growth (~31 %) was seen in the GEM-treated group. This could be because of its lower tumor accumulation and susceptibility to cytidine deaminase inactivation (Han, et al. (2017) ACS Nano 11(2): 1281-1291). To gain further insight into the antitumor effect, the tumor tissue sections were analyzed using hematoxylin and eosin (H&E), cleaved caspase-3 (CC3), and CD8+ staining.

The H&E staining of the tumor section revealed large areas (dotted line) of apoptotic cells in the PCQ(r)6-SS-GEM12 group (Fig. 7A) with similar results seen with CC3 staining (Fig. 7A and B). GEM treatment increases the CD8+ cell infiltration (Du, et al. (2020) Int. Immunopharmacol., 86: 106694; Zhang, et al. (2019) Biomaterials 189:48-59). Consistent with these observations, Figs. 7A and 7C show increased CD8⁺ staining, indicating a higher infiltration of CD8⁺ cytotoxic T-cells in the PCQ(r)6-SS-GEM12 group.

Antimetastatic effect of the prodrug nanoparticles

Metastasis is the foremost cause of cancer-related deaths (O’Reilly, et al. (2021) J. Clin. Oncol., 39(15):el6250; Lin, et al. (2021) Adv. Sci., 8(4):2002834). Inhibition of metastasis, along with the inhibition of primary tumor growth, is a promising strategy to treat PDAC. Anti-metastatic activity was evaluated at the end of the therapeutic study by counting the number of macroscopic metastases in different organs (Fig. 7D). The placebo (PBS) group showed a broadly metastasized disease with lesions in the liver, lung, kidney, intestine, stomach, peritoneum, and diaphragm. The prodrug nanoparticles showed a remarkable inhibition of metastasis, with complete inhibition of the metastasis to the liver, lung, kidney, peritoneum, and diaphragm. It is worth mentioning that the liver, the primary site for PDAC metastasis, was completely free of metastatic lesions following PCQ(r)6- SS-GEM12 treatment. Without being bound by theory, the higher anti-metastatic effect of PCQ(r)6-SS-GEM12 was attributed to the antimigration effect of PCQ which was further reinforced by the cytotoxic effect of GEM. The PCQ(r)6 group demonstrated strong inhibition of metastasis, which aligns with the results from the cell migration study (Fig. 2). While both GEM and (HCQ +GEM) treatments also reduced the extent of metastasis, this effect is primarily due to the cytotoxic effect of GEM.

Toxicity profile ofPCQ(r)6-SS-GEM12

To examine potential toxic effects associated with the treatments, the body weight was measured during the treatment, and tissues from major organs were isolated and stained with H&E and examined for any histological abnormality. There was no significant change in body weight during the treatment (Fig. 7E). Although biodistribution data (Fig. 6B) indicated that the kidney was the second- highest accumulation site, H&E staining and the analysis of blood urea nitrogen and creatinine revealed no signs of renal abnormality (Table 4). Whole blood analysis and blood biochemistry confirmed no adverse hematological effects as evidenced by normal levels of lymphocytes, monocytes, neutrophils, white blood cells, platelets, red blood cells, and hemoglobin (Table 4). These findings collectively indicate no side effects or minimal side effects associated with the treatment.

Table 4: A summary of whole blood analysis and blood biochemistry results. LYM: lymphocytes. Mono: monocytes. NEUT: neutrophile. WBC: white blood cell. PLT: platelets. RBC: red blood cells. HgB: hemoglobulin. BUN: blood urea nitrogen.

RNA sequencing

To uncover the underlying mechanism of tumor growth inhibition, RNA- sequencing (RNA-Seq) analysis and transcriptome profiling of the tumors were performed. Differential gene expression analysis revealed an upregulation of 1418 genes and a downregulation of 1162 genes in the prodrug nanoparticle group vs PBS group (Fig. 8A). Comparison of (PCQ (r)6 + GEM) vs PBS and GEM vs PBS showed an upregulation of 2848, 1272, and the downregulation of 1444, and 994 genes, respectively (Fig. 8B-C). A Venn diagram was constructed to identify the uniquely deregulated genes in the PCQ(r)6-SS-GEM12 group (Fig. 8D). The diagram showed 574 exclusive differentially expressed genes (DEGs) for PCQ(r)6- SS-GEM12 along with 2006 common DEGs. To further investigate the biologically related gene sets that are specifically associated with downregulated DEGs, a pathway enrichment analysis utilizing the Reactome database was used and the most significant pathways are displayed for the GEM, PCQ(r)6 + GEM, and PCQ(r)6-SS- GEM12 groups (Figs. 8E-8G). The pathway enrichment analysis displayed the downregulation of genes associated with molecular signatures related to translation inhibition for (PCQ(r)6 + GEM) and PCQ(r)6-SS-GEM12 treated groups. Without being bound by theory, this is likely due to the induction of endoplasmic reticulum stress upon PCQ(r)6-SS-GEM12 treatment, which triggers the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a). Phosphorylated eIF2a reduces the overall rate of translation initiation, thereby repressing protein synthesis (Palam, et al. (2015) Cell Death Dis., 6:el913). This observation aligns with the finding of repression of global translation in GEM-treated PDAC cells (Palam, et al. (2015) Cell Death Dis., 6:el913). Moreover, the PCQ(r) 6-SS-GEM12 treatment group exhibited downregulation of genes associated with the citric acid cycle (TCA), complex I, and the respiratory electron transport (ATP) pathway (Fig. 8E). The inhibition of these key metabolic pathways likely attenuated oxidative phosphorylation (OXPHOS), a potential antitumor target (Ashton, et al. (2018) Clin. Cancer Res., 24(l l):2482-2490; Reyes-Castellanos, et al. (2020) Biomedicines 8(8):270; Luo, et al. (2017) Oncogene 36(25):3609-3617). Therefore, combined inhibition of translational machinery and OXPHOS could be a unique contributing factor to the remarkable antitumor effect of PCQ(r)6-SS-GEM12.

In summary, anti-metastatic CQ-based copolymers were synthesized. It was surprisingly discovered that random copolymers with low CQ content exhibit superior anti-metastatic effects compared to their gradient counterparts. The novel PCQ(r)6-SS-GEM12 prodrug nanoparticles demonstrated favorable safety and anticancer activity in vitro, along with enhanced tumor accumulation and deep tumor penetration in vivo. In a metastatic PDAC model, the treatment showed significant anti -metastatic and antitumor efficacy. RNA sequencing revealed that the combined inhibition of translational machinery and OXPHOS were possible reasons for the observed activity. The PCQ(r)6-SS-GEM12 prodrug nanoparticles contain two FDA-approved drugs, CQ and GEM, demonstrating its clinical relevance. In addition, the prodrug strategies are a cornerstone of modem drug development, with nearly 20% of FDA-approved drugs employing this approach (Min, et al. (2023) J. Med. Chem., 66(24): 16546-16567). Furthermore, the clinical trial of several polymeric nanoparticle-based drug delivery systems, such as NC- 6040, SP1049C, and Nanoxel-M, underscores the translational ability ofPCQ(r)6- SS-GEM12 (Beach, et al. (2024) Chem. Rev., 124(9):5505-5616).

A number of publications and patent documents are cited throughout the foregoing specification in order to describe the state of the art to which this invention pertains. The entire disclosure of each of these citations is incorporated by reference herein.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A copolymer comprising a plurality of a first monomer, a plurality of a second monomer, and a plurality of a third monomer, wherein said first monomer comprises an antimetastatic agent, wherein the mole percent of the first monomer in the copolymer is about 1% to about 10%, wherein said second monomer comprises a therapeutic agent, wherein said therapeutic agent is attached to the backbone of the copolymer via a cleavable linker, wherein the mole percent of the second monomer in the copolymer is at least about 1%, and wherein said third monomer is hydrophilic.

2. The copolymer of claim 1, wherein said copolymer is linear.

3. The copolymer of claim 1, wherein said copolymer is a random copolymer.

4. The copolymer of claim 1, wherein said antimetastatic agent is chloroquine or hydroxychloroquine.

5. The copolymer of claim 1, wherein said therapeutic agent is a chemotherapeutic agent.

6. The copolymer of claim 5, wherein said chemotherapeutic agent is gemcitabine.

7. The copolymer of claim 1, wherein said cleavable linker comprises a disulfide bond.

8. The copolymer of claim 1, wherein the first monomer has the structure:

9. The copolymer of claim 1, wherein said first monomer has the structure:

10. The copolymer of claim 1, wherein the second monomer has the structure

wherein X is a therapeutic agent, wherein each n is independently 0 to 10, and wherein m is 0 to 2.

5 11. The copolymer of claim 1, wherein the second monomer has the structure

wherein each n is independently 0 to 10, and wherein m is 0 to 2.

5 12. The copolymer of claim 1, wherein the second monomer has the structure

13. The copolymer of claim 1, wherein said third monomer has the structure wherein m is 0 to 2.

14. The copolymer of claim 1, wherein said third monomer has the structure: 15. The copolymer of claim 1, wherein said first monomer has the structure:

wherein the second monomer has the structure

16. The copolymer of claim 15, wherein said third monomer has the structure:

17. A nanoparticle comprising a copolymer of any one of claims 1-16.

18. A composition comprising a copolymer of any one of claims 1-16 and a pharmaceutically acceptable carrier.

19. A composition comprising a nanoparticle of claim 17 and a pharmaceutically acceptable carrier.

20. A method of treating, inhibiting, and/or preventing a disease or disorder in a subject in need thereof, said method comprises administering a copolymer of any one of claims 1-16 to said subject.

21. The method of claim 20, wherein said disease or disorder is cancer.

22. The method of claim 21, wherein said cancer is pancreatic cancer.

23. A method of treating, inhibiting, and/or preventing a disease or disorder in a subject in need thereof, said method comprises administering a nanoparticle of claim 17 to said subject.

24. The method of claim 23, wherein said disease or disorder is cancer.

25. The method of claim 24, wherein said cancer is pancreatic cancer.