Small polymeric carriers for delivery of agents

Abstract

To provide a polymer for delivery of agents, and a formulation comprising the polymer.SOLUTION: There is provided a polymer including: (1) a polymer skeleton as alkylene; (2) a plurality of first pendant groups including nucleosides; (3) a plurality of first linking moieties that covalently bond the first pendant groups to the polymer skeleton; (4) a plurality of second pendant groups including polyalkyleneoxides; and (5) a plurality of second linking moieties that covalently bond the second pendant groups to the polymer skeleton, wherein the first linking moieties contain a group selected from the group of those in the figure and the second linking moieties contain a hydrolytically labile group selected from the group consisting of an ester group, an ortho ester group, a thioether ester group, an anhydride group, an amide group, and a carbonate group.SELECTED DRAWING: Figure 3A

Description

[Technical field]

[0001] Government Interests This invention was made with Government support under Grant Nos. CA174305 and CA223788 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 744,928, filed October 12, 2018, the disclosure of which is incorporated herein by reference. [Background technology]

[0003] The following information is provided to aid the reader in understanding the technology disclosed below and the environment in which such technology may typically be used. Terms used herein are not intended to be limited to any particular narrow interpretation unless otherwise specified herein. References described herein may facilitate understanding of their technology or background. The disclosures of all references cited herein are incorporated by reference.

[0004] Various small molecules have been developed as potential candidates for therapeutic treatment, such as anti-cancer or anti-viral therapy of patients. However, many drawbacks, such as low water solubility, poor bioavailability, and high toxic side effects, limit the clinical application of such therapeutics or compounds. Formulating these drugs into nano-sized micelles improves solubility and provides controllable drug delivery. This is a promising strategy to enhance therapeutic efficacy and reduce systemic toxicity. It is known that the biodistribution profile and tumor penetration of micelles can be tailored by optimizing particle size. Reducing the size of nanoparticles can increase the circulation time and tumor accumulation of drugs. However, smaller nanoparticles usually result in lower drug payload and loading efficiency.

[0005] In addition, it may be desirable to deliver drugs or compounds other than therapeutic drugs via nanoparticle carrier drugs. Such drugs may have some of the same drawbacks or delivery problems as those described above for therapeutic drugs. For example, it may be desirable to deliver diagnostic drugs or compounds, such as contrast agents or compounds for use in imaging techniques (e.g., diagnostic and imaging techniques such as X-ray techniques, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound imaging, light-based imaging, and positron emission tomography (PET), including angiography, venography, and urography), either independently of or in combination with other drugs or compounds (e.g., therapeutic drugs). Summary of the Invention

[0006] In one embodiment, the polymer comprises a hydrophobic polymer backbone (e.g., formed by radical polymerization), a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer.

[0007] At least one of the first plurality of pendant groups and / or the second plurality of pendant groups may, for example, independently be attached to the hydrophobic polymer backbone via a linker. In some embodiments, the linker comprises at least one group that interacts via π-π stacking. In some embodiments, the first plurality of pendant groups are attached to the hydrophobic polymer backbone via a first linker comprising at least a first group that interacts via π-π stacking. The at least a first group that interacts via π-π stacking may comprise, for example, an aromatic group. The at least a first group that interacts via π-π stacking may comprise, for example, a benzyl group.

[0008] In some embodiments, the hydrophobic polymer backbone is formed via radical polymerization of vinyl monomers. The hydrophobic polymer backbone may be formed, for example, via free radical polymerization. The hydrophobic polymer backbone may be formed, for example, via reversible deactivation radical polymerization.

[0009] In some embodiments, the at least one group comprising multiple hydroxyl groups is a hydrophilic group. The at least one group comprising multiple hydroxyl groups may be adapted to form hydrogen bonds with an aqueous environment, for example. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises a sugar group. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises a nucleoside. The at least one group comprising multiple hydroxyl groups may comprise, for example, a nucleoside analog, a sugar alcohol, ascorbic acid, 2-deoxy-D-glucose, triethanolamine, pentaerythritol, tris(hydroxymethyl)aminomethane, dopamine, catechin, or cholic acid. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises glycerol, di-glycerol, erythritol, xylitol, arabitol, fucitol, ribitol, sorbitol, maltitol, isomalt, mannitol, saccharides, oligosaccharides, polysaccharides, ascorbic acid, gluconic acid, glucoronic acid, or glycosamine. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises glucose, fructose, sucrose, galactose, maltose, trehalose, lactulose, or lactose. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises ascorbic acid or 2-deoxy-D-glucose. In some embodiments, the nucleoside is a ribonucleoside, a deoxyribonucleoside, or a nucleoside analog therapeutic agent. The nucleoside may be, for example, selected from the group consisting of gemcitabine, azacytidine, cytarabine, or biologically active derivatives thereof. The nucleoside may be, for example, a therapeutic agent.

[0010] In some embodiments, the at least one group comprising the plurality of hydroxyl groups comprises a therapeutic or diagnostic agent, and the second plurality of pendant groups are attached to the hydrophobic polymer backbone via an in vivo labile linkage. The in vivo labile linkage may comprise, for example, at least one of a reduction-sensitive bond, a pH-sensitive bond, a ROS-sensitive bond, or a protease-sensitive bond. The in vivo labile linkage may comprise, for example, at least one of an ester bond, an orthoester bond, a thioether ester bond, an anhydride bond, an amide bond, a carbonate bond, a disulfide bond, a hydrazone bond, a cic-acotinyl bond, an acetal bond, a maleic acid carboxydimethyl bond, an imine bond, an oxime bond, a silyl ether bond, a ketal bond, a thioketal bond, or a protease-cleavable peptide. As described above, the linkage may further comprise, for example, at least one group that interacts via π-π stacking. The at least one group that interacts via π-π stacking may comprise, for example, an aromatic group (e.g., a benzyl group).

[0011] In some embodiments, the polymers herein form micelles with diameters less than 100 nm, diameters of 50 nm or less, diameters of 30 nm or less, diameters of 20 nm or less, hi some embodiments, the polymers form micelles with diameters in the range of 10-30 nm or in the range of 10-20 nm.

[0012] In another aspect, the formulation herein comprises a plurality of polymers (as described above), for example, formed by radical polymerization, thereby having a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone, the first plurality of pendant groups comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone, the second plurality of pendant groups comprising at least one hydrophilic polymer. The formulation may further comprise at least a first compound. The formulation may further comprise at least a second compound different from the first compound. The first compound and the second compound may, for example, be independently a first hydrophobic compound and a second hydrophobic compound. One or both of the first compound and the second compound may, for example, be independently a small molecule compound. Such a small molecule compound may, for example, have a molecular weight of less than 1.5 kDa or less than 1.0 kDa.

[0013] The first compound and / or the second compound may, for example, independently be a therapeutic compound or a diagnostic compound. The first diagnostic compound may, for example, comprise a contrast enhancing agent for use in imaging techniques.

[0014] In some embodiments, the first compound is a first therapeutic compound, such as a chemotherapeutic compound, an antiviral compound (e.g., for use in treating HIV and / or other viral infections), an antibiotic compound, an antifungal compound, an anticancer compound, an anti-rejection compound, an analgesic compound, an antioxidant compound, an immunomodulatory compound, or an anti-inflammatory compound. The first therapeutic compound may be, for example, JP4-039, paclitaxel, docetaxel, FK506 (tacrolimus), cyclosporine A, protoporphyrin, GW4064 (3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole), rose bengal, epigallocatechin gallate, simvastatin, curcumin, indomethacin, JQ1, I-BET 151, I-BET 762, resiquimod, riluzole, tamoxifen, NLG-919 (indoleamine 2,3-dioxygenase (IDO) pathway inhibitor), sunitinib, imatinib, erlotinib, gefitinib, cetuximab, ac Myc inhibitors (10058-F4 (5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone), etc.), cladribine, fludarabine, nelarabine, troxacitabine, capecitabine, 2'-fluoro-2'-deoxyadenosine, acyclovir, tenofovir, lamivudine, entecavir, GS-441524, GS-5734, 2'-C-methyladenosine, 7-deoxyadenosine, The compound may be 2'-C-methyl-adenosine, 2'-C-methylguanosine, INX-08189, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-ethynyl adenosine, NITD008, NITD449, NITD203, 4'-C-azidocytidine, valapiravir, RO-9187, BCX4430, ribavirin, 6-azauridine or 9-deazaadenosine.Antiviral agents that may be used include, for example, acyclovir, tenofovir, lamivudine, entecavir, GS-441524, GS-5734, 2'-C-methyl adenosine, 7-deaza-2'-C-methyl-adenosine, 2'-C-methylguanosine, INX-08189, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-ethynyl adenosine, NITD008, NITD449, NITD203, 4'-C-azidocytidine, valapiravir, RO-9187, BCX4430, ribavirin, 6-azauridine, and 9-deazaadenosine.

[0015] In some embodiments, the second therapeutic compound is independently selected from the group consisting of JP4-039, paclitaxel, docetaxel, FK506 (tacrolimus), cyclosporine A, protoporphyrin, GW4064 (3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole), rose bengal, epigallocatechin gallate, simvastatin, curcumin, indomethacin, JQ1, I-BET 151, I-BET 762, resiquimod, riluzole, tamoxifen, NLG-919 (indoleamine 2,3-dioxygenase (IDO) pathway inhibitor), sunitinib, imatinib, erlotinib, gefitinib, cetuximab, ac Myc inhibitors (10058-F4 (5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone) etc.), cladribine, fludarabine, nelarabine, troxacitabine, capecitabine, 2'-fluoro-2'-deoxyadenosine, acyclovir, tenofovir, lamivudine, entecavir, GS-441524, GS-5734, 2'-C-methyladenosine, 7- Deaza-2'-C-methyl-adenosine, 2'-C-methylguanosine, INX-08189, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-ethynyl adenosine, NITD008, NITD449, NITD203, 4'-C-azidocytidine, valapiravir, RO-9187, BCX4430, ribavirin, 6-azauridine and 9-deazaadenosine.

[0016] As described above, at least one of the first plurality of pendant groups and / or the second plurality of pendant groups is, for example, independently, attached to the hydrophobic polymer backbone via a linker. In some embodiments, the linker comprises at least one group that interacts via π-π stacking. In some embodiments, the first plurality of pendant groups is attached to the hydrophobic polymer backbone via a first linker comprising at least a first group that interacts via π-π stacking. The at least a first group that interacts via π-π stacking may comprise, for example, an aromatic group. The at least a first group that interacts via π-π stacking may comprise, for example, a benzyl group.

[0017] In some embodiments, the hydrophobic polymer backbone is formed via radical polymerization of vinyl monomers. The hydrophobic polymer backbone may be formed, for example, via free radical polymerization. The hydrophobic polymer backbone may be formed, for example, via reversible deactivation radical polymerization.

[0018] In some embodiments, the at least one group comprising multiple hydroxyl groups is a hydrophilic group. The at least one group comprising multiple hydroxyl groups may be adapted to form hydrogen bonds with an aqueous environment, for example. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises a sugar group. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises a nucleoside. The at least one group comprising multiple hydroxyl groups may comprise, for example, a nucleoside analog, a sugar alcohol, ascorbic acid, 2-deoxy-D-glucose, triethanolamine, pentaerythritol, tris(hydroxymethyl)aminomethane, dopamine, catechin, or cholic acid. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises glycerol, di-glycerol, erythritol, xylitol, arabitol, fucitol, ribitol, sorbitol, maltitol, isomalt, mannitol, saccharides, oligosaccharides, polysaccharides, ascorbic acid, gluconic acid, glucoronic acid, or glycosamine. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises glucose, fructose, sucrose, galactose, maltose, trehalose, lactulose, or lactose. In some embodiments, the at least one group comprising multiple hydroxyl groups comprises ascorbic acid or 2-deoxy-D-glucose. In some embodiments, the nucleoside is a ribonucleoside, a deoxyribonucleoside, or a nucleoside analog therapeutic agent. The nucleoside may be selected from the group consisting of, for example, gemcitabine, azacitidine, cytarabine, or biologically active derivatives thereof. The nucleoside can be, for example, a therapeutic agent.

[0019] In some embodiments, the at least one group comprising a plurality of hydroxyl groups is a therapeutic or diagnostic agent, and the second plurality of pendant groups are attached to the hydrophobic polymer backbone via an in vivo labile linkage. The in vivo labile linkage may comprise, for example, at least one of a reduction-sensitive bond, a pH-sensitive bond, a ROS-sensitive bond, or a protease-sensitive bond. The in vivo labile linkage may comprise, for example, at least one of an ester bond, an orthoester bond, a thioether ester bond, an anhydride bond, an amide bond, a carbonate bond, a disulfide bond, a hydrazone bond, a cic-acotinyl bond, an acetal bond, a maleic acid carboxydimethyl bond, an imine bond, an oxime bond, a silyl ether bond, a ketal bond, a thioketal bond, or a protease-cleavable peptide. As described above, the linkage may further comprise, for example, at least one group that interacts via π-π stacking. The at least one group that interacts via π-π stacking may comprise, for example, an aromatic group (e.g., a benzyl group).

[0020] In some embodiments, the polymers herein form micelles having diameters less than 100 nm, diameters of 50 nm or less, diameters of 30 nm or less, diameters of 20 nm or less. In some embodiments, the polymers form micelles having diameters in the range of 10-30 nm or in the range of 10-20 nm. The micelles may have a loading capacity of the first therapeutic compound of, for example, at least 4-30% by weight, at least 8-30% by weight, or at least 8-20% by weight.

[0021] In another aspect, the formulation for in vivo delivery of a compound herein comprises a plurality of polymers (as described above) comprising a hydrophobic polymer backbone (e.g., formed by radical polymerization), a first plurality of pendant groups attached to the hydrophobic polymer backbone, the first plurality of pendant groups comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone, the second plurality of pendant groups comprising at least one hydrophilic polymer. The formulation may further comprise at least a first compound. The formulation may further comprise at least a second compound different from the first compound. The first compound and the second compound may, for example, be independently a first hydrophobic compound and a second hydrophobic compound. The first compound and / or the second compound may, for example, be independently a therapeutic compound or a diagnostic compound. One or both of the first compound and the second compound may, for example, be a small molecule compound. Such a small molecule compound may, for example, have a molecular weight of less than 1.5 kDa or less than 1.0 kDa. The polymers may, for example, form micelles.

[0022] In another aspect, a method of formulating a composition for delivery of a first compound includes mixing a plurality of polymers (as described above) comprising a hydrophobic polymer backbone (e.g., formed by radical polymerization), a first plurality of pendant groups attached to the hydrophobic polymer backbone, the first plurality of pendant groups comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone, the second plurality of pendant groups comprising at least one hydrophilic polymer, with a plurality of the first compounds. The formulation may further comprise at least a second plurality of compounds different from the first compounds. As described above, the first compound and the second compound may, for example, be independently a first hydrophobic compound and a second hydrophobic group. The first compound and / or the second compound may, for example, be independently a therapeutic compound or a diagnostic compound. One or both of the first compound and the second compound may, for example, be a small molecule compound. Such a small molecule compound may, for example, have a molecular weight of less than 1.5 kDa or less than 1.0 kDa. The polymers may, for example, form micelles.

[0023] In a further aspect, a method of delivering a first compound includes administering a formulation comprising a plurality of polymers (as described above) and a plurality of the first compounds, the plurality of polymers comprising a hydrophobic polymer backbone (e.g., formed by radical polymerization), a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer. The formulation may further comprise at least a second plurality of compounds different from the first compounds. The first compound and the second compound may be, for example, hydrophobic compounds. The first compound and / or the second compound may be, for example, independently therapeutic or diagnostic compounds. The first compound and the second compound may be, for example, small molecule compounds. Such small molecule compounds may have, for example, a molecular weight of less than 1.5 kDa or less than 1.0 kDa. In some embodiments, the plurality of polymers may form micelles, and the first compound and / or the second compound may be loaded into the micelles.

[0024] The present systems, methods and compositions, together with their attributes and attendant advantages, will best be appreciated and understood by considering the following detailed description in conjunction with the accompanying drawings. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows a schematic representation of a representative embodiment of a generalized structure of an amphiphilic polymer.

[0026] [Diagram 2] FIG. 2 illustrates generally another exemplary embodiment of a generalized structure of an amphiphilic polymer.

[0027] [Figure 3A]FIG. 3A shows an idealized schematic of a micellar carrier structure formed from multiple polymers herein that is loaded with multiple therapeutic agents, compounds or drugs.

[0028] [Figure 3B] FIG. 3B shows a schematic depiction of the possible mechanism of micelle aggregation by POEG-co-PVD polymers in comparison with the formation of small micelles and improved drug loading by PGEM polymers.

[0029] [Figure 4] FIG. 4 shows a representative linker group L1 from FIGS.

[0030] [Diagram 5] FIG. 5 shows a representative linker group L2 from FIGS.

[0031] [Figure 6] FIG. 6 shows the synthesis scheme of the POEG-co-PVDGEM polymer herein.

[0032] [Figure 7] FIG. 7 shows the characterization of drug-loaded micelles, where panel A shows the particle size of PTX-loaded PGEM micelles, panel B shows the particle size of NLG-loaded PGEM micelles, panel C shows the particle size of PTX / NLG co-loaded PGEM micelles, panel D shows the TEM image of PTX-loaded PGEM micelles, panel E shows the TEM image of NLG-loaded PGEM micelles, and panel F shows the TEM image of PTX / NLG co-loaded PGEM micelles using negative staining. Scale bars are 100 nm in panels D to F, and PTX and NLG are 3.8% and 7.4%, respectively, in the DLC study.

[0033] [Figure 8A]Figure 8A shows the cumulative PTX release profile from PTX / NLG co-loaded PGEM micelles, where PBS containing 0.5% (w / v) TWEEN® 80 (a non-ionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid) was used as the release medium and reported values ​​are the mean ± SD of triplicate samples.

[0034] [Figure 8B] Figure 8B shows the cumulative NLG release profile from PTX / NLG co-loaded PGEM micelles, where PBS containing 0.5% (w / v) TWEEN 80 was used as the release medium and values ​​reported are the mean ± SD of triplicate samples.

[0035] [Figure 9A] Figure 9A shows the MTT cytotoxicity assay of PGEM prodrug micelles in PANC02 cell line with free PGEM as control, where cells were treated with different micelles for 96 h and values ​​reported are the mean ± SD of triplicate samples.

[0036] [Figure 9B] Figure 9B shows the MTT cytotoxicity study of various formulations in PANC02 cell line, where cells were treated with different micelles for 96 h and values ​​reported are the mean ± SD of triplicate samples.

[0037] [Figure 10] FIG. 10 shows the in vitro inhibitory effect of IDO activity when PANC02 cells were treated with IFN-γ together with free NLG919, PTX / PGEM, NLG / PGEM, and PTX+NLG / PGEM, and kynurenine (Kyn) was measured in the supernatant two days later.

[0038] [Figure 11]FIG. 11 shows the in vivo therapeutic effect, where Panel A shows that PANC02 cells were subcutaneously injected 12 days prior to treatment with various formulations including saline, PGEM, PTX / PGEM, NLG / PGEM, PTX+NLG / PGEM, and a mixture of Taxol and free GEM, with five intravenous injections every three days. Panel B shows the relative tumor volume change in mice treated with various formulations. Panel C shows the tumor weights of mice receiving various treatments and the tumor inhibition rate (IR) of various formulations. Panel D shows the photographs of excised tumors from each treatment group.

[0039] [Figure 12A] Figure 12A shows flow cytometry analysis of CD4+FoxP3+Treg immune cells in tumor tissues after treatment with various formulations. The percentage of tumor-infiltrating immune cells was correspondingly quantified, and the results are reported as mean ± SD, *p<0.05, **p<0.01 (vs control group), #p<0.05 (vs taxol + free GEM).

[0040] [Figure 12B] Figure 12B shows flow cytometry analysis of CD4+IFNγ+ T immune cells in tumor tissues after treatment with various formulations. The percentage of tumor-infiltrating immune cells was correspondingly quantified, and the results are reported as mean ± SD, *p<0.05 (vs. control group).

[0041] [Figure 12C] Figure 12C shows flow cytometry analysis of CD8+IFNγ+T immune cells in tumor tissues after treatment with various formulations. The percentage of tumor-infiltrating immune cells was quantified correspondingly, and the results are reported as mean ± SD, *p<0.05, (vs control group), #p<0.05 (vs taxol + free GEM).

[0042] [Figure 13] FIG. 13 shows the synthesis scheme of azacytidine-conjugated polymer (PAza).

[0043] [Figure 14A] FIG. 14A shows DLS particle size study and TEM images of blank PAza micelles.

[0044] [Figure 14B] FIG. 14B shows DLS particle size study and TEM images of DOX-loaded PGAza micelles.

[0045] [Figure 15A] Figure 15A shows a synthetic scheme for synthesizing P2DG polymer, where 2DG is 2 deoxy-D-glucose.

[0046] [Figure 15B] FIG. 15B shows DLS particle size study of P2DG, TEM images of blank P2DG micelles, and their fluorescence intensity study.

[0047] [Figure 16A] FIG. 16A shows DLC particle size studies of P2DG loaded with PTX (panel A), DOX (panel B), NLG919 (panel C) and sunitinib (SUN; panel D).

[0048] [Figure 16B] FIG. 16B shows TEM images of blank P2DG and P2DG loaded with sunitinib (SUN), PTX and PTX / SUN. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present devices, systems, methods and compositions, together with their attributes and attendant advantages, will best be appreciated and understood by considering the following detailed description in conjunction with the accompanying drawings.

[0050] It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the exemplary embodiments described. Thus, the following more detailed description of the exemplary embodiments, as illustrated in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely representative of exemplary embodiments.

[0051] References throughout this specification to "one embodiment" or "an embodiment" (or the like) mean that a particular feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" or equivalent phrases in various places throughout this specification are not necessarily all referring to the same embodiment.

[0052] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to provide a thorough understanding of the embodiments. However, one of ordinary skill in the art will recognize that various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring understanding.

[0053] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds and their equivalents known to those of skill in the art, and a reference to "the virus" is a reference to one or more such compounds and their equivalents known to those of skill in the art. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each value falling within the range. Unless otherwise indicated herein, each individual value and intermediate range is incorporated herein as if each were individually set forth herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless otherwise clearly indicated to the contrary in the text.

[0054] As used herein, the term "polymer" refers to a chemical compound that is composed of multiple small molecules or monomers that are arranged in a repeating configuration to form a larger molecule. Thus, a polymer is a compound that has multiple repeat units (or monomeric units), and includes the term "oligomer" (a polymer with only a few repeat units). The term "copolymer" refers to a polymer that contains two or more different repeat units, including terpolymers (containing three different repeat units), etc. Polymers may be naturally occurring or synthetically formed. The use of the term "polymer" includes homopolymers and copolymers. The term "copolymer" is used herein to include any polymer with two or more different monomers. Copolymers may include, for example, alternating copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, graft copolymers, etc. Examples of polymers include, for example, polyalkylene oxides.

[0055] As used herein, the term "pendant" refers to a group or moiety attached to the backbone of a long molecule, such as a polymer, as described above. A pendant group can be either (1) a short chain or low molecular weight group, or (2) a high molecular weight group, such as a long chain or polymer. A pendant group may also be referred to as a side group. A long chain or high molecular weight pendant group may also be referred to as a "pendant chain" or a "side chain."

[0056] In some embodiments, systems, formulations, methods and compositions herein are provided for co-delivery of a small molecule therapeutic or drug (e.g., a chemotherapeutic agent, therapeutic agent or drug) and a nucleic acid-based therapeutic or drug.

[0057] 1 and 2 show schematic diagrams of amphiphilic polymers herein. The amphiphilic polymers may be formed, for example, via radical polymerization, thereby having a hydrophobic polymer backbone. The hydrophobic polymer backbone may be formed, for example, by free radical polymerization or by reversible deactivation radical polymerization or RDRP (previously referred to as controlled radical polymerization or CRP).

[0058] Reversible deactivation radical polymerization (RDRP) techniques include, for example, nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP) and reversible addition-fragmentation transfer (RAFT), as well as others that have evolved in the last two decades (including cobalt-mediated transfer). RDRP offers a route to obtain polymers and copolymers containing radically polymerizable / copolymerizable monomers with predefined molecular weight, composition, structure and narrow / controlled molecular weight distribution. Because RDRP processes can provide compositionally uniform and well-defined polymers with predefined molecular weight, narrow / controlled molecular weight distribution and a high degree of α- and ω-chain end functionalization, they have been the subject of much research, as reported in several review articles and ACS symposia. See, for example, Qiu, J.; Charleux, B.; Matyjaszewski, K., Prog. Polym. Sci. 2001, 26, 2083; Davis, KA, Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1; Matyjaszewski, K., Ed. Controlled Radical Polymerization; ACS: Washington, DC, 1998; ACS Symposium Series 685.; Matyjaszewski, K., Ed.; Controlled / Living Radical Polymerization. Progress in ATRP, NMP, and RAFT; ACS: Washington, DC, 2000; ACS Symposium Series 768; and Matyjaszewski, K., Davis, TP, Eds. Handbook Wiley: Hoboken, 200, the disclosures of which are incorporated herein by reference.

[0059] The hydrophobic polymer backbone may be formed via radical polymerization of various radically polymerizable monomers. Such monomers may include pendant groups, such as those described above, prior to polymerization. Alternatively, such pendant groups may be attached after polymerization. Exemplary monomers for use herein include styrene, acrylic acid, methacrylic acid, acrylonitrile, vinyl monomers and their derivatives. In some embodiments, the degree of polymerization of the hydrophobic polymers herein is, for example, less than 500, less than 200, or less than 100.

[0060] In some embodiments, the polymer comprises a first plurality of pendant groups (X) (i.e., polyols) attached to the hydrophobic polymer backbone and comprising a plurality (i.e., at least two) hydroxyl groups (OH), and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer (P). In some embodiments, at least one of the first plurality of pendant groups and the second plurality of pendant groups are attached to the hydrophobic polymer backbone via a linker. The linker may comprise at least one group that interacts via π-π stacking. The first plurality of pendant groups may, for example, be a first linking group (L 1 The first linking group (L 1 The first linking group (L) may include at least a first group that interacts with the first linking group (L) via, for example, π-π stacking. 1 ) may be absent and the group X may be directly attached to the hydrophobic polymer backbone. The second plurality of pendant groups may also include a second linking group (L 2 The second linking group (L 2 The second linking group (L) may be absent, and the hydrophilic polymer may be directly attached to the hydrophobic polymer backbone. 2 The first linking group (L) may, for example, independently include at least a second group that interacts via π-π stacking. 1 ) and / or a second linking group (L 2) may include, for example, an aromatic group. In general, an aromatic group is a cyclic molecule that contains a resonance bond that exhibits increased stability compared to other geometric or connecting arrangements with the same set of atoms. Aromatic groups include, for example, benzyl and naphthyl groups. In some embodiments herein, the aromatic group herein is a benzyl group.

[0061] Suitable polyols for use herein include, for example, sugar groups, nucleosides, sugar alcohols, sterols, and other groups. In some embodiments, representative polyols include nucleosides, nucleoside analogs, ascorbic acid, 2-deoxy-D-glucose, triethanolamine, pentaerythritol, tris(hydroxymethyl)aminomethane, dopamine, catechin, cholic acid, glycerol, diglycerol, erythritol, xylitol, arabitol, fucitol, ribitol, sorbitol, maltitol, isomalt, mannitol, sugars, oligosaccharides, polysaccharides, ascorbic acid, gluconic acid, glucoronic acid, or glycosamine. Representative sugars include, for example, glucose, fructose, sucrose, galactose, maltose, trehalose, lactulose, or lactose. In some embodiments, the polyol nucleoside herein is a ribonucleoside, a deoxyribonucleoside, or a nucleoside analog therapeutic agent (e.g., gemcitabine, azacytidine, or cytarabine). In some embodiments, the polyol is ascorbic acid or 2-deoxy-D-glucose.

[0062] The hydrophilic oligomer or hydrophilic polymer may be, for example, selected from the group consisting of polyalkylene oxide, polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyoxazoline, polysaccharide, and polypeptide. In some embodiments, at least one hydrophilic polymer is a polyalkylene oxide. The polyalkylene oxide may be, for example, polyethylene glycol. The polyethylene glycol or other hydrophilic polymers herein may have, for example, a molecular weight of at least 500 Da. In some embodiments, the polyethylene glycol of the other hydrophilic polymers herein has a molecular weight in the range of 100 Da to 5 KDa, or in the range of 500 Da to 2 KDa.

[0063] The pendant groups, such as the first and / or second pendant groups herein, may be attached to the hydrophobic polymer backbone via a linking group that includes a labile moiety or bond. The (in vivo) labile group or moiety may include, for example, at least one of a hydrolytically unstable group, a reduction-sensitive bond, a pH-sensitive bond, a ROS-sensitive bond, or an enzyme / protease-sensitive bond. The labile linking group may be unstable, for example, under acidic pH conditions. The pH-sensitive or acid-labile bond may include, for example, carboxydimethyl maleate, hydrazine, imine, acetal, oxime, silyl ether, cis-asonityl, ketal, or another pH or acid-labile bond or linkage. The use of a labile bond that is sensitive to acidic conditions may be used to cleave the pendant group, for example, in the acidic tumor environment. In some embodiments, the labile linking group is sensitive to reduction, such as a disulfide bond. In some embodiments, hydrolytically unstable groups include ester, orthoester, thioetherester, anhydride, amide (e.g., peptide) or carbonate groups. ROS-sensitive labile bonds or linkages include, for example, thioketal bonds. Enzyme or protease-sensitive bonds or linkages include, for example, protease-cleavable peptides containing the sequence CGLDD that are unstable in response to the presence of matrix metalloproteinases MMP-2 or MMP-9.

[0064] FIG. 3A shows an idealized schematic diagram of a micellar carrier structure formed of multiple polymers herein loaded with multiple therapeutic agents, compounds or drugs (i.e., a first compound, a second compound, etc., which may be, for example, independently, anti-cancer compounds, anti-viral compounds, etc., as described in more detail below). As used herein, a therapeutic agent, compound or drug is a biologically active substance that has an effect on the body (e.g., a medicinal or therapeutic effect, an addictive effect, a performance enhancing effect, or another effect). The therapeutic agent may be, for example, an antibody, an antibiotic, an anti-viral agent, an anti-fungal agent, an anti-cancer agent, an immunomodulatory agent, a chemotherapeutic agent, an anti-rejection agent, an analgesic agent, or an anti-inflammatory agent. Small molecule drugs suitable for use herein include, but are not limited to, JP4-039, paclitaxel, docetaxel, FK506 (tacrolimus), cyclosporine A, protoporphyrin, GW4064 (3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole), rose bengal, epigallocatechin gallate, simvastatin, curcumin, indomethacin, JQ1, I-BET 151, I-BET 762, resiquimod, riluzole, tamoxifen, NLG-919 (indoleamine 2,3-dioxygenase (IDO) pathway inhibitor), sunitinib, imatinib, erlotinib, gefitinib, cetuximab, ac Myc inhibitors (10058-F4 (5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone), etc.), cladribine, fludarabine, nelarabine, troxacitabine, capecitabine, 2'-fluoro-2'-deoxyadenosine, acyclovir, tenofovir, lamivudine, entecavir, GS-441524, GS-5734, 2'-C-methyladenosine, 7-deoxyadenosine, These include aza-2'-C-methyl-adenosine, 2'-C-methylguanosine, INX-08189, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-ethynyl adenosine, NITD008, NITD449, NITD203, 4'-C-azidocytidine, valapiravir, RO-9187, BCX4430, ribavirin, 6-azauridine and 9-deazaadenosine.

[0065] Without being limited by mechanism, and referring to Figure 3A, it is hypothesized that inwardly oriented hydrophobic domains are created during micelle formation via the hydrophobic backbone of the polymers herein, which may adopt a folded conformation by orienting via intrachain hydrophobic interactions. If pendant aromatic groups are present, they may enhance hydrophobicity to facilitate the formation of hydrophobic domains and π-π interactions with one or more hydrophobic therapeutic or other compounds loaded within the hydrophobic domains. It is further hypothesized that outwardly oriented hydrophilic domains are formed by hydrophilic polymer side chains.

[0066] Without being limited to the mechanism, and referring to FIG. 3B, the formation of relatively large micelles by conventional amphiphilic polymers can be explained by multi-micelle aggregates. Small micelles formed in the early stage are not stable enough and rapidly aggregate into larger multi-micelle aggregates through inter-micelle interactions. In the amphiphilic polymers herein, the pendant groups containing multiple hydroxyl groups at the interface can stabilize the relatively small micelles by forming hydrogen bonds with the aqueous environment. This mechanism can also explain why the small micelles formed by amphiphilic polymers exhibit high drug loading capacity and excellent stability. The hydrophobic core (e.g., formed from multiple benzyl or other aryl rings and a hydrophobic backbone) can encapsulate hydrophobic drugs through π-π stacking and hydrophobic interactions. It is hypothesized that some hydroxyl groups oriented inwardly of the micelle core can also further enhance the drug loading capacity and stability by forming hydrogen bonds with the drug.

[0067] The covalently attached pendant group X, which contains multiple hydroxyl groups, may be a biologically active group (e.g., a therapeutic agent). If group X is biologically active, it may be desirable for group X to be attached via a labile bond, as described above (i.e., L 1(X may include a labile bond). When X is inert or inactive, the group X may be attached, for example, via a stable bond (i.e., a bond that is not readily cleavable or labile in vivo). The pendant groups X (which may be the same or different throughout the polymer) of the polymers herein may be hydrophilic. Thus, micelles formed from the polymers herein may deliver both hydrophobic (loaded into the micelle or other structure formed by the polymers herein) and hydrophilic (covalently attached to the polymers herein) therapeutic (or other) agents. This has been very difficult to achieve prior to the present compositions.

[0068] In some embodiments, the polymers herein contain a polyol group X, which is a sugar or a nucleoside / nucleoside analog. Many nucleoside analogs (e.g., gemcitabine, azacytidine, cytarabine) are common drugs used, for example, in the treatment of cancer. However, these drugs are rapidly excreted and distributed nonspecifically, often resulting in side effects, poor therapeutic efficacy, and resistance. When nucleoside analogs are conjugated to the amphiphilic polymers herein, the bioavailability of these therapeutic compounds or drugs is improved. Furthermore, these nucleoside analog-conjugated polymers herein can serve as a platform for the delivery / co-delivery of various hydrophobic drugs. In some embodiments, the polyol group has a molecular weight of 1000 or less.

[0069] Nanoparticles formulated from the polymers herein may be prepared, for example, by facile film hydration methods, and may have small diameters / sizes (e.g., less than 100 nm, 50 nm or less, 30 nm or less, 20 nm or less, in the range of 10-20 nm or in the range of 10-30 nm), high drug loading capacity (e.g., 28% for paclitaxel) and excellent stability. In some embodiments, the drug loading capacity is in the range of 4-30% by weight, in the range of 8-30% by weight, or in the range of 8-20% by weight. In vivo data show that formulations herein have significantly improved therapeutic efficacy compared to free drug or the corresponding free drug combinations. Additionally, many formulations herein show immune stimulatory effects that contribute to overall antitumor activity.

[0070] As noted above, in some embodiments, L 2 L may contain labile bonds, such that after delivery to a target area (e.g., tumor cells), the hydrophilic polymer may be cleaved from the backbone, resulting in disassembly of the micelle, thereby facilitating the release of the loaded or encapsulated drug. 1 and L 2 Representative linking groups are shown, for example, in Figures 4 and 5, respectively.

[0071] In some exemplary embodiments, the carriers and formulations herein are effective in treating, for example, pancreatic cancer. As one of the most difficult cancers to treat, pancreatic cancer is predicted to be the second leading cause of cancer-related deaths by 2030. The standard chemotherapy drug for first-line treatment of pancreatic cancer is gemcitabine (also called GEM), a cytidine analogue that acts through inhibition of DNA replication. However, gemcitabine has only achieved limited clinical benefit due to its rapid excretion by cytidine deaminase (CDA) metabolism and low vascular permeability. To improve treatment outcomes, preclinical and clinical studies have evaluated the combination of GEM with other chemotherapy or targeted therapy (e.g., nab-paclitaxel, cisplatin, erlotinib, etc.). Increasing evidence indicates that paclitaxel (PTX), in addition to its direct cytotoxic effect on tumor cells, may reduce CDA expression and increase GEM accumulation in tumors, resulting in a synergistic tumor-killing effect with GEM. In addition, treatment with PTX and / or GEM can stimulate antitumor immune responses by presenting antigens released from dying tumor cells or by directly killing immunosuppressive cells, which also contributes to overall antitumor activity.

[0072] However, overall success remains poor due to various negative feedback mechanisms in the highly immunosuppressive pancreatic tumor microenvironment (TME), such as immune checkpoints cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1), which aid in tumor evasion from normal immune surveillance. Indoleamine 2,3-dioxygenase (IDO) is another well-characterized immunosuppressive enzyme overexpressed in pancreatic cancer. Evidence indicates that high IDO expression correlates with a worse prognosis in pancreatic cancer patients. IDO functions through the degradation of tryptophan to kynurenine, which is toxic to effector T cells, induces the recruitment of T regulatory cells (Tregs), and suppresses antitumor immune responses. Thus, inhibition of the IDO pathway in combination with chemotherapy represents an attractive strategy for the treatment of pancreatic cancer. NLG919 has been shown to be effective in treating ECs. 50 It is a potent IDO1 inhibitor with low EC 50 is the concentration that elicits a response halfway between the baseline and maximum responses after a specified exposure time and is commonly used as a measure of drug potency. Co-delivery of NLG919 and chemotherapeutic agents to tumors is challenging due to its poor solubility, limited oral bioavailability, and different physical properties than chemotherapeutic agents.

[0073] NLG919 prodrug (a micelle system of PEG2K-Fmoc-NLG) was developed as a dual-functional carrier to encapsulate PTX for combination therapy. See, for example, US Patent Application Publication No. 2018 / 0214563. PTX has been found to synergize with NLG919 to inhibit tumor growth by directly killing tumor cells and simultaneously enhancing antitumor immune responses in breast tumor models. This system is suitable for co-delivery of water-insoluble hydrophobic drugs. However, co-delivery of hydrophobic drugs such as PTX and water-soluble drugs such as GEM is more challenging. Furthermore, as mentioned above, many conventional micelle systems have a relatively large hydrodynamic size, which may hinder their penetration into poorly vascularized and stroma-rich tumors such as pancreatic tumors.

[0074] It is well established that many tumor blood vessels are leaky due to their large pore size compared to normal blood vessels, which leads to preferential accumulation of particles in the nano-sized range (4-200 nm) in tumor tissues. However, the cut-off pore size of tumor vasculature varies greatly depending on the tumor type. Pancreatic cancer is known to be poorly vascularized with much smaller pore size (~50-60 nm) compared to other types of cancer. In addition, pancreatic cancer has a dense stroma that further limits the penetration of larger sized nanoparticles. Previous studies have found that only nanoparticles smaller than 50 nm can penetrate poorly permeable pancreatic tumors. It has also been reported that small sized (~10 nm) nanoparticles have shown superior tumor penetration compared to larger nanoparticles in other tumor models. However, small sized nanoparticles often have limited drug loading due to their lower drug loading capacity and efficiency compared to larger nanoparticles. The development of small nanocarriers capable of deep tumor penetration yet highly effective in co-delivering various drugs (GEM / PTX, NLG, etc.) for immunochemotherapy of pancreatic cancer remains a challenge.

[0075] Again, due to the biological complexity of pancreatic cancer and the highly immunosuppressive TME, there is a dire need to develop new combinations of immunotherapeutic and chemotherapeutic agents and related formulations for pancreatic cancer immunochemotherapy. Compared with free drug combinations, designing combined drugs within a single nanocarrier can improve pharmacokinetic and biodistribution profiles, reduce side effects, and allow multiple drugs to be simultaneously delivered to the tumor site at optimal dosages, thus improving synergistic therapeutic effects.

[0076] In a representative embodiment herein, "ultra-small" nanocarriers assembled from redox-responsive gemcitabine (GEM)-conjugated polymer POEG-co-PVDGEM (PGEM) are described to co-deliver, for example, the IDO inhibitor NLG919 and the chemotherapeutic drugs PTX and GEM into pancreatic tumors for immunochemotherapy. The GEM-conjugated polymer (PGEM) nanocarriers were capable of co-loading a wide variety of hydrophobic drugs with high loading capacity and excellent stability. The PGEM nanocarriers were able to efficiently penetrate the tumor core and inhibit tumor growth. Incorporation of PTX into the PGEM carriers was shown to downregulate CDA expression and inhibit tumor growth synergistically with the co-delivered GEM. Incorporation of NLG into the carriers enhanced IFN-γ expression, which was shown to inhibit tumor growth synergistically with the co-delivered GEM. + CD4 + and IFN-γ + CD8 + A more immune-active tumor microenvironment was induced with increased T cells and decreased Treg cells, thereby improving the therapeutic efficacy. Co-delivery of GEM / PTX / NLG through nanocarriers further improved the therapeutic efficacy with a high tumor inhibition rate of 90.4%, indicating its great potential as an effective combination therapy for pancreatic cancer immunochemotherapy.

[0077] Again, particle size of injectable carriers is a very important physicochemical parameter to consider since it plays a key role in cellular uptake, biodistribution and tumor penetration. Also, as mentioned above, it has been reported that nanoparticles in the diameter range of 4-200 nm can efficiently accumulate in tumors as a result of long circulation time and enhanced permeability and retention (EPR) effect. Nanoparticles smaller than 4 nm are rapidly excreted by the kidney, whereas nanoparticles larger than 200 nm tend to be taken up by the reticuloendothelial system (RES). Furthermore, accumulating evidence shows that nanomedicines with small size (10-30 nm) exhibit superior tumor penetration and enhanced antitumor activity, especially against pancreatic cancer. Representative PGEM carriers herein exhibit desirable particle size (~13 nm) to avoid RES uptake and renal excretion, which is also beneficial for accumulation and penetration in pancreatic tumor tissues (<50 nm).

[0078] Moreover, the exemplary PGEM carriers herein overcome the limitations of ultrasmall polymer carriers in drug loading. It is generally believed that reducing the size of polymeric micelles can be achieved by increasing the ratio of hydrophilic / hydrophobic blocks. However, such high ratios produce thick shells with "small" cores, resulting in low encapsulation capacity and poor stability of drug-loaded micelles. Surprisingly, the exemplary PGEM carriers herein were able to load a wide variety of hydrophobic drugs, such as curcumin, NLG919, doxorubicin, dasatinib, and c-Myc inhibitor 10058-F4, with excellent stability and high loading capacity. When loading PTX, a capacity of 24.2 wt% was achieved. Furthermore, the PGEM carriers herein allow high simultaneous loading of multiple drugs with different properties, such as hydrophilic PGEM and hydrophobic PTX / NLG. Conjugation of hydrophilic GEM molecules to the POEG-co-PVD polymer significantly reduced the particle size (from about 160 nm to about 13 nm) while improving the loading capacity and formulation stability.

[0079] Again, CDA is overexpressed in pancreatic tumors, which plays an important role in GEM resistance. Approximately 80% of administered GEM is metabolized by extracellular and intracellular CDA to the inactive form 2',2'-difluorodeoxyuridine (dFdU), which greatly limits the therapeutic efficacy of GEM. PTX can reduce CDA protein levels instead of RNA levels, possibly mediated by PTX-induced ROS, and this reduction in CDA expression led to higher accumulation of active GEM forms in tumors. Consistent with these studies, our data showed that PTX synergized with GEM in killing PANC02 cells, and that CDA expression was significantly downregulated when PTX was incorporated into PGEM carriers. This contributed to the improved antitumor efficacy of PTX / PGEM compared to PGEM in vitro and in vivo.

[0080] Meanwhile, various immunosuppressive mechanisms and molecules, such as TGF-β, galectin-1 and IDO, have been identified in the pancreatic TME, which has opened a new window for immunochemotherapy of pancreatic cancer. Among them, IDO is an attractive immunotherapy target, which is overexpressed in pancreatic cancer but not in healthy pancreatic tissue. The IDO inhibitor indoximod in combination with chemotherapy agents including nab-PTX and GEM for advanced PDAC has progressed to phase Ib trials. NLG919 has a clear mechanism and low EC 50 NLG919 is another potent IDO inhibitor with a PGEM carrier. NLG919 formulated in a PGEM carrier maintained its biological effect of inhibiting IDO activity, and co-delivery of PTX and NLG did not affect the IDO inhibitory effect of NLG. Immunological analysis showed that NLG / PGEM treatment reduced CD4 + / CD8 + T cells and IFN-γ positive CD4 + / CD8 + It has been shown to generate a more immunoactive TME with increased T cells. The improved therapeutic efficacy of NLG / PGEM over PGEM is mainly due to enhanced antitumor immune responses, since the incorporation of NLG did not improve the cytotoxicity of PGEM. PGEM itself may also enhance antitumor immune responses by producing fewer immunosuppressive Treg cells and more IFN-γ production, indicating that PGEM is an immunostimulatory carrier. GEM has been reported to suppress Treg cells in a dose-dependent manner. PTX / PGEM showed better efficacy in activating the immune system compared to the combination of Taxol+GEM, which may be the result of more effective delivery of PTX and GEM to tumors, for example, via the carriers herein. Compared to NLG / PGEM, co-delivery of PTX and NLG via PGEM did not result in significant changes in the immune tumor microenvironment, but provided the highest therapeutic efficacy. This suggests that the combination of PTX / NLG is desirable.

[0081] FIG. 3A shows a schematic of the proposed mechanism of action of the representative PGEM carrier herein. PGEM micelles loaded with PTX and NLG simultaneously penetrate tumor tissue efficiently due to their small particle size. PGEM showed tumor killing and immune stimulating effects itself. PTX and NLG were easily released from the PGEM carrier in response to intracellular GSH. The released PTX could reduce the expression of CDA and exert synergy with GEM in killing tumors. The released NLG919 could further reverse IDO1 function and increase T cell responses against tumors.

[0082] Representative studies have shown that the PTX / NLG / PGEM regimen provided by the PGEM carrier herein is highly effective in treating pancreatic cancer in the PANC02 cell line model, as further described below. PANC02 is a unique tumor model that is highly resistant to almost all known clinical chemotherapeutic agents and can be used as a close mimic of human pancreatic cancer.

[0083] As shown in Figure 6, vinylbenzyl monomers with disulfide bonds (VD monomers) were synthesized by reaction of vinylbenzyl chloride with 4,4'-dithiodibutyric acid. POEG-co-PVD polymers were then synthesized by RAFT copolymerization of VD monomers with OEG950 monomers. POEG-co-PVD GEM polymers were obtained by coupling GEM to the POEG-co-PVD polymer backbone (e.g., using EDC / HOBt coupling reactions known in the chemical arts).

[0084] The structures of the VD monomer, POEG-co-PVD and POEG-co-PVDGEM polymers are shown in 1 The POEG-co-PVD polymers were characterized by HNMR. The average degree of polymerization (DP) of the OEG950 monomer was calculated to be 9 based on the conversion of the OEG950 monomer at the end of the polymerization. The DP of the VD monomer was calculated to be 1. c and I dAfter conjugating GEM to the POEG-co-PVD polymer, 1 Proton peaks corresponding to GEM were observed in the H NMR spectrum, and the number of GEM units per polymer molecule was determined to be I c and I d The loading capacity of GEM was also determined by HPLC-UV analysis with alkaline hydrolysis method. The gemcitabine loading on POEG-co-PVDGEM polymer carrier was determined to be 8.9% w / w.

[0085] The molecular weights and distributions of the POEG-co-PVD and POEG-co-PVDGEM polymers were also determined by GPC. n The M of POEG-co-PVD and POEG-co-PVDGEM were 11,600 and 9,200, respectively. Both polymers showed low polydispersity of 1.12. The M of POEG-co-PVDGEM was determined by GPC. n Note that, decreased after GEM conjugation to the POEG-co-PVD polymer backbone. It is well known that GPC separates polymers by hydrodynamic size and not molar mass. Therefore, the decrease in the measured polystyrene relative molecular weight of POEG-co-PVDGEM indicated the compaction of the polymer chains in THF after GEM conjugation. The GPC and NMR results indicate the successful synthesis of POEG-co-PVDGEM (PGEM) copolymers with defined and controllable architectures.

[0086] Both POEG-co-PVD and PGEM polymers could form nanoparticles in aqueous solution by a simple film hydration method. POEG-co-PVD micelles showed a diameter of 161 nm. After GEM conjugation, PGEM polymer formed small nanoparticles with a diameter reduced to 13.1 nm, indicating that the GEM structure played an important role in the formation of small-sized nanoparticles.

[0087] TEM images of PGEM nanoparticles show a spherical morphology with an average diameter of 12.5 ± 2.0 nm, which is consistent with DLS measurements. The critical micelle concentration of PGEM was evaluated by fluorescence spectroscopy using Nile Red as a probe. The critical micelle concentration value (CMC; the concentration above which micelles are formed) of PGEM micelles was 0.0072 mg / mL, which resulted in good colloidal stability after dilution in the blood circulation.

[0088] PTX-loaded micelles were prepared by the film hydration method with different carrier / drug mass ratios. As shown in Table 1, the loading capacity of PTX increased with decreasing the carrier / PTX mass ratio, and a surprisingly high loading capacity of 24.2% was achieved. All formulations showed very small particle size, which is suitable for penetrating, for example, pancreatic tumor tissue. The formulation stability at room temperature was also evaluated by measuring the change in size. At a PTX loading capacity of about 5%, the formulations could be stable for more than 20 days without any precipitation being observed. Moreover, the micelle solution was stable after repeated freeze-drying. [Table 1]

[0089] In addition to PTX, the PGEM carriers could be loaded with a variety of other drugs, some of which are difficult compounds to formulate, such as curcumin, NLG919, doxorubicin, dasatinib, and the c-Myc inhibitor 10058-F4 (see Table 2 below). All of these micelle formulations exhibited ultra-small size, high drug loading capacity, and excellent stability. Furthermore, the PGEM carriers could simultaneously encapsulate two different drugs, such as PTX and NLG919. The size distribution and morphology of PTX-loaded micelles, NLG-loaded micelles, or PTX / NLG-coloaded micelles were characterized by DLS and TEM (see Figure 7). All of these formulations exhibited spherical morphology with a uniform particle size of about 15 nm. [Table 2]

[0090] The PTX and NLG release profiles of PTX+NLG / PGEM micelles were evaluated by dialysis. As shown in Figure 8A, Taxol showed a faster release of PTX, with nearly 75% of PTX released within 72 hours. In comparison, PTX+NLG / PGEM micelles showed a more favorable release kinetics of PTX, with only 35% of PTX released slowly within 72 hours. In the presence of 10 mM GSH, PTX release from PTX+NLG / PGEM micelles was promoted, with 47% of PTX released in 72 hours. The NLG release profiles of PTX+NLG / PGEM micelles with and without 10 mM GSH are shown in Figure 8B. Approximately 72% of NLG was released from PTX+NLG / PGEM micelles in 72 hours, whereas 84% ​​of NLG was released in the presence of 10 mM GSH. These results indicate that the highly redox environment in tumor cells may promote the release of PTX and NLG from the carriers as a result of disulfide bond cleavage by intracellular GSH.

[0091] The combined effect of free GEM and PTX was studied in two pancreatic cancer cell lines, PANC02 and H7. Compared with single drugs, the combination of PTX and GEM significantly improved the killing effect of tumor cells. The combination coefficients (CI) for PANC02 and H7 cells were calculated to be 0.5 and 0.6, respectively, suggesting a synergistic effect (CI<1) between PTX and GEM in both cells.

[0092] The cytotoxicity of PGEM prodrug micelles was investigated in PANC02 cells. From Figure 9A, it can be seen that both free GEM and PGEM showed a concentration-dependent cell killing effect. The IC of PGEM prodrug micelles 50 IC of free GEM from PANC02 cells 50 This may be the result of incomplete cleavage of GEM from the PGEM polymer in cells during the short treatment time. The half-maximal inhibitory concentration, or IC 50is a measure of the effectiveness of a substance in inhibiting a particular biological or biochemical function. The cytotoxicity of the various nanoformulations was also evaluated in PANC02 cells (see Figure 9B). NLG-loaded micelles showed similar cytotoxicity compared to the PGEM carrier. PTX-loaded micelles and PTX / NLG co-loaded micelles showed superior cytotoxicity compared to free PTX, PGEM and NLG-loaded micelles. A similar trend was observed for the various formulations of H7 cells, showing that the incorporation of NLG did not alter the cell killing efficacy of PGEM, but the incorporation of PTX in the formulation improved the cell killing efficacy.

[0093] As mentioned above, CDA is overexpressed in pancreatic cancer and mediates the metabolism of cytidine analogue drugs (e.g. gemcitabine (GEM) and the like), resulting in resistance to treatment with these drugs. Therefore, reducing the expression of CDA will open up a new therapeutic window for pancreatic cancer. CDA expression in PANC02 cells treated with various formulations was investigated by Western blot. Compared with the control group, CDA expression was not changed by free GEM and PGEM, but CDA expression was significantly downregulated after treatment with PTX / PGEM formulation or PTX / NLG / PGEM formulation. In addition, NLG / PGEM formulation also reduced CDA protein levels to some extent.

[0094] The IDO inhibitory activity of the formulations NLG / PGEM and PTX+NLG / PGEM was investigated in PANC02 cells by detecting the decrease in the level of kynurenine (Kyn) by colorimetric assay. Figure 10 shows the Kyn inhibition rate of PANC02 cells after treatment with free NLG, NLG / PGEM and PTX+NLG / PGEM at various NLG concentrations. All compositions inhibited IDO function in a NLG concentration-dependent manner. NLG / PGEM and PTX+NLG / PGEM formulations showed similar IDO inhibitory effects compared to free NLG919.

[0095] The in vivo biodistribution of PGEM micelles was investigated by near-infrared fluorescence optical imaging in a PANC02 model using POEG-co-PVD nanoparticles as a control (~160 nm). To image tissues, a highly permeable hydrophobic fluorescent dye, DiR, was loaded within the micelles. The DiR signal increased in the tumor area over time and was strongest 24 hours after treatment with both micelles. Compared to POEG-co-PVD, PGEM micelles showed less uptake by the liver, spleen, and kidneys.

[0096] It has been reported that nanoparticles with small size have deep tumor penetration. Therefore, the tumor penetration ability of the ultrasmall PGEM carriers herein was determined by fluorescence imaging in comparison with larger nanoparticles self-assembled by POEG-co-PVD polymer backbone (~160 nm). Rhodamine and fluorescein were loaded as fluorescent probes into PGEM carriers and POEG-co-PVD carriers, respectively. To minimize individual differences, the two kinds of dye-loaded nanoparticles were mixed in the same amount and co-injected intravenously in the same mice. The green fluorescent signal from fluorescein / POEG-co-PVD nanoparticles became weaker in the tumor core, while the strong red fluorescent signal from rhodamine / PGEM nanoparticles was still clearly observed. This indicated the deeper penetration ability of PGEM carriers. To exclude the possibility that different intensity of fluorescence may interfere with the results, the fluorescent probes were exchanged and fluorescein was loaded using PGEM carriers and rhodamine was loaded by using POEG-co-PVD. Similarly, fluorescein-loaded PGEMs exhibited higher tumor uptake and deeper tumor diffusion than rhodamine-loaded POEG-co-PVDs.

[0097] To further study the combined effect of immunotherapy and chemotherapy in pancreatic cancer, an immunocompetent mouse model with PANC02 tumors was used. PANC02 cells were subcutaneously injected into C57BL / 6 mice. Twelve days later, tumor-bearing mice were intravenously (IV) injected with taxol in combination with saline, PGEM carrier, PTX / PGEM, NLG / PGEM, PTX / NLG / PGEM micelles, and free GEM (see FIG. 11, Panel A).

[0098] PGEM prodrug micelles showed similar antitumor activity as the group combined with taxol and free GEM (see Figure 11, panel B). PTX / PGEM micelles showed much higher antitumor activity than PGEM carrier, which was consistent with the in vitro MTT cytotoxicity results (see Figures 9A and 9B). NLG / PGEM micelles showed even higher antitumor activity compared to PTX / PGEM micelles, indicating that the introduction of IDO inhibitor NLG919 into PGEM carriers may improve antitumor immune responses. This is of great importance for pancreatic cancer treatment. Among all the formulations, PTX / NLG-loaded micelles showed the highest antitumor activity. The body weight of the mice was also monitored, and mice treated with various formulations showed no significant changes in body weight. After the mice were sacrificed, the tumor weight was measured (see Figure 11, panel C). Mice treated with PTX / NLG / PGEM micelles showed the lowest tumor weight and the highest tumor inhibition rate (90.4%), which further confirmed its improved therapeutic efficacy over other formulations.

[0099] FIG. 11, panel D, shows the photographs of tumors excised after various treatments. Mice treated with PTX / NLG / PGEM micelles showed the smallest tumor size. In hematoxylin and eosin (H&E) stained images (not shown) of tumor sections after various treatments, large nuclei were observed in tumor cells in saline treatment, while shrunken nuclei were observed in tumor tissue in other treatments. Among them, mice treated with PTX+NLG / PGEM showed the largest extent of necrosis of tumor cells.

[0100] Biochemical parameters, including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and blood urea nitrogen (BUN) levels, were evaluated as indicators of liver and kidney function. Mice treated with PTX+NLG / PGEM formulations showed normal levels of these parameters, suggesting that there was no obvious damage to liver and kidney function after treatment with PTX+NLG / PGEM micelles.

[0101] Changes in the PANC02 tumor microenvironment after various treatments were investigated by flow cytometric analysis of immune cell populations within the tumor. Total CD4 + No significant changes in the number of T cells were observed. After treatment with PGEM, NLG / PGEM, or PTX / NLG / PGEM, the number of CD8 + The relative number of T cells was significantly increased. Among these groups, tumors in the NLG / PGEM-treated group were CD8 + The relative number of T cells was the highest. Treg cells are a subtype of T cells that contribute to the immunosuppressive microenvironment. All treatments, except for the combination of Taxol and free GEM, were able to significantly reduce the number of Treg cells in the tumor tissue (see Figure 12A). IFN-γ is a potent molecule produced by T cells and plays an important role in the elimination of solid tumors

[37] . IFN-γ in tumors + CD4 + T cells (see Figure 12B) and IFN-γ + CD8 + The relative number of T cells (see FIG. 12C) was found to be significantly increased after treatment with PGEM, PTX / PGEM, NLG / PGEM, or PTX / NLG / PGEM. The PTX / PGEM group showed significantly higher IFN-γ expression than the Taxol and free GEM group. + CD4 + and IFN-γ + CD8 + The relative number of T cells was higher and the number of Treg cells was lower. In addition, compared to PGEM, the incorporation of NLG in the formulation increased IFN-γ+ CD4 + and IFN-γ + CD8 + The number of T cells was further increased. Overall, our formulation was able to induce a more immunoactive tumor microenvironment and generate an enhanced antitumor immune response.

[0102] The carrier platform herein is not a specific or limited system of GEM. The carrier herein may be formed by, for example, many structural analogues of GEM, or other hydrophilic compounds / drugs. Thus, the carrier system strategy herein may be extended to the simultaneous delivery of multiple distinct hydrophilic and hydrophobic drugs for various combination therapies.

[0103] Other embodiments herein are described and characterized in Figures 13-16B. Figure 13 shows a synthesis scheme of azacitidine conjugated polymers (PAza). PAza polymers were obtained by conjugating azacitidine to a POEG-co-PVD polymer backbone using, for example, EDC / HOBt coupling reactions known in the chemical arts. Figure 14A shows DLS particle size study and TEM images of blank PAza micelles. Blank PAza micelles show a particle size of about 13 nm and a spherical morphology. Figure 14B shows DLS particle size study and TEM images of DOX-loaded PAza micelles. DOX-loaded PAza micelles also show a slightly larger spherical morphology with a size of 16 nm.

[0104] FIG. 15A shows a synthetic scheme for synthesizing 2DG conjugated polymer (P2DG), where 2DG is 2-deoxy-D-glucose. The P2DG polymer was obtained by conjugating 2DG to a POEG-co-PVD polymer backbone using, for example, EDC / HOBt coupling reaction known in chemical arts. FIG. 15B shows DLS particle size study, TEM image of blank P2DG micelles, and its fluorescence intensity study. The DLS particles showed a particle size of about 11.48 nm and a spherical morphology. In addition, the DLS particles showed good stability due to low critical micelle concentration (CMC, 22.6 mg / L). FIG. 16A shows DLC particle size study of P2DG loaded with PTX (panel A), DOX (panel B), NLG919 (panel C) and sunitinib (panel D). All of these nanoparticles were small in size. Figure 16B shows TEM images of blank P2DG and P2DG loaded with sunitinib (SUN), PTX and a combination of PTX / SUN. All of these nanoparticles showed spherical morphology. These results indicate that P2DG polymers could be loaded with various bioactive agents, such as anticancer and / or antiviral agents.

[0105] Experimental Example

[0106] material Vinylbenzyl chloride, 4,4'-dithiodibutyric acid, oligo(ethylene glycol) methacrylate (OEG950 monomer, average M n=950), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 2,2-azobis(isobutyronitrile) (AIBN), trypsin-EDTA solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Dulbecco's modified Eagle's medium (DMEM) were all purchased from Sigma-Aldrich (MO, USA). AIBN was purified by recrystallization in absolute ethanol. 1-Hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl (EDC) were purchased from GL Biochem (Shanghai, China). Diisopropylethylamine (DIPEA) was purchased from Acros Organics. Paclitaxel was purchased from AK Scientific (CA, USA). Doxorubicin hydrochloride (DOX·HCl) and gemcitabine (GEM) were purchased from LC Laboratories (MA, USA). Fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Invitrogen (NY, USA).

[0107] Characterization The structures of the monomers and polymers were analyzed by a Varian 400 FT-NMR spectrometer (400.0 MHz). 1 The polymer was characterized by H NMR spectroscopy. The molecular weight of the polymer (M n and M w ) and polydispersity index (M w / M n ) was determined by gel permeation chromatography (GPC) with a Waters 2414 refractive index detector. A series of commercially available polystyrene standards was used for the calibration curve. The mean particle size, size distribution and morphology of the micelles were measured by dynamic light scattering (DLS, Malvern Zeta Sizer) and transmission electron microscopy (TEM).

[0108] Synthesis of VD monomer Vinylbenzyl chloride (305.2 mg, 2 mM), 4,4'-dithiodibutyric acid (2.38 g, 10 mM) and K2CO3 (0.69 g, 5 mM) were dissolved in 10 mL DMF and reacted with stirring at 50 °C. After 16 h, the mixture was cooled to room temperature, followed by the addition of 80 mL of CH2Cl2. The mixture was centrifuged at 4500 rpm for 12 min, and the supernatant was washed three times with water and then dried over anhydrous sodium sulfate. The VD monomer was obtained by column chromatography purification using ethyl acetate / petroleum ether (v / v, 1 / 2 to 1 / 1) as the eluent.

[0109] Synthesis of POEG-co-PVD polymer 4-Cyano-4-(thiobenzoylthio)pentanoic acid (6 mg, 0.0215 mmol), AIBN (2 mg, 0.0124 mmol), VD monomer (300 mg, 0.95 mmol), OEG950 monomer (400 mg, 0.42 mmol) and 2 mL of dry tetrahydrofuran were added into a Schlenk tube. After deoxygenation by three free-pump-thawing cycles, the mixture was stirred at 80° C. under N2 protection for 18 h. The reaction was then quenched by liquid nitrogen and precipitated in ether three times to obtain the POEG-co-PVD polymer. Conversion (OEG950モノマー) = 45.9%; conversion rate (VDモノマー) =50.0%.

[0110] Synthesis of POEG-co-PVDGEM polymer As-synthesized POEG-co-PVD polymer (120 mg, 0.17 mmol-COOH), GEM (179 mg, 0.68 mmol), HOBT (270 mg, 2 mmol) and EDC (450 mg, 2.35 mmol) were dissolved in 30 mL of DMSO with the addition of 300 μL of DIPEA. After stirring at 37 °C for 72 h, the mixture was dialyzed against DMSO and water for 2-3 days. After lyophilization, the POEG-co-PVD GEM polymer was obtained.

[0111] Determination of GEM loading of POEG-co-PVDGEM polymer The GEM loading of the POEG-co-PVDGEM polymer was quantified by alkaline hydrolysis method using 1N NaOH [32,33]. The amount of GEM in the polymer was measured by high-performance liquid chromatography (HPLC) equipped with a UV detector using methanol / water (04:96 v / v) as the mobile phase.

[0112] Preparation and characterization of drug-loaded micelles Blank and drug-loaded micelles were prepared by film hydration method. POEG-co-PVDGEM polymers and anticancer drugs (e.g., PTX, NLG919, DOX, dasatinib, 10058-F4) were mixed in dichloromethane / methanol with different carrier / drug ratios. After complete removal of the organic solvent, a thin film was formed. The thin film was then hydrated with PBS solution to obtain PTX-loaded POEG-co-PVDGEM micelles. PTX and NLG co-loaded micelles were prepared in a similar manner.

[0113] The drug loading capacity (DLC) and drug loading efficiency (DLE) were determined by HPLC and calculated according to the following formula:

[0114] DLC (%) [weight of drug loaded / (weight of polymer + drug used)] × 100

[0115] DLE(%) = (weight of drug loaded / weight of drug entered) x 100

[0116] Critical micelle concentration (CMC) of POEG-co-PVDGEM micelles. The CMC values ​​of POEG-co-PVDGEM micelles were measured using Nile red as a fluorescent probe

[59] . POEG-co-PVDGEM micelles (1 mg / mL) were prepared by a film hydration method, diluted to different concentrations, and then added to each vial containing Nile red. After overnight incubation, the fluorescence intensity of the solutions was measured by a fluorescence spectrometer.

[0117] In vitro PTX and NLG release The release of PTX and NLG from POEG-co-PVDGEM micelles was investigated by dialysis at 37°C. PTX / NLG co-loaded POEG-co-PVDGEM micelles were transferred into a dialysis bag with MWCO of 3500 Da and incubated in 50 mL PBS containing 0.5% (w / v) Tween 80 with gentle shaking. At specific time intervals, the PTX and NLG concentrations in the dialysis bag were measured by HPLC.

[0118] MTT assay The combined effect of PTX and GEM, and the cytotoxicity of blank and drug-loaded micelles were investigated by MTT assay using mouse pancreatic cancer cell lines PANC02 and H7 cells. Cells were seeded in 96-well plates at a density of 5000 cells / well and incubated with 100 μL of Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS for 24 h. Cells were treated with various concentrations of free PTX, GEM or micelle solution for 96 h. Then, 20 μL of MTT solution (5 mg / mL) was added to each well and cells were incubated for another 4 h. After removing the medium, 100 μL of DMSO was added to each well, thereby dissolving the MTT formazan crystals. The optical density was measured using a microplate reader and the cell viability was calculated with untreated cells as the control group.

[0119] In vitro IDO inhibition. The IDO assay was used to evaluate the IDO inhibitory activity of drug-loaded micelles

[15] . Briefly, PANC02 cells were seeded in 96-well plates (5 × 10 3After overnight culture, recombinant human IFN-γ was added to each well at a concentration of 50 ng / mL. Cells were then treated with various concentrations of micellar or free NLG919 for 48 h. Supernatants (150 μL) were transferred to a new 96-well plate, followed by the addition of 75 μL of 30% trichloroacetic acid. After incubation at 50° C. for 30 min, N-formylkynurenine was hydrolyzed to kynurenine. Supernatants were transferred to a new 96-well plate and treated with an equal volume of Ehrlich's reagent (2% p-dimethylaminobenzaldehyde in glacial acetic acid, w / v) for 10 min for colorimetric analysis at 490 nm.

[0120] In vivo biodistribution DiR-loaded POEG-co-PVD micelles and PGEM micelles with a DiR concentration of 0.5 mg / mL were injected into PANC02 tumor-bearing mice. At the indicated time points, the mice were imaged with an IVIS 200 system (Perkin Elmer, USA) at an exposure time of 60 seconds with excitation at 730 nm and emission at 835 nm. The mice were sacrificed for imaging. The tumors and various organs were excised for ex vivo imaging.

[0121] In vivo tumor penetration Fluorescent probes, rhodamine and fluorescein, were loaded onto PGEM and POEG-co-PVD carriers, respectively

[60] . These nanoparticles (4 mg each) were mixed in 200 μL of PBS and co-injected into mice via the tail vein. 15 hours after injection, tumors were excised and frozen sections were made at a thickness of 7 μm. These sections were stained with DAPI to label cell nuclei. Fluorescent signals were examined under a fluorescence microscope (OLYMPUS America, Melville, NY).

[0122] in vivo therapeutic research The syngeneic PANC02 pancreatic tumor model was established using 2 × 10 5 The tumor volume was confirmed by inoculating PANC02 cells into the flank of C57BL / 6 mice. 3When tumor volume reached 100 μg / kg, mice were divided into six groups (n=5) and treated every 3 days with PBS, POEG-co-PVDGEM micelles, PTX / POEG-co-PVDGEM micelles, NLG / POEG-co-PVDGEM micelles, TX / NLG / POEG-co-PVDGEM micelles, and a combination of taxol and free GEM for a total of 5 times. The doses of GEM, PTX, and NLG were kept at 20 mg / kg, 10 mg / kg, and 20 mg / kg. Tumor volume and mouse body weight were measured every 3 days. Tumor volume (V) was calculated by the following formula: V=(tumor length)×(tumor width). 2 After the experiment was completed, tumor tissues were excised, fixed in 10% formaldehyde, and embedded in paraffin. Tissues were sliced ​​at 5 μm, stained with hematoxylin and eosin (H&E), and observed under a Zeiss Axiostar plus Microscope (Pennsylvania, USA).

[0123] Quantification of tumor-infiltrating lymphocytes by flow cytometry C57BL / 6 mice bearing PANC02 tumors received various treatments intravenously every 3 days for 3 doses. 24 hours after the last treatment, tumors and spleens were excised. Single cell suspensions were filtered and red blood cells were lysed. Cells were then stained with various antibodies for flow cytometry analysis using FlowJo software (Tree Star).

[0124] statistical analysis Data are expressed as mean ± standard deviation (SD). Differences between groups were compared by one-way analysis of variance (ANOVA), and p<0.05 was considered statistically significant.

[0125] The foregoing description and accompanying drawings present some representative embodiments at present. Of course, various modifications, additions and alternative designs will become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope of the invention. The scope of the invention is now suggested by the following claims, rather than the foregoing description. All changes and modifications that come within the meaning and range of equivalence of the claims are to be embraced within their scope.

Claims

1. 1. A polymer comprising a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer.

2. 10. The polymer of claim 1, wherein at least one of the first plurality of pendant groups or the second plurality of pendant groups is attached to the hydrophobic polymer backbone via a linkage.

3. The polymer of claim 2 , wherein the linking moiety comprises at least one group that interacts via π-π stacking.

4. 3. The polymer of claim 2, wherein the first plurality of pendant groups are attached to the hydrophobic polymer backbone via a first linking group that includes at least a first group that interacts via pi-pi stacking.

5. The polymer of claim 4, wherein at least a first group that interacts through π-π stacking comprises an aromatic group.

6. The polymer of claim 5, wherein at least a first group that interacts through π-π stacking comprises a benzyl group.

7. The polymer of claim 1 , wherein the hydrophobic polymer backbone is formed via radical polymerization of vinyl monomers.

8. The polymer of claim 7 , wherein the hydrophobic polymer backbone is formed via free radical polymerization.

9. The polymer of claim 7 , wherein the hydrophobic polymer backbone is formed via reversible deactivation radical polymerization.

10. The polymer of claim 1 , wherein at least one of the groups comprising a plurality of hydroxyl groups is a hydrophilic group.

11. 11. The polymer of claim 10, wherein at least one group comprising the plurality of hydroxyl groups is adapted to form hydrogen bonds with an aqueous environment.

12. The polymer of claim 11 , wherein the at least one group comprising a plurality of hydroxyl groups comprises a sugar group.

13. 13. The polymer of claim 12, wherein the at least one group containing multiple hydroxyl groups comprises a nucleoside.

14. 12. The polymer of claim 11, wherein the at least one group containing multiple hydroxyl groups comprises a nucleoside analog, a sugar alcohol, ascorbic acid, 2-deoxy-D-glucose, triethanolamine, pentaerythritol, tris(hydroxymethyl)aminomethane, dopamine, catechin, or cholic acid.

15. 12. The polymer of claim 11, wherein the at least one group containing a plurality of hydroxyl groups comprises glycerol, di-glycerol, erythritol, xylitol, arabitol, fucitol, ribitol, sorbitol, maltitol, isomalt, mannitol, a saccharide, an oligosaccharide, a polysaccharide, ascorbic acid, gluconic acid, glucoronic acid, or glycosamine.

16. 12. The polymer of claim 11, wherein the at least one group containing multiple hydroxyl groups comprises glucose, fructose, sucrose, galactose, maltose, trehalose, lactulose, or lactose.

17. The polymer of claim 13 , wherein the nucleoside is a ribonucleoside, a deoxyribonucleoside, or a nucleoside analog therapeutic drug.

18. 12. The polymer of claim 11, wherein the at least one group containing multiple hydroxyl groups comprises ascorbic acid or 2-deoxy-D-glucose.

19. 14. The polymer of claim 13, wherein the nucleoside is selected from the group consisting of gemcitabine, azacytidine, cytarabine or biologically active derivatives thereof.

20. The polymer of claim 13 , wherein the nucleoside is a therapeutic agent.

21. 12. The polymer of claim 11, wherein at least one of the hydroxyl-containing groups comprises a therapeutic or diagnostic agent, and the second pendant groups are attached to a hydrophobic polymer backbone via linkages that are labile in vivo.

22. 22. The polymer of claim 21, wherein the in vivo labile linkage comprises at least one of a reduction-sensitive bond, a pH-sensitive bond, a ROS-sensitive bond, or a protease-sensitive bond.

23. 22. The polymer of claim 21, wherein the in vivo labile linkage comprises at least one of an ester bond, an orthoester bond, a thioether ester bond, an anhydride bond, an amide bond, a carbonate bond, a disulfide bond, a hydrazone bond, a cic-acotinyl bond, an acetal bond, a maleate carboxydimethyl bond, an imine bond, an oxime bond, a silyl ether bond, a ketal bond, a thioketal bond, or a protease cleavable peptide.

24. The polymer of claim 21 , wherein the linking moiety further comprises at least one group that interacts via π-π stacking.

25. The polymer of claim 24, wherein at least one of the groups that interact through pi-pi stacking comprises an aromatic group.

26. 26. The polymer of claim 25, wherein at least one group that interacts through pi-pi stacking comprises a benzyl group.

27. 27. The polymer of any one of claims 1 to 26, wherein the plurality of polymers form micelles having a diameter of less than 100 nm.

28. The polymer of any one of claims 1 to 26, wherein the plurality of polymers form micelles having a diameter of 50 nm or less.

29. 27. The polymer of any one of claims 1 to 26, wherein the plurality of polymers form micelles having a diameter of 30 nm or less.

30. 27. The polymer of any one of claims 1 to 26, wherein the plurality of polymers form micelles having a diameter of 20 nm or less.

31. 27. The polymer of any one of claims 1 to 26, wherein the plurality of polymers form micelles having diameters in the range of 10 to 30 nm.

32. 27. The polymer of any one of claims 1 to 26, wherein the plurality of polymers form micelles having diameters in the range of 10 to 20 nm.

33. 1. A formulation comprising: a plurality of polymers comprising a hydrophobic polymer backbone; a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one group comprising a plurality of hydroxyl groups; and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer; and at least a first compound.

34. 34. The formulation of claim 33, further comprising at least a second compound.

35. 35. The formulation of claim 34, wherein at least one of the first compound and the second compound is a therapeutic compound or a diagnostic compound.

36. 36. The formulation of claim 35, wherein at least one of the first compound and the second compound is a small molecule compound.

37. 35. The formulation of claim 34, wherein at least one of the first compound and the second compound has a molecular weight of less than 1.5 kDa.

38. 34. The formulation of claim 33, wherein the first compound is a hydrophobic compound.

39. 35. The formulation of claim 34, wherein the first compound and the second compound are hydrophobic compounds.

40. 34. The formulation of claim 33, wherein the first compound is a first diagnostic compound.

41. 41. The formulation of claim 40, wherein the first diagnostic compound comprises a contrast enhancing agent for use in an imaging technique.

42. 34. The formulation of claim 33, wherein the first compound is a first therapeutic compound.

43. 43. The formulation of claim 42, wherein the first therapeutic compound is a chemotherapeutic compound, an antiviral compound, an antibiotic compound, an antifungal compound, an anticancer compound, an anti-rejection compound, an analgesic compound, an antioxidant compound, an immunomodulatory compound, or an anti-inflammatory compound.

44. The first therapeutic compound is selected from the group consisting of JP4-039, paclitaxel, docetaxel, FK506 (tacrolimus), cyclosporine A, protoporphyrin, GW4064 (3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole), rose bengal, epigallocatechin gallate, simvastatin, curcumin, indomethacin, JQ1, I-BET 151, I-BET 762, resiquimod, riluzole, tamoxifen, NLG-919 (indoleamine 2,3-dioxygenase (IDO) pathway inhibitor), sunitinib, imatinib, erlotinib, gefitinib, cetuximab, Myc inhibitors (such as 10058-F4 (5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone)), cladribine, fludarabine, nelarabine, troxacitabine, capecitabine, 2'-fluoro-2'-deoxyadenosine, acyclovir, tenofovir, lamivudine, entecavir, GS-441524, GS-5734, 2'-C-methyladenosine, 7-deaza-2' 44. The formulation of claim 43, which is -C-methyl-adenosine, 2'-C-methylguanosine, INX-08189, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-ethynyl adenosine, NITD008, NITD449, NITD203, 4'-C-azidocytidine, valapiravir, RO-9187, BCX4430, ribavirin, 6-azauridine or 9-deazaadenosine.

45. 43. The formulation of claim 42, further comprising at least a second compound.

46. 46. ​​The formulation of claim 45, wherein the second compound is a second therapeutic compound, wherein the second therapeutic compound is a chemotherapeutic compound, an antiviral compound, an antibiotic compound, an antifungal compound, an anticancer compound, an anti-rejection compound, an analgesic compound, an antioxidant compound, an immunomodulatory compound, or an anti-inflammatory compound.

47. 47. The formulation of claim 46, wherein the second compound is a small molecule therapeutic compound.

48. 48. The formulation of claim 47, wherein the second compound has a molecular weight of less than 1 kDa.

49. 47. The formulation of claim 46, wherein the second compound is a hydrophobic compound.

50. the second compound is selected from the group consisting of JP4-039, paclitaxel, docetaxel, FK506 (tacrolimus), cyclosporine A, protoporphyrin, GW4064 (3-(2,6-dichlorophenyl)-4-(3′-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole), rose bengal, epigallocatechin gallate, simvastatin, curcumin, indomethacin, JQ1, I-BET 151, I-BET 762, resiquimod, riluzole, tamoxifen, NLG-919 (indoleamine 2,3-dioxygenase (IDO) pathway inhibitor), sunitinib, imatinib, erlotinib, gefitinib, cetuximab, Myc inhibitors (10058-F4 (5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone) etc.), cladribine, fludarabine, nelarabine, troxacitabine, capecitabine, 2'-fluoro-2'-deoxyadenosine, acyclovir, tenofovir, lamivudine, entecavir, GS-441524, GS-5734, 2'-C-methyladenosine, 7-deaza-2'-C-methyladenosine, 47. The formulation of claim 46, wherein the agonist is selected from the group consisting of tyl-adenosine, 2'-C-methylguanosine, INX-08189, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-ethynyl adenosine, NITD008, NITD449, NITD203, 4'-C-azidocytidine, valapiravir, RO-9187, BCX4430, ribavirin, 6-azauridine, and 9-deazaadenosine.

51. The first compound is selected from the group consisting of JP4-039, paclitaxel, docetaxel, FK506 (tacrolimus), cyclosporine A, protoporphyrin, GW4064 (3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole), rose bengal, epigallocatechin gallate, simvastatin, curcumin, indomethacin, JQ1, I-BET 151, I-BET 762, resiquimod, riluzole, tamoxifen, NLG-919 (indoleamine 2,3-dioxygenase (IDO) pathway inhibitor), sunitinib, imatinib, erlotinib, gefitinib, cetuximab, a c Myc inhibitors (10058-F4 (5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone), etc.), cladribine, fludarabine, nelarabine, troxacitabine, capecitabine, 2'-fluoro-2'-deoxyadenosine, acyclovir, tenofovir, lamivudine, entecavir, GS-441524, GS-5734, 2'-C-methyladenosine, 7-deaza-2'-C-methyl 51. The formulation of claim 50, wherein said agonists are independently selected from the group consisting of adenosine, 2'-C-methylguanosine, INX-08189, 2'-C-methylcytidine, 2'-C-methyluridine, 2'-C-ethynyl adenosine, NITD008, NITD449, NITD203, 4'-C-azidocytidine, valapiravir, RO-9187, BCX4430, ribavirin, 6-azauridine and 9-deazaadenosine.

52. 34. The formulation of claim 33, wherein at least one of the first plurality of pendant groups or at least one of the second plurality of pendant groups is attached to the hydrophobic polymer backbone via a linkage.

53. The formulation of claim 52, wherein the linking moiety comprises at least one group that interacts via π-π stacking.

54. 34. The formulation of claim 33, wherein the first plurality of pendant groups are attached to the hydrophobic polymer backbone via a first linking group that includes at least a first group that interacts via pi-pi stacking.

55. The formulation of claim 54, wherein at least a first group that interacts via pi-pi stacking comprises an aromatic group.

56. The formulation of claim 55, wherein at least a first group that interacts via π-π stacking comprises a benzyl group.

57. 34. The formulation of claim 33, wherein the hydrophobic polymer backbone is formed via radical polymerization of vinyl monomers.

58. 58. The formulation of claim 57, wherein the hydrophobic polymer backbone is formed via free radical polymerization.

59. 58. The formulation of claim 57, wherein the hydrophobic polymer backbone is formed via reversible deactivation radical polymerization.

60. 34. The formulation of claim 33, wherein at least one of the groups comprising a plurality of hydroxyl groups is a hydrophilic group.

61. 61. The formulation of claim 60, wherein at least one group comprising the plurality of hydroxyl groups is adapted to form hydrogen bonds with an aqueous environment.

62. 62. The formulation of claim 61, wherein at least one group comprising a plurality of hydroxyl groups comprises a sugar group.

63. 62. The formulation of claim 61, wherein the at least one group comprising multiple hydroxyl groups comprises a nucleoside.

64. 62. The formulation of claim 61, wherein the at least one group containing multiple hydroxyl groups comprises a nucleoside analog, a sugar alcohol, ascorbic acid, 2-deoxy-D-glucose, triethanolamine, pentaerythritol, tris(hydroxymethyl)aminomethane, dopamine, catechin, or cholic acid.

65. 62. The formulation of claim 61, wherein the at least one group comprising a plurality of hydroxyl groups comprises glycerol, di-glycerol, erythritol, xylitol, arabitol, fucitol, ribitol, sorbitol, mannitol, a saccharide, an oligosaccharide, a polysaccharide, ascorbic acid, gluconic acid, glucoronic acid, or glycosamine.

66. 62. The formulation of claim 61, wherein the at least one group containing multiple hydroxyl groups comprises glucose, fructose, sucrose, galactose, maltose, trehalose, lactulose or lactose.

67. 64. The formulation of claim 63, wherein the nucleoside is a ribonucleoside, a deoxyribonucleoside, or a nucleoside analog therapeutic drug.

68. 62. The formulation of claim 61, wherein the at least one group containing a plurality of hydroxyl groups comprises ascorbic acid or 2-deoxy-D-glucose.

69. 64. The formulation of claim 63, wherein the nucleoside is selected from the group consisting of gemcitabine, azacytidine, cytarabine or biologically active derivatives thereof.

70. 64. The formulation of claim 63, wherein the nucleoside is a therapeutic agent.

71. 34. The formulation of claim 33, wherein at least one of the hydroxyl-containing groups is a therapeutic or diagnostic agent and the second pendant groups are attached to the hydrophobic polymer backbone via linkages that are labile in vivo.

72. 72. The formulation of claim 71, wherein the in vivo labile linkage comprises at least one of a reduction-sensitive bond, a pH-sensitive bond, a ROS-sensitive bond, or a protease-sensitive bond.

73. 73. The formulation of claim 72, wherein the in vivo labile linkage comprises at least one of an ester bond, an orthoester bond, a thioether ester bond, an anhydride bond, an amide bond, a carbonate bond, a disulfide bond, a hydrazone bond, a cic-acotinyl bond, an acetal bond, a maleic acid carboxydimethyl bond, an imine bond, an oxime bond, a silyl ether bond, a ketal bond, a thioketal bond, or a protease cleavable peptide.

74. The formulation of claim 72, wherein the linker further comprises at least one group that interacts via π-π stacking.

75. The formulation of claim 74, wherein at least one of the groups that interact via pi-pi stacking comprises an aromatic group.

76. The formulation of claim 75, wherein at least one group that interacts via pi-pi stacking comprises a benzyl group.

77. 77. The formulation of any one of claims 33-76, wherein the plurality of polymers form micelles having a diameter of less than 100 nm.

78. 77. The formulation of any one of claims 33-76, wherein the plurality of polymers form micelles having a diameter of 50 nm or less.

79. 77. The formulation of any one of claims 33-76, wherein the plurality of polymers form micelles having a diameter of 30 nm or less.

80. 77. The formulation of any one of claims 33-76, wherein the plurality of polymers form micelles having a diameter of 20 nm or less.

81. 77. The formulation of any one of claims 33-76, wherein the plurality of polymers form micelles having diameters in the range of 10-30 nm.

82. 77. The formulation of any one of claims 33-76, wherein the plurality of polymers form micelles having diameters in the range of 10-20 nm.

83. 80. The formulation of claim 79, wherein the micelles have a loading capacity of the first therapeutic compound of at least 4-30% by weight.

84. 80. The formulation of claim 79, wherein the micelles have a loading capacity of the first therapeutic compound of at least 8-30% by weight.

85. 80. The formulation of claim 79, wherein the micelles have a loading capacity of the first therapeutic compound of at least 8-20% by weight.

86. 1. A formulation for delivering a compound in vivo comprising a plurality of polymers comprising a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer.

87. The formulation of claim 86, wherein the multiple polymers form a micelle.

88. 1. A method of formulating a composition for delivering a first compound, the method comprising mixing a plurality of polymers comprising a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer, with a plurality of the first compounds.

89. 90. The method of claim 88, wherein the plurality of polymers form micelles.

90. 90. The method of claim 89, wherein the first compound is a therapeutic agent or a diagnostic agent.

91. 1. A method of delivering a first compound, the method comprising administering a formulation comprising a plurality of polymers comprising a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one group comprising a plurality of hydroxyl groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer, and a plurality of said first compounds.

92. 92. The method of claim 91, wherein the plurality of polymers form micelles and the first compound is loaded into the micelles.

93. 93. The method of claim 92, wherein the first compound is a therapeutic or diagnostic agent.

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