Targeted polymeric nanoparticles loaded with dual therapeutic agents and methods thereof

Abstract

Provided herein are nanoparticles comprising a triblock copolymer and a targeting moiety for targeted delivery of one or more therapeutic agents, and formulations thereof. Also provided herein are layered drug delivery vehicles. Further provided herein are methods, or uses thereof, for treating cancers, preferably pancreatic and brain cancers, by administration of the nanoparticles disclosed herein.

Description

Attorney Docket No.: 31134 / 70234 / PC TARGETED POLYMERIC NANOPARTICLES LOADED WITH DUAL THERAPEUTIC AGENTS AND METHODS THEREOF STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under CA150190, awarded by National Institutes of Health. The government has certain rights in the invention. FIELD

[0002] The disclosure relates to polymeric nanoparticle formulations that can carry therapeutic agents for disease and deliver the therapeutic agents in a targeted manner for improved therapeutic outcomes. BACKGROUND

[0003] The use of single chemotherapeutic agents has shown limitations caused by their poor stability and bioavailability, systemic toxicity, severe side effects, and drug resistance. Instead of single-agent chemotherapy, a multi-drug chemotherapeutic regimen approach has become the mainstay of treatment to enhance combined delivery and overcome resistance.

[0004] Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer- related deaths in the United States, with a relatively low 5-year survival rate of around 12.5% in 2024. Surgical resection of the tumor is a viable treatment option. Still, it can only cure a small percentage (15%) of patients who are diagnosed at an early stage with resectable and borderline resectable PDAC. Unfortunately, most PDAC patients are diagnosed with advanced local stage or distant metastatic form of the disease, making palliative chemotherapy the only treatment approach.

[0005] Gemcitabine monotherapy (Gem), the initial standard of care treatment for PDAC, exhibited modest efficacy with minimal improvement in overall response rate (ORR), progression-free survival (PFS), and overall survival (OS); yet led to side effects specifically in elderly patients. Hence, to improve treatment outcomes, investigators explored the combination of Gem with other chemotherapeutic agents. In 2013, the FDA approved albumin-bound PTX (nab-PTX) for treating PDAC in combination with Gem. In a large-scale phase III clinical trial, the nab-PTX-Gem cohort, with a Gem / PTX ratio of approximately 10:1 w / w, demonstrated an improved OS compared to the control arm, which was single agent Gem. However, the treatment resulted in a high percentage of side effects, including neutropenia, leukopenia, fatigue, and peripheral neuropathy. These findings demonstrated that combining Gem and nab-PTX can improve efficacy and increase toxicities. SUMMARYAttorney Docket No.: 31134 / 70234 / PC

[0006] Polymeric nanoparticles loaded with one or more therapeutic agents can provide for delivery of the therapeutic agent through active tumor targeting achieved by conjugating different tissue-specific ligands to the surface of the nanoparticles, such as antibodies, peptides, aptamers, and vitamins. Targeted drug delivery offers several advantages over counterparts, including selective delivery of drug-loaded nanoparticles, enhanced drug accumulation, and increased cellular uptake at tumor sites. Site-directed delivery can also allow for reduced side-effects or toxicity issues by allowing target delivery that limits systemic interaction with the therapeutic agent and / or by reducing the amount of therapeutic agent needed for efficacy. Moreover, the tumor-targeted peptide-conjugated polymer nano- formulations with hydrophilic and hydrophobic blocks in accordance with the disclosure, also referred to herein as targeted amphiphilic copolymers can improve drug bioavailability of hydrophilic and hydrophobic drugs. Polymeric nanoparticles in accordance with the disclosure encapsulating two or more therapeutic agents can provide methods for simultaneously delivering the two or more therapeutic agents.

[0007] A polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can comprise a polymeric material comprising a triblock copolymer having a structure according to Formula (I) or Formula (II):the one or more therapeutic agents are encapsulated by the triblock copolymer; wherein t, v, w, and z are each independently 6 to 500; y is 0 to 500; A is OC1-6alkylene; RBis C(O)NH– C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N(RN)2, C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RBis substituted with 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O; each of RCand RC′ are independently selected from C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2, C(O)NH– C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RCis substituted with 0 to 2 substituents selected from C1-3alkyl and =O; D is –C(O)C1-6alkylene–O substituted with 0 to 2 C1-3alkyl substituents; X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then RBis the targeting moiety; the END group is C1-6alkyl or C6-12arylAttorney Docket No.: 31134 / 70234 / PC and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; each RNis independently H or C1-3alkyl; wherein the nanoparticle has an average particle diameter in the range of 100 nm to 500 nm; each therapeutic agent has a content in the range of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle; the nanoparticle has a Zeta potential in the range of -35 mV to +40 mV; and the nanoparticle has a polydispersity index in the range of 0.150 to 0.300.

[0008] In accordance with the disclosure, the polymeric material can have a structure according to formula (I); wherein t is 6; v and w are each 100 to 500; y is 100 to 500; A is OC3alkylene;X is NRN; each CAP group is a targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl, and C0-3alkylene–CN; each RNis H; and the targeting moiety is a tumor-targeted peptide.

[0009] A polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can comprise a polymeric material comprising a triblock copolymer having a structure according to Formula (III):more therapeutic agents encapsulated by the polymeric material; wherein t, v, w, and z are each independently 6 to 500; y is 0 to 500; E is a hydrophilic polymer; each of F, G and G′ are independently selected from a targeting moiety, a hydrophobic polymer, or a lipophilic polymer, provided that at least one hydrophilic polymer and lipophilic polymer are each present; X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then F is the targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; each RNis independently H or C1-3alkyl; the nanoparticle has an average particle diameter in the range of 100 nm to 500 nm; each therapeutic agent has a content in the range of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle; the nanoparticle has a Zeta potential in the range of -35 mV to +40 mV; and the nanoparticle has a polydispersity index in the range of 0.150 to 0.300.

[0010] A polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can comprise a polymeric material comprising a triblockAttorney Docket No.: 31134 / 70234 / PC copolymer having a structure according to Formulaand one or more therapeutic agents encapsulated by the polymeric material; wherein J is a targeting moiety; Q is a hydrophilic polymer (optionally a saccharide); and M is a hydrophobic polymer having a structure according to Formula (IV’):and q, are independently selected from 1 to 20; the nanoparticle has an average particle diameter in the range of 100 nm to 500 nm; each therapeutic agent has a content in the range of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle; the nanoparticle has a Zeta potential in the range of -35 mV to +40 mV; the nanoparticle has a polydispersity index in the range of 0.150 to 0.300; and the nanoparticle has a saccharide content in the range of 40 wt.% to 60 wt.%, based on the entire weight of the nanoparticle.

[0011] A layered drug delivery system in accordance with the disclosure can comprise alternating ones of one or more therapeutic agent layers and one or more hydrogel layers, each therapeutic agent layer comprising a plurality of the nanoparticles of the disclosure.

[0012] A method of treating cancer, or use thereof, in accordance with the disclosure can comprise administering the nanoparticles of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Scheme 1 shows a scheme showing the synthesis of 6-O-methacryl-2,3,4,6-tetra- O-acetyl-D-mamnopyranoside (MAcMn).

[0014] Figure 1A shows an1H NMR spectrum of MAcMn in CDCl3.

[0015] Figure 1B shows an13C NMR spectrum of MAcMn in CDCl3.

[0016] Figure 2A shows an FT-IR spectrum of MAcMn.

[0017] Figure 2B shows a mass spectrum of MAcMn.

[0018] Scheme 2 shows a scheme showing the synthesis of tumor-targeted peptide conjugated amphiphilic triblock copolymer (TTP-PEG6-b-P(NVCL-co-HEA-g-LA).Attorney Docket No.: 31134 / 70234 / PC

[0019] Figure 3 shows a1H-NMR spectra of t-But-OOC-PEG6-b-P(NVCL-co-HEA); HOOC-PEG6-b-P(NVCL-co-HEA-g-LA); and TTP-PEG6-b-P(NVCL-co-HEA-g-LA), respectively.

[0020] Figure 4 is a chromatogram of gel permeation chromatography performed on HOOC-PEG6-b-P(NVCL-co-HEA-g-LA) polymer according to the disclosure.

[0021] Figure 5 is a graph of stacked FT-IR spectra for (A) t-But-OOC-PEG6-b-P(NVCL- co-HEA); (B) HOOC-PEG6-b-P(NVCL-co-HEA-g-LA); (C) TTP-PEG6-b-P(NVCL-co-HEA-g- LA) according to the disclosure.

[0022] Figure 6A is a hydrodynamic diameter histogram of the unloaded polymer nanoparticles as measured by dynamic light scattering (DLS).

[0023] Figure 6B is a hydrodynamic diameter histogram of the polymer nanoparticles loaded with Gem, as measured by dynamic light scattering (DLS).

[0024] Figure 6C is a hydrodynamic diameter histogram of the polymer nanoparticles loaded with PTX, as measured by dynamic light scattering (DLS).

[0025] Figure 6D is a hydrodynamic diameter histogram of the polymer nanoparticles loaded with Gem and PTX, as measured by dynamic light scattering (DLS).

[0026] Figure 6E is a TEM image of the unloaded polymer nanoparticles.

[0027] Figure 6F is a TEM image of the polymer nanoparticles loaded with Gem.

[0028] Figure 6G is a TEM image of the polymer nanoparticles loaded with PTX.

[0029] Figure 6H is a TEM image of the polymer nanoparticles loaded with Gem and PTX.

[0030] Figure 6I shows the stability of the empty polymer nanoparticles and dual drug- loaded polymeric nanoparticles over 30 days.

[0031] Figure 6J shows the cumulative release of Gem and PTX both with and without (W / O) GSH.

[0032] Figure 7 shows the in vitro, cellular uptake of Rhodamine-b labeled PNPs in cell lines. PANC-1 and KPC cells were treated with rhodamine-b loaded control polymer (CP) or TTP-conjugated polymer (TP) at two different time points: 2h and 4h. Nuclei are blue (DAPI), and rhodamine-b PNPs are red. Images were captured by confocal fluorescence microscopy under blue and red channels. TP-treated cells showed significantly higher uptake of rhodamine-B for both time points than CP-treated cells in two cell lines. Bar Length = 50 µm.

[0033] Figure 8A shows the in vitro cellular cytotoxicity in PANC-1 of Gem, PTX, Gem + PTX, without the polymeric nanoparticle formulation.Attorney Docket No.: 31134 / 70234 / PC

[0034] Figure 8B shows the in vitro cellular cytotoxicity in KPC of Gem, PTX, Gem + PTX, without the polymeric nanoparticle formulation.

[0035] Figure 8C shows the in vitro cellular cytotoxicity of empty or single and dual drug- loaded polymeric nanoparticles loaded with gemcitabine and Paclitaxel in PANC-1 pancreatic cancer cell lines, using Gem, PTX, Gem + PTX, and empty polymeric nanoparticle formulation.

[0036] Figure 8D shows the in vitro cellular cytotoxicity of empty or single and dual drug- loaded polymeric nanoparticles loaded with gemcitabine and Paclitaxel in KPC pancreatic cancer cell lines, using Gem, PTX, Gem + PTX, and empty polymeric nanoparticle formulation.

[0037] Figure 9 shows the biodistribution of tumor-targeted peptide polymer nanoparticles (PNPs) in an orthotopic pancreatic cancer model treated with control - no treatment: NIR dye formulation; CP: Polymer formulation without TTP; TP with TTP, were injected. (top) IVIS images taken at 24 h after administration. (bottom) Ex vivo imaging of the tumors and major organs examined after 24 h.

[0038] Figure 10A is a schematic of the experimental plan used in clinical studies with mice bearing PANC-1 orthotopic pancreatic tumors.

[0039] Figure 10B is a graph showing tumor volume (mm3) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with nanoparticles according to the disclosure.

[0040] Figure 10C is a graph showing tumor weight (mg) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with nanoparticles according to the disclosure.

[0041] Figure 10D is a graph showing survival (%) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with nanoparticles according to the disclosure.

[0042] Figure 11A is a schematic of the experimental plan used in clinical studies with mice bearing KPC orthotopic pancreatic tumors.

[0043] Figure 11B is a graph showing tumor volume (mm3) in mice bearing KPC orthotopic pancreatic tumors after treatment with nanoparticles according to the disclosure.

[0044] Figure 11C is a graph showing tumor weight (mg) in mice bearing KPC orthotopic pancreatic tumors after treatment with nanoparticles according to the disclosure.

[0045] Figure 11D is a graph showing survival (%) in mice bearing KPC orthotopic pancreatic tumors after treatment with nanoparticles according to the disclosure.

[0046] Figure 12A shows representative images of the H&E-stained tumor sections from different treatment groups.Attorney Docket No.: 31134 / 70234 / PC

[0047] Figure 12B shows quantification of Ki67 positive nuclei in PANC-1 tumor sections where * and *** denotes p <0.05 and p<0.001 compared to untreated, respectively.

[0048] Figure 12C shows quantification of Ki67 positive nuclei in KPC tumor sections where * and *** denotes p <0.05 and p<0.001 compared to untreated, respectively.

[0049] Figure 13 is an image of hematoxylin and eosin (H+E) staining of the major organs (lung, heart, kidney spleen, and liver) of mice in the (A) control (B) Gem+PTX (C) P(Gem) (D) P(PTX) and (E) P(Gem+PTX).

[0050] Scheme 3 shows the synthesis of (tBu-OOH-PEG6-b-P(NVCL-co-Vac).

[0051] Figure 14A shows effects on tumor weight of orally administered combinations of TTP-polymeric loaded paclitaxel(P) Gemcitabine(G) and cisplatin© treatments in mice bearing KPC orthotopic pancreatic tumors with P(C), P(G+P) and P(C+G+P), relative to treatment with free paclitaxel, gemcitabine and cisplatin (C+G+P), as well as unloaded particles (Control). Paclitaxel(P) 1 mg / kg, gemcitabine(G) 2 mg / kg and Cisplatin(C) 2mg / kg, were used. **** denotes p < 0.0001 compared to the indicated respective group.

[0052] Figure 14B shows effects on tumor volume of orally administered combinations of TTP-polymeric loaded paclitaxel(P) Gemcitabine(G) and cisplatin© treatments in mice bearing KPC orthotopic pancreatic tumors with P(C), P(G+P) and P(C+G+P), relative to treatment with free paclitaxel, gemcitabine and cisplatin (C+G+P), as well as unloaded particles (Control). Paclitaxel(P) 1 mg / kg, gemcitabine(G) 2 mg / kg and Cisplatin(C) 2mg / kg, were used. **** denotes p < 0.0001 compared to the indicated respective group.

[0053] Figure 15 shows a1H-NMR spectra of (PEG6-b-P(NVCL-co-Vac)

[0054] Figure 16A shows a hydrodynamic diameter histogram of unloaded polymeric nanoparticles.

[0055] Figure 16B shows a hydrodynamic diameter histogram of polymeric nanoparticles loaded with PTX.

[0056] Figure 17A shows the In vitro cellular cytotoxicity of empty and PTX loaded polymeric nanoparticles in PANC-1 cancer cells.

[0057] Figure 17B shows the In vitro cellular cytotoxicity of empty and PTX loaded polymeric nanoparticles in KPC-GL cancer cells.

[0058] Scheme 4 shows the synthesis of (PEG-b-P(NVCL-co- DMEAM-co-BA).

[0059] Figure 18 shows a1H-NMR spectra of (PEG-b-P(NVCL-co-DMEAM-co-BA).Attorney Docket No.: 31134 / 70234 / PC

[0060] Figure 19A shows a hydrodynamic diameter histogram of unloaded polymeric nanoparticles.

[0061] Figure 19B shows a hydrodynamic diameter histogram of polymeric nanoparticles loaded with PTX.

[0062] Scheme 5 shows the synthesis of PEG-b-P(AHAM-co-DMEAM).

[0063] Figure 20A shows a hydrodynamic diameter histogram of unloaded polymeric nanoparticles.

[0064] Figure 20B shows a hydrodynamic diameter histogram of polymeric nanoparticles loaded with PTX.

[0065] Figure 21A shows the In vitro cellular cytotoxicity of empty and PTX loaded polymeric nanoparticles in PANC-1 cancer cells.

[0066] Figure 21B shows the In vitro cellular cytotoxicity of empty and PTX loaded polymeric nanoparticles in KPC-GL cancer cells.

[0067] Scheme 6 shows the synthesis of (PEG-S-S-PLA).

[0068] Figure 22A shows a hydrodynamic diameter histogram of unloaded polymeric nanoparticles.

[0069] Figure 22B shows a hydrodynamic diameter histogram of polymeric nanoparticles loaded with PTX.

[0070] Figure 23A shows the In vitro cellular cytotoxicity of empty and PTX loaded polymeric nanoparticles in PANC-1 cancer cells.

[0071] Figure 23B shows the In vitro cellular cytotoxicity of empty and PTX loaded polymeric nanoparticles in KPC-GL cancer cells.

[0072] Scheme 7 shows the synthesis of (TP-PEG6-b-P( NVCL-co-GMA).

[0073] Figure 24A shows a hydrodynamic diameter histogram of unloaded polymeric nanoparticles.

[0074] Figure 24B shows a hydrodynamic diameter histogram of polymeric nanoparticles loaded with PTX.

[0075] Scheme 8 shows the synthesis of ROS-S-MnP.

[0076] Figure 25 shows1H-NMR spectra of (top) S-P(MAcMn) in CDCl3; (middle) S- P(MHMn); and (bottom) ROS-S-MnP in D2O.

[0077] Figure 26 shows an13C NMR spectrum of S-P(MAcMn) in CDCl3.Attorney Docket No.: 31134 / 70234 / PC

[0078] Figure 27A shows FT-IR spectra of (top) S-P(MAcMn) in CDCl3; (middle) S- P(MHMn); and (bottom) ROS-S-MnP.

[0079] Figure 27B shows TGA thermograms of (top) S-P(MAcMn) in CDCl3; (middle) S- P(MHMn); and (bottom) ROS-S-MnP. TGA was performed with 10 °C / min temperature increment under nitrogen.

[0080] Figure 27C shows DSC curves.of (top) S-P(MAcMn) in CDCl3; (middle) S- P(MHMn); and (bottom) ROS-S-MnP. DSC was performed with 10 °C / min temperature increment under nitrogen.

[0081] Figure 28A shows the average hydrodynamic diameter histograms of the S-MP micelles, obtained through dynamic light scattering (DLS) intensity measurement.

[0082] Figure 28B shows the average hydrodynamic diameter histograms of the PTX loaded S-MP micelles, obtained through dynamic light scattering (DLS) intensity measurement.

[0083] Figure 28C is an image of a transmission electron micrograph of empty S-MP micelles.

[0084] Figure 28D is an image of a transmission electron micrograph of PTX loaded S-MP micelles.

[0085] Figure 28E shows the average hydrodynamic diameter histograms of the S-MP micelles in 1 mM H2O2, obtained through dynamic light scattering (DLS) intensity measurement.

[0086] Figure 28F shows the zeta potential of S-MP(empty), both alone and in 1mM H2O2, and S-MP(PTX).

[0087] Figure 28G shows the stability of the S-MP(PTX) micelles, alone and in 1mM H2O2.

[0088] Figure 28H shows cumulative PTX release from the S-MP(PTX) micelles, alone and in 1mM H2O2.

[0089] Figure 29 shows the in vitro, cellular uptake of Rhodamine-b labeled S-MP in PDAC cell lines. PANC-1 and AsPC cells were treated with rhodamine-b alone for 6h, and rhodamine-b loaded S-MP at three different time points: 1h, 2h, and 4h. Nuclei are blue (DAPI), and rhodamine-b PNPs are red. Images were captured by confocal fluorescence microscopy under blue and red channels. Bar Length = 50 µm.

[0090] Figure 30A shows the in vitro cellular cytotoxicity of PTX-loaded S-MP micelles with control Paxclitaxel in a PANC-1 cancer cell line.Attorney Docket No.: 31134 / 70234 / PC

[0091] Figure 30B shows the in vitro cellular cytotoxicity of PTX-loaded S-MP micelles with control Paxclitaxel in a AsPC cancer cell line.

[0092] Figure 30C shows the reactive oxygen species (ROS) generation by ROS assay. PANC-1 cells treated with free PTX, S-MP(PTX) with IC50concertation and 50-uM NAC for +Ve control; then incubated for 2 h and measured mean fluorescence intent % with respective control in a PANC-1 cancer cell line.

[0093] Figure 30D shows the reactive oxygen species (ROS) generation by ROS assay. AsPC cells treated with free PTX, S-MP(PTX) with IC50concertation and 50-uM NAC for +Ve control; then incubated for 2 h and measured mean fluorescence intent % with respective control in a AsPC cancer cell line.

[0094] Figure 31A shows a colorogenic assay of PANC-1 cells. S-MP PTX and Free PTX were grown in PANC-1 cells for 14 days.

[0095] Figure 31B shows a colorogenic assay of AsPC cells. S-MP PTX and Free PTX were grown in AsPC cells for 14 days.

[0096] Figure 31C shows colony formation percentage after treatment, relative to control in PANC-1 cells.

[0097] Figure 31D shows colony formation percentage after treatment, relative to control in AsPC cells.

[0098] Figure 32A shows an IVIS image taken at 24 h of the biodistribution of hydrogel- coated tumor-targeted peptide polymer nanoparticles (PNPs) in an orthotopic KPC pancreatic cancer model (TP), relative to hydrogel-coated polymer nanoparticles without tumor-targeted peptide (NTP) and only NIR-dye only formulation (Con).

[0099] Figure 32B shows an IVIS image taken at 48 h of the biodistribution of hydrogel- coated tumor-targeted peptide polymer nanoparticles (PNPs) in an orthotopic KPC pancreatic cancer model (TP), relative to hydrogel-coated polymer nanoparticles without tumor-targeted peptide (NTP) and only NIR-dye only formulation (Con).

[0100] Figure 32C shows an IVIS image taken at 72 h of the biodistribution of hydrogel- coated tumor-targeted peptide polymer nanoparticles (PNPs) in an orthotopic KPC pancreatic cancer model (TP), relative to hydrogel-coated polymer nanoparticles without tumor-targeted peptide (NTP) and only NIR-dye only formulation (Con).

[0101] Figure 33 shows effects on body weight of orally administered combinations of PNP-polymeric loaded paclitaxel(P), gemcitabine(G) and cisplatin(C) treatments in mice bearing KPC orthotopic pancreatic tumors with P(C), P(G+P) and P(C+G+P), relative toAttorney Docket No.: 31134 / 70234 / PC treatment with free paclitaxel, gemcitabine and cisplatin (C+G+P) as well as unloaded PNPs (Control). Paclitaxel(P) 1 mg / kg, gemcitabine(G) 4 mg / kg and Cisplatin(C) 2mg / kg, were used.

[0102] Figure 34A shows is a schematic of the experimental plan used in clinical studies of hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) with mice bearing KPC orthotopic pancreatic tumors.

[0103] Figure 34B shows a graph showing tumor weight (mg) in mice bearing KPC orthotopic pancreatic tumors after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) according to the disclosure. **** denotes p < 0.0001 compared to the P(Cis+Gem+Ptx) indicated respective group.

[0104] Figure 34C shows a graph showing tumor volume (mL) in mice bearing KPC orthotopic pancreatic tumors after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) according to the disclosure. **** denotes p < 0.0001 compared to the P(Cis+Gem+Ptx) indicated respective group.

[0105] Figure 34D shows a graph showing survival (%) in mice bearing KPC orthotopic pancreatic tumors after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) according to the disclosure.

[0106] Figure 35A shows Ex vivo imaging of tumors examined in mice bearing PANC-1 orthotopic pancreatic tumors 24 h after treatment with tumor-targeted peptide polymer nanoparticles (PNPs).

[0107] Figure 35B shows a graph showing tumor weight (mg) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) according to the disclosure. **** denotes p < 0.0001 compared to the P(Cis+Gem+Ptx) indicated respective group.

[0108] Figure 35C shows a graph showing tumor volume (mL) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) according to the disclosure. **** denotes p < 0.0001 compared to the P(Cis+Gem+Ptx) indicated respective group.

[0109] Figure 36A shows a graph showing tumor volume (mL) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with unloaded nanoparticles.

[0110] Figure 36B shows a graph showing tumor volume (mL) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) loaded with rapamycin(R) and vinorelbine(V). PNPs were administered at 2.5 mg / Kg.Attorney Docket No.: 31134 / 70234 / PC

[0111] Figure 36C shows a graph showing tumor volume (mL) in mice bearing PANC-1 orthotopic pancreatic tumors after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) loaded with rapamycin(R) and vinorelbine(V). PNPs were administered at 5 mg / Kg.

[0112] Figure 36D shows a graph showing tumor volume (mL) in a Renca mouse model after treatment with hydrogel-coated tumor-targeted peptide polymer nanoparticles (PNPs) loaded with rapamycin(R) and vinorelbine(V). PNPs were administered at 2.5 mg / Kg and 5 mg / Kg.

[0113] Figure 37A shows the average hydrodynamic diameter histograms of the targeted- polymer nanoparticles, obtained through dynamic light scattering (DLS) intensity measurement.

[0114] Figure 37B shows the average hydrodynamic diameter histograms of the Neuropilin-1(#9) loaded targeted-polymer nanoparticles, obtained through dynamic light scattering (DLS) intensity measurement.

[0115] Figure 37C shows the average hydrodynamic diameter histograms of the cabozantinib(Cabo) loaded targeted-polymer nanoparticles, obtained through dynamic light scattering (DLS) intensity measurement.

[0116] Figure 37D shows the average hydrodynamic diameter histograms of the Neuropilin-1 and cabozantinib(#9+Cabo) loaded targeted-polymer nanoparticles, obtained through dynamic light scattering (DLS) intensity measurement.

[0117] Figure 38A shows the In vitro cellular cytotoxicity of free Neuropilin-1(#9), cabozantinib(Cabo), and Neuropilin-1 with cabozantinib(#9+Cabo) in GBM-22 cancer cells.

[0118] Figure 38B shows the In vitro cellular cytotoxicity of free Neuropilin-1(#9), cabozantinib(Cabo), and Neuropilin-1 with cabozantinib(#9+Cabo) in GBM-1A cancer cells.

[0119] Figure 38C shows the In vitro cellular cytotoxicity of polymeric nanoparticles loaded with Neuropilin-1(#9), cabozantinib(Cabo), and Neuropilin-1 with cabozantinib(#9+Cabo) in GBM-22 cancer cells.

[0120] Figure 38D shows the In vitro cellular cytotoxicity of polymeric nanoparticles loaded with Neuropilin-1(#9), cabozantinib(Cabo), and Neuropilin-1 with cabozantinib(#9+Cabo) in GBM-1A cancer cells.

[0121] Figure 39A shows a colorogenic assay of GBM-22 cells. Tumor-targeted peptide polymer nanoparticles were loaded with Neuropilin-1(#9) and cabozantinib(Cabo).Attorney Docket No.: 31134 / 70234 / PC TP(#9+Cabo), TP(#9), TP(Cabo), free #9, and free cabo were grown in GBM-22 cells for 14 days.

[0122] Figure 39B shows colony formation percentage after treatment, relative to control in GBM-22 cells. **** denotes p < 0.0001 compared to the P(#9+Cabo) indicated respective group.

[0123] Figure 40 shows the biodistribution of tumor-targeted peptide polymer nanoparticles (PNPs) in an orthotopic GBM cancer model intravenously treated with unloaded nanoparticles (Control), NIR dye only formulation, polymer formulation without tumor-targeted peptide, and tumor-targeted peptide polymer nanoparticles (PNPs) with dye. (top) IVIS images taken at 24 h after administration. (bottom) Ex vivo imaging of the tumors and major organs examined after 24 h. DETAILED DESCRIPTION

[0124] Provided herein are polymeric nanoparticles which has a triblock copolymer structure that can be tailored based on the therapeutic agents of interest and target for deliver. The nanoparticles of the disclosure can provide for controlled to release the therapeutic agents via changes in via pH and / or electric potential. The polymeric nanoparticles of the disclosure can allow for targeted delivery of therapeutic agents, enhanced accumulation of therapeutic agents, increased cellular uptake of therapeutic agents at tumor sites, and / or improved bioavailability of therapeutic agents.

[0125] A polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can include a polymeric material that encapsulates the one or more therapeutic agents. The polymeric material is a triblock copolymer having a hydrophilic (optionally polyethylene glycol (PEG)) block that enhances biocompatibility and solubility, a hydrophobic block that improves the incorporation and release of therapeutic agents, and a thermo-responsive or lipophilic and stimulative block that stabilizes the polymeric nanoparticles.

[0126] For example, the triblock copolymer can have a structure according to formula (I) or formula (II):Attorney Docket No.: 31134 / 70234 / PC(II), wherein: the one or more therapeutic agents are encapsulated by the triblock copolymer; t, v, w, and z are each independently 6 to 500; y is 0 to 500; A is OC1-6alkylene; RBis C(O)NH–C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N(RN)2, C(O)NH–C1- 6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RBis substituted with 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O; each of RCand RC′ are C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2, C(O)NH–C1-6alkylene– N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RCis substituted with 0 to 2 substituents selected from C1-3alkyl and =O; D is –C(O)C1-6alkylene–O substituted with 0 to 2 C1-3alkyl substituents; X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then RBis the targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; and each RNis independently H or C1-3alkyl.

[0127] The therapeutic nanoparticles of the disclosure can have an average particle diameter in the range of 100 nm to 500 nm. The therapeutic agent can be provided in an amount of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle. The nanoparticles of the disclosure can have a Zeta potential in the range of -35 mV to +40 mV. The nanoparticles of the disclosure can have a polydispersity index in the range of 0.150 to 0.300.

[0128] Alternatively, a polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can include a polymeric material encapsulating one or more therapeutic agents where the polymer material has a triblock copolymer having a structure according to formula (III):Attorney Docket No.: 31134 / 70234 / PCwherein: t, v, w, and z are each independently 6 to 500; y is 0 to 500; E is a hydrophilic polymer; each of F, G and G′ are a targeting moiety, a hydrophobic polymer, or a lipophilicpolymer, provided that at least one hydrophilic polymer and lipophilic polymer are each present, X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then F is the targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; and each RNis independently H or C1-3alkyl.

[0129] The nanoparticle can have an average particle diameter in the range of 100 nm to 500 nm. Each therapeutic agent can be present in an amount of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle. The nanoparticle can have a Zeta potential in the range of -35 mV to +40 mV. The nanoparticle can have a polydispersity index in the range of 0.150 to 0.300.

[0130] In a further alternative, a polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can include a polymeric material encapsulating one or more therapeutic agents where the polymer material has a triblock copolymer having a structure according to formula (IV):wherein: J is a targeting moiety, said moiety targeted to reactive oxygen species; Q is a hydrophilic polymer and optionally, a targeting moiety; and M is a hydrophobic polymer having a structure according to Formula (IV’):Attorney Docket No.: 31134 / 70234 / PCwherein: X is halo; and each of n, m, p, and q, are independently selected from 1 to 20;

[0131] The nanoparticle can have an average particle diameter in the range of 100 nm to 500 nm. Each therapeutic agent can be present in an amount of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle. The nanoparticle can have a Zeta potential in the range of -35 mV to +40 mV. The nanoparticle can have a polydispersity index in the range of 0.150 to 0.300. The hydrophilic polymer has a content in the range of 40 wt.% to 60 wt.%, based on the entire weight of the nanoparticle.

[0132] It has been observed that by including a hydrophilic block, hydrophilic blocks, and lipophilic and stimulative or lipophilic and thermo-responsive blocks in the polymeric material, polymeric nanoparticles can be produced with the enhanced ability to incorporate and release therapeutic agents.

[0133] The hydrophilic block can enhance biocompatibility and solubility of the copolymer. The hydrophilic block can enhance the solubility of the nanoparticles in formulations and under physiological conditions and provide flexibility to the nanoparticles to incorporate and release the one or more therapeutic agents. The hydrophilic block can be a PEG polymer. The hydrophilic (optionally PEG) block can include one or more hydrophilic (or PEG) co- monomers and a thiocarbonylthio moiety suitable for RAFT polymerization. For example, the hydrophilic PEG block can

[0134] The hydrophilic block is biocompatible and can be protein-repellant. The hydrophilic block can allow the nanoparticles to be conjugated to a targeting moiety to deliver therapeutic agents to specific cells. The hydrophilic block can be a saccharide. The saccharide block is hydrophilic, and when an aldehyde containing sugar such as glucose or mannose, can be conjugated to a targeting moiety to enhance the targeting efficiency of the nanoparticles. It has been found that saccharide blocks can act as both hydrophilic blocks and targeting moieties themselves.Attorney Docket No.: 31134 / 70234 / PC

[0135] The hydrophobic block can enhance incorporation and release of therapeutic agents. The hydrophobic block can be formed from one or more co-monomers, for example, selected from penta fluoro-phenylacrylate (PFHEA), 2-hydroxyethyl meth / acrylate (HEA), oxiran-2-ylmethyl acrylate, lauryl acrylate, butyl acrylate, triethoxysilyl)propylcarbamate ethyl acrylate, and 2-ethylhexyl acrylate (EHA ). The co-monomers used for the hydrophobic block are biocompatible and can be protein-repellant. The hydrophobic block can allow the nanoparticles to circulate in the bloodstream for an extended period with minimum opsonization.

[0136] The lipophilic and stimulative block (if present) can increase the relative hydrophobicity and stability of the nanoparticle. The lipophilic and stimulative block can be formed by reacting a co-monomer with the hydrophilic block. The lipophilic and stimulative block can be formed from one or more co-monomers selected from, for example, lipoic acid (LA), 3,3’-disulfanediyldipropionic acid, N-(2-(diethylamino)ethyl)acrylamide, carboxy betaine acrylamide, carboxybetaine acrylamide, glycerol carbonate methacrylate, and saccharide based acrylates, such as glycose and mannose acrylates, and (4- (bromomethyl)phenyl)boronic acid. The lipophilic and stimulative block can allow the nanoparticles to increase or decrease in charge to accommodate conditions in the targeted cell. The lipophilic and stimulative block can allow the nanoparticles to circulate in the bloodstream for an extended period of time.

[0137] The lipophilic and thermo-responsive block (if present) can enhance the incorporation and release of therapeutic agents from the nanoparticles. The thermo- responsive block can be formed from one or more co-monomers selected from, for example, N-vinyl caprolactam (NVCL), . The thermo-responsive blocks can increase the stability of the nanoparticles and enhance the targeted delivery of therapeutic agents. Generally, the thermo-responsive block contains a reactive moiety, such as a large ring for NVCL, which can undergo reactions in response to cell conditions, i.e. temperature. This can allow the nanoparticles to release therapeutic agents only in response to changes in cell temperature. The thermo-responsive block can allow the nanoparticles to circulate in the bloodstream for an extended period of time.

[0138] The nanoparticles are conjugated to a targeting moiety through the hydrophilic block. Nanoparticles conjugated to targeting moieties can effectively deliver therapeutic agents to specific cells by binding to sites expressed on the surface of targeted cells. Accordingly, the nanoparticles of the disclosure can be adopted to target a cell of interest, reducing the amount of therapeutic agent required and reducing undesirable effects of treatment.Attorney Docket No.: 31134 / 70234 / PC

[0139] Additionally, the behavior of the nanoparticles can be varied by changing the identity of the blocks in the polymeric material. It has been observed that functional groups with acidic protons, such as carboxylic acids, as well as functional groups with basic electron pairs, such as amines can impart control over the release of therapeutic agents based on the pH and reduction potential of the environment around the nanoparticles. For example, if the polymeric material includes any acidic or basic functional groups, then when immersed in the appropriate pH, the polymeric material will be ionized upon deprotonation or protonation, respectively. Likewise, functional groups can be ionized by an appropriate reduction potential. Polymeric Material of Formulae (I) and (II)

[0140] A polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can include a polymeric material comprising a triblock copolymer having a structure according to Formula (I) or Formula (II):(II); and one or more therapeutic agents encapsulated by the polymeric material; wherein: t, v, w, and z are each independently 6 to 500; y is 0 to 500; A is OC1-6alkylene; RBis C(O)NH–C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N(RN)2, C(O)NH–C1- 6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RBis substituted with 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O; each of RCand RC′ are C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2, C(O)NH–C1-6alkylene– N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RCis substituted with 0 to 2 substituents selected from C1-3alkyl and =O; D is –C(O)C1-6alkylene–O substituted with 0 to 2 C1-3alkyl substituents;Attorney Docket No.: 31134 / 70234 / PC X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then RBis the targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; and each RNis independently H or C1-3alkyl.

[0141] In the polymeric nanoparticles of Formulae (I) and (II), each of t, v, w, and z can independently be 6 to 500. In various cases, t is 6 to 50, and each of v, w, and z are 100 to 500.

[0142] In the polymeric nanoparticles of Formulae (I) and (II), y can be 0 to 500. In various cases, y is 0. In various cases, y is 100 to 300.

[0143] In the polymeric nanoparticles of Formulae (I) and (II), A can be OC1-6alkylene. In various cases, A is OC1-3alkylene. In various cases, A is OC3alkylene.

[0144] In the polymeric nanoparticles of Formulae (I), RBcan be C(O)NH–C1-6alkylene– COOH, C(O)NH–C1-6alkylene–N(RN)2, C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RBis substituted with 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O. In various cases, RBis C(O)NH–C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O. In various cases, RBis C(O)NH– C1-6alkylene–COOH, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and substituted with 0 to 2 substituents selected from C1-3alkyl and =O. In various cases, RBis C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, substituted with 0 to 2 B(OH)2.

[0145] In the polymeric nanoparticles of Formula (I), each of RCand RC′ can independently be C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O– C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2, C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RCis substituted with 0 to 2 substituents selected from C1-3alkyl and =O. In various cases, RCis C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and RCis substituted with 0 to 2 substituents selected from C1-3alkyl and =O. In various cases, RCis C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, or C(O)O–C1-6alkyl, wherein the heterocycloalkyl has 1-2 ring heteroatomsAttorney Docket No.: 31134 / 70234 / PC selected from N, O, and S and is substituted with 0 to 2 C1-3alkyl substituents. In various cases, RCis NHC(O)C1-6alkylene–N(RN)2or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and is substituted with 0 to 2 =O substituents. In various cases, RC′ is C(O)O–C1-6alkylene–OC(O)–C1-6alkylene–C3-8heterocycloalkyl, having 1-2 ring heteroatoms selected from N, O, and S and is substituted with 0 to 2 =O substituents.

[0146] In the polymeric nanoparticles of Formula (II), D can be –C(O)C1-6alkylene–O substituted with 0 to 2 C1-3alkyl substituents. In various cases, D is –C(O)C1-3alkylene–O. In various cases, D is substituted with C1alkyl.

[0147] In the polymeric nanoparticles of Formulae (I) and (II), each X can be NRNor O. In various cases, each X is NRN.

[0148] In the polymeric nanoparticles of Formulae (I) and (II), each CAP group can be a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then RBis the targeting moiety. In various cases, each CAP group is a targeting moiety. In various cases, each CAP group is C1-6alkyl. In various cases, each CAP group is C3-6alkyl.

[0149] In the polymeric nanoparticles of Formulae (I) and (II), the END group can be C1- 6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0- 3alkylene–CN. In various cases, the END group is C1-6alkyl. In various cases, the END group is substituted with 0 to 2 C1-3alkyl substituents.

[0150] In the polymeric nanoparticles of Formulae (I) and (II), each RNcan independently be H or C1-3alkyl. In various cases, each RNis independently H or C2alkyl.

[0151] In various cases, t is 6, v and w are each 100 to 500, y is 100 to 500, A is OC3alkylene,X is NRN, each CAP group is a targeting moiety, the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN, and each RNis H. w are each 100 to 500, A is OC3alkylene, RBiseach CAP group is a targeting moiety, the END group is C6aryl, and each RNis H.Attorney Docket No.: 31134 / 70234 / PC

[0153] In various cases, t is 6, v and w are each 100 to 500, A is OC3alkylene, RBiseach CAP group is t-butyl, the END group is C6aryl, each RNis H, and the targeting moiety is RB.

[0154] In various cases, t is 6, v and w are each 100 to 500, A is OC3alkylene, RBiseach CAP group is a targeting moiety, the END group is C3alkyl substituted with C1alkyl and CN, and each RNis H or C1-3alkyl.

[0155] In various cases, t is 6, z is 100 to 500, A is OC3alkylene,, each X is NRN, each CAP group is a targeting moiety, and each RNis H. are each 100 to 500, A is OC3alkylene, RBisthe CAP group is a targeting moiety, the END group is C6aryl, and each RNis H. Polymeric Material of Formula (III)

[0157] A polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can include a polymeric material comprising a triblock copolymer having a structure according to Formula (III):one or more therapeutic agents encapsulated by the polymeric material; wherein: t, v, w, and z are each independently 6 to 500; y is 0 to 500; E is a hydrophilic polymer;Attorney Docket No.: 31134 / 70234 / PC each of F, G and G′ are a targeting moiety, a hydrophobic polymer, or a lipophilicpolymer, provided that at least one hydrophobic polymer and lipophilic polymer are each present, X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then F is the targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; and each RNis independently H or C1-3alkyl.

[0158] In the polymeric nanoparticles of Formula (III), each X can be NRNor O. In various cases, each X is NRN.

[0159] In the polymeric nanoparticles of Formula (III), each RNcan independently be H or C1-3alkyl. In various cases, each RNis independently H or C2alkyl.

[0160] In the polymeric nanoparticles of Formula (III), E is a hydrophilic polymer. In various cases, E is a PEG polymer. In various cases, E is OC1-6alkylene.

[0161] In the polymeric nanoparticles of Formula (III), each of F, G and G′ can be atargeting moiety, a hydrophobic polymer, or a lipophilic polymer, provided that at least one hydrophobic polymer and lipophilic polymer are each present. In various cases, F is hydrophobic polymer and each of G and G′ are a lipophilic polymer. In various cases, F is a targeting moiety.

[0162] In the polymeric nanoparticles of Formula (III), each CAP group can a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then F is the targeting moiety. In various cases, each CAP group is the targeting moiety. In various cases, each CAP group is C1-6alkyl and F is the targeting moiety.

[0163] In the polymeric nanoparticles of Formula (III), the END group can be C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene– CN. In various cases, the END group is C1-6alkyl with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN.

[0164] In the polymeric nanoparticles of Formula (III), the hydrophobic polymer can be any polymer that is predominantly hydrophobic. In various cases, the hydrophobic polymer is C(O)NH–C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N(RN)2, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and F is substituted with 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O.Attorney Docket No.: 31134 / 70234 / PC

[0165] In the polymeric nanoparticles of Formula (III), the lipophilic polymer can be any polymer that has neutral and charged regions at physiological pH. In various cases, each lipophilic polymer unit is C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and G or G′ is substituted with 0 to 2 substituents selected from C1-3alkyl and =O.

[0166] In the polymeric nanoparticles of Formula (III), the targeting moiety can be on the CAP group or on one of F, G, and G′. In various cases, the targeting moiety is on the CAP group. In various cases, the targeting moiety is C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene– C6-12aryl. Polymeric Material of Formula (IV)

[0167] A polymeric nanoparticle for targeted delivery of one or more therapeutic agents in accordance with the disclosure can include a polymeric material comprising a triblock copolymer having a structure according to Formula (IV):one or more therapeutic agents encapsulated by the polymeric material; wherein: J is a targeting moiety, said moiety targeted to reactive oxygen species; each Q is a hydrophilic polymer and optionally comprises a targeting moiety; and M is a hydrophobic polymer having a structure according to Formula (IV’):wherein: X is halo; and each of n, m, p, and q, are independently selected from 1 to 20.Attorney Docket No.: 31134 / 70234 / PC

[0168] In the polymeric nanoparticles of Formula (IV), J can be a targeting moiety. In various cases,

[0169] In the polymeric nanoparticles of Formula (IV), each Q can be a hydrophilic polymer and optionally comprises a targeting moiety. In various cases, Q is a hydrophilic polymer and a targeting moiety. In various cases, Q is a saccharide. In various cases, Q is glucose or mannose. In various cases, Q is D-glucose or D-mannose.

[0170] In the polymeric nanoparticles of Formula (IV), M can have a structure according to

[0171] In the polymeric nanoparticles of Formulae (IV) and (IV’), X can be halo. In various cases, X is Cl or Br.

[0172] In the polymeric nanoparticles of Formulae (IV) and (IV’), each of n, m, p, and q can be independently selected from 1 to 20. In various cases, each of m, n, p, and q are the same. In various cases, at least one of m, n, p, and q is not the same. In various cases, each of m, n, p, and q are independently selected from 1-10. In various cases, each of m, n, p, and q are independently selected from 11-20.

[0173] In the polymeric nanoparticles of Formulae (IV) and (IV’), the hydrophilic polymer can have a content in the range of 40 wt.% to 60 wt.%, based on the entire weight of the nanoparticle. In various cases, the hydrophilic polymer can have a content in the range of 45 wt.% to 55 wt.%, based on the entire weight of the nanoparticle.

[0174] In any of the polymeric nanoparticles of the disclosure, the nanoparticle can have an average particle diameter in the range of 100 nm to 500 nm. For example, the nanoparticle can have an average particle diameter of at least about 125, 150, 175, 200, 225, 250, or 275 nm and / or up to about 400 or 500 nm. In various cases, the particle has an average particle diameter in the range of 100 nm to 300 nm. The particle size can represent a weight-, number-, surface area-, or volume-average size for a particle size distribution of the nanoparticles.Attorney Docket No.: 31134 / 70234 / PC

[0175] Generally, the polymeric nanoparticles of the disclosure can have Zeta potential in the range of -35 mV to +40 mV. In various cases, the nanoparticle has a Zeta potential in the range of -35 mV to + 35 mV. In various cases, the nanoparticle has a neutral potential, i.e. the Zeta potential is in the range of -10 mV to +10 mV. In various cases, the nanoparticle has a positive Zeta potential, i.e. the Zeta potential is greater than +10 mV. In various cases, the nanoparticle has a negative Zeta potential, i.e. the Zeta potential is less than –10 mV.

[0176] Generally, any of the polymeric nanoparticles of the disclosure can have a polydispersity index in the range of 0.150 to 0.300. In various cases, the nanoparticle has a polydispersity index in the range of 0.150 to 0.250.

[0177] In any of the polymeric nanoparticles of the disclosure, each therapeutic agent is present in an amount of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle. In various cases, each therapeutic agent is present in an amount of 2 wt.% to 8 wt.%, based on the entire weight of the nanoparticle. In any of the polymeric nanoparticles of the disclosure, the total amount of therapeutic agent can be 1 wt.% to 10 wt.%. In various cases, the total amount of therapeutic agent is 1 wt.% to 5 wt.%.

[0178] In various cases, the average particle diameter can be 100 nm to 200 nm, and each therapeutic agent can be present in an amount of 3 wt.% to 6 wt.%, based on the entire weight of the particle.

[0179] In any of the polymeric nanoparticles of the disclosure, each therapeutic agent can independently be an snRNA, a peptide, a nucleic acid, a nucleotide, or a pharmaceutically active compound. In various cases, at least one therapeutic agent is a peptide. In various cases, at least one peptide is an antibody. In various cases, at least one therapeutic agent is a pharmaceutically active compound. In various cases, the one or more therapeutic agents are Gemcitabine and Paclitaxel.

[0180] In any of the polymeric nanoparticles of the disclosure, a targeting moiety is a group that binds to sites expressed on the surface of targeted cells. Nanoparticles conjugated to targeting moieties can effectively deliver therapeutic agents to specific cells by binding to sites expressed on the surface of targeted cells. Accordingly, the nanoparticles of the disclosure can be tailored to target specific cell lines, reducing the amount of therapeutic agent delivered and undesirable side effects of treatment. In various cases, the targeting moiety is a tumor-targeted peptide. In various cases, the targeting moiety is a functional group that can react with reactive oxygen species (ROS). In various cases, the targeting moiety is a saccharide. In various cases, the targeting moiety is glucose or mannose. In various cases, the targeting moiety is D-glucose or D-mannose.Attorney Docket No.: 31134 / 70234 / PC

[0181] A ROS targeted nanoparticle can leverage the fact that higher ROS levels in tumors can be a reliable predictor of tumor-targeted drug delivery locations. This is because ROS levels are approximately 100 times higher in inflammatory and tumor locations as compared to normal tissues. (Liu, J., et al., J. Hematology & Oncology, 2023.16(1): p.116; Tao, W. et al., Asian J Pharm Sci, 2018.13(2): p.101-112). Accordingly, ROS targeted nanoparticles can also accumulate passively at tumor locations due to the increased permeability and retention (EPR) effect. Specifically, glycopolymer-based nanotechnology (synthetic polymer backbones with saccharide moieties attached as pendants) can target sugar-binding proteins (lectins), which are overexpressed on cancer cell surfaces. (Wojtczak, K. et al., ChemMedChem, 2022.17(12): p. e202200081; Suzuki, K., et al.,. J Control Release, 2019.301: p.28-41). Once targeted, the nanoparticles can deliver therapeutic agents to cancer cells via receptor-mediated endocytosis. The binding between single saccharides and lectins is usually modest, but the "glycocluster effect" can considerably improve it. Multivalent carbohydrate ligands can increase the binding constant, leading to the development of saccharide-targeted systems. (Gupta, A. et al., J. Nanopart. Res., 2022.24(11): p.228). These systems can overcome conventional chemotherapeutic agents' poor specificity and toxicity. Moreover, due to the weak interaction between the mannose receptor and the single mannose, the nanoparticles containing multiple mannose residues can provide a more substantial binding and a higher presentation of the ligands. Additionally, mannosylated moieties can also increase hydrophilicity, thereby facilitating the encapsulation of drugs, enhancing the bioavailability and effects of treatment with therapeutic agents and reduced cytotoxicity.

[0182] Generally, the polymeric nanoparticles of the disclosure exhibit controlled release of therapeutic agents over a period of 1 month to 1 year. The release of the polymeric nanoparticles can be controlled based on the blocks present in the polymeric material. In particular, the blocks of the polymeric material can be ionized to affect the release of the therapeutic agent. As the polymeric material is ionized, the affinity of the therapeutic agents for the polymeric material will decrease.

[0183] In any of the polymeric nanoparticles of the disclosure, the polymeric material can be sensitive to low pH (i.e., pH <7), high pH (i.e., pH >7), as well as reduction potentials. As the pH decreases, more basic electron pairs, such as amines will be protonated and become cationic, and as the pH increases, the functional groups with acidic protons, such as carboxylic acids, will be deprotonated and become anionic. Likewise, as the reduction potential of the environment decreases, functional groups will be reduced and become anionic, and as the reduction potential of the environment decreases, functional groups will be oxidized and become cationic. As the polymeric material changes in response toAttorney Docket No.: 31134 / 70234 / PC environmental conditions, the nanoparticle cannot incorporate the therapeutic agent, which in turn, results in release of the therapeutic agent from the nanoparticle.

[0184] The immersion and loading of any nanoparticles of the disclosure can be performed according to methods known in the art (Rao, N.N.M., et al., J. Appl. Polym. Sci, 2020.137(32)).

[0185] In accordance with the disclosure, the polymeric nanoparticles can be coated with a hydrogel. For example, the hydrogel can be a biocompatible hydrogel matrix. Examples of biocompatible hydrogel matrix materials include, but are not limited to, functionalized sodium alginate, modified hyaluronic acid, and ROS responsive PEGylated hydrogels such as PEG-b-PPGb--PCL-based hydrogels. Thus, in some cases, the hydrogel is functionalized sodium alginate, modified hyaluronic acid, or ROS responsive PEGylated hydrogel. The hydrogel coating can enhance the therapeutic functionality of the polymeric nanoparticles of the disclosure, for example, by improving stability of the loaded therapeutic agents, enabling site-specific delivery, and / or promoting synergistic activity between the co- encapsulated therapeutic agents.

[0186] Furthermore, the hydrogel coating can provide a protective barrier against enzymatic degradation and pH fluctuations in the gastrointestinal tract, thereby improving the bioavailability and uptake of the drugs at the target tumor site. This integrated hydrogel- nanoparticle platform can provide a non-invasive treatment method that can enhance therapeutic outcomes. In some cases, the hydrogel formulation can be used in the treatment of pancreatic ductal adenocarcinoma (pDAC). Layered Drug Delivery System of the Disclosure

[0187] Additionally, a layered delivery vehicle can be provided in which layers of polymeric nanoparticles loaded with one or more therapeutic agents are separated by hydrogel layers. In some cases, the one or more hydrogel layers comprises a functionalized sodium alginate, a modified hyaluronic acid, or an ROS responsive PEGylated hydrogel. In some cases, the one or more hydrogel layers comprises a functionalized sodium alginate. In some cases, the one or more hydrogel layers comprises a modified hyaluronic acid. In some cases, the one or more hydrogel layers comprises an ROS responsive PEGylated hydrogel.

[0188] Each layer of polymeric nanoparticles can have the same or different therapeutic agents encapsulated in the nanoparticles. Even within the same layer, it is contemplated that the polymeric nanoparticles can be loaded with and encapsulate different therapeutic agents. In some cases, the one or more therapeutic agent layers comprises the same oneAttorney Docket No.: 31134 / 70234 / PC or more therapeutic agents. In some cases, the one or more therapeutic agent layers comprises different therapeutic agents.

[0189] Generally, the number of layers is not limiting. The outermost layer of the drug delivery system can include a hydrogel layer or a therapeutic agent layer. In some cases, the outermost layer of the drug delivery system is a hydrogel layer. The layered delivery vehicle can be useful in withstanding gastrointestinal degradation, facilitating mucoadhesion, and / or enabling sustained and localized release of therapeutic agents upon oral administration.

[0190] In some cases, the layered delivery vehicle can be used in the treatment of cancer. In some cases, the layered delivery vehicle can be used in the treatment of pancreatic cancer or brain cancer. In some cases, the cancer is ductal adenocarcinoma (pDAC) or glioblastoma. Chemical Definitions

[0191] As used herein, the term “alkyl” refers to straight chained and branched saturated hydrocarbon groups containing one to thirty carbon atoms, for example, one to twenty carbon atoms, or one to ten carbon atoms. The term Cnmeans the alkyl group has “n” carbon atoms. For example, C6alkyl refers to an alkyl group that has 6 carbon atoms. C1-7alkyl refers to an alkyl group having a number of carbon atoms encompassing the entire range (i.e., 1 to 6 carbon atoms), as well as all subgroups (e.g., 1-5, 2-5, 3-6, 1, 2, 3, 4, 5, and 6 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and t-butyl (1,1-dimethylethyl). Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0192] As used herein, the term “alkylene” refers to a bivalent saturated aliphatic radical. The term Cnmeans the alkylene group has "n" carbon atoms, e.g., a C1alkylene is CH2. For example, C1-6alkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range, as well as all subgroups, as previously described for "alkyl" groups.

[0193] As used herein, the term “aryl” refers to an aromatic carbocycle, and can be monocyclic or polycyclic (e.g., fused bicyclic and fused tricyclic) carbocyclic aromatic ring systems. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, phenanthrenyl, biphenylenyl, indanyl, indenyl, anthracenyl, fluorenyl, tetralinyl. Unless otherwise indicated, an aryl group can be an unsubstituted aryl group or a substituted aryl group.Attorney Docket No.: 31134 / 70234 / PC

[0194] As used herein, the term "heterocycle" refers to a non-aromatic ring which contains one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur. Additionally, heterocycles of the disclosure can be monocyclic, bicyclic, bridged, fused or spirocyclic. For example, a heterocycle can be a monocyclic, bicyclic, bridged, fused, or spirocyclic 4-8 membered ring having 1 or 2 or 3 heteroatoms selected from N, O, and S. As another example, a heterocycle can be a 8-10 membered bicyclic, bridged, fused, or spirocyclic group having 1 or 2 or 3 ring heteroatoms selected from N, O, and S in the bicyclic ring. Nonlimiting examples of heterocycle groups include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, and oxazepane.

[0195] As used herein, the term "halo" is defined as fluoro, chloro, bromo, and iodo. Accordingly, a “haloalkyl” refers to an alkyl group substituted with one or more halo atoms.

[0196] As used herein, a “saccharide” is understood to interchangeable with the terms “sugar” and “carbohydrate” and can refer to a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, as well as oligosaccharides and polysaccharides. Examples of saccharides include, but are not limited to, mannose, glucose, fructose, etc.

[0197] As used herein, a “substituted” functional group is a functional, group having at least one hydrogen radical that is substituted with a non-hydrogen radical (i.e., a substituent). Examples of non-hydrogen radicals (or substituents) include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkyl, alkynyl, ether, aryl, heteroaryl, heterocycle, hydroxyl, oxy (or oxo), alkoxyl, ester, thioester, acyl, carboxyl, cyano, nitro, amino, sulfhydryl, and halo. When a substituted alkyl group includes more than one non-hydrogen radical, the substituents can be bound to the same carbon or different carbon atoms. Pharmaceutical Formulations

[0198] Also provided herein are pharmaceutical formulations that include nanoparticles of the disclosure loaded with an effective amount of one or more therapeutic agents and one or more pharmaceutically acceptable excipients. As used herein, the term “formulation” is used interchangeable with “composition.”

[0199] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and / or ameliorating a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and / or substantially lessening the chances of a disease or condition in a subject. As used herein, the terms “patient” and “subject” may be used interchangeably and mean animals, such as dogs, cats, cows, horses, and sheep (i.e., non-human animals) and humans. ParticularAttorney Docket No.: 31134 / 70234 / PC patients or subjects are mammals (e.g., humans). The terms “patient” and “subject” include males and females.

[0200] As used herein, the term “excipient” means any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API), suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.

[0201] The nanoparticles of the disclosure can be administered alone or as part of a pharmaceutically acceptable composition or formulation. In addition, the nanoparticles can be administered all at once, as for example, by a bolus injection, multiple times, e.g. by a series of tablets, or delivered substantially uniformly over a period of time, as for example, using transdermal delivery. It is also noted that the dose of the therapeutic agent can be varied over time.

[0202] The nanoparticles disclosed herein, if desired, can be administered to a subject or patient by any suitable route, e.g. orally, topically, rectally, parenterally, (for example, subcutaneous injections, intravenous, intramuscular, intrasternal, and intrathecal injection or infusion techniques), or as a buccal, inhalation, or nasal spray. The administration can be to provide a systemic effect (e.g. eneteral or parenteral). All methods that can be used by those skilled in the art to administer a pharmaceutically active agent are contemplated. In some cases, the disclosed formulations can be administered orally or intravenously.

[0203] Suitable oral compositions or formulations in accordance with the disclosure include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, gels, syrups or elixirs. Compositions or formulations suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions.

[0204] Compositions or formulations suitable for oral use can comprise hydrogels and / or be in the form of a hydrogel. For example, the hydrogel can be a biocompatible hydrogel matrix. Examples of biocompatible hydrogels include, but are not limited to, functionalized sodium alginate, modified hyaluronic acid, and ROS responsive PEGylated hydrogels such as PEG-b-PPGb--PCL-based hydrogels.

[0205] Hydrogel formulations can also include polymeric nanoparticles loaded with multiple therapeutic agents, providing a layered delivery vehicle which can withstand gastrointestinal degradation, facilitate mucoadhesion, and enable sustained and localized release of therapeutic agents upon oral administration.Attorney Docket No.: 31134 / 70234 / PC

[0206] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the therapeutic agents, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0207] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the loaded nanoparticles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0208] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.Attorney Docket No.: 31134 / 70234 / PC

[0209] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0210] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0211] Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

[0212] The nanoparticles of the disclosure can be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of therapeutic agent calculated to produce the desired therapeutic effect, optionally inAttorney Docket No.: 31134 / 70234 / PC association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.

[0213] The nanoparticles of the disclosure can be administered to a subject or patient at dosage levels in the range of about 0.1 to about 3,000 mg per day. For a normal adult human having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kilogram body weight is typically sufficient. The specific dosage and dosage range that will be used can potentially depend on a number of factors, including the requirements of the subject or patient, the severity of the condition or disease being treated, and the pharmacological activity of the therapeutic agent being administered. The determination of dosage ranges and optimal dosages for a particular subject or patient is within the ordinary skill in the art. Methods of Treatment

[0214] The nanoparticles, formulations, and layered drug delivery systems of the disclosure can be used to treat cancer. The nanoparticles disclosed herein can deliver chemotherapy drugs to oncogenic cells through specific selection of the targeting moiety based on the cancer of interest. Chemotherapy effectiveness can be assessed in a manner as described in the Examples section below.

[0215] Thus, provided herein are methods of treating cancer. In these methods, a subject is administered the compound or pharmaceutical composition to treat cancer. Additionally, pharmaceutical compositions comprising nanoparticles of the disclosure, provide a means of administering a drug to a subject and treating these conditions. As used herein, the terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, or ameliorating a disease or condition, and / or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated. As used herein, the terms "treat," "treating," "treatment," and the like may include "prophylactic treatment," which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously-controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition. The term "treat" and synonyms contemplate administering a therapeutically effective amount of a compound of the disclosure to an individual in need of such treatment. Within the meaning of the disclosure, "treatment" also includes relapse prophylaxis or phase prophylaxis, as well as the treatment of acute or chronic signs, symptoms and / orAttorney Docket No.: 31134 / 70234 / PC malfunctions. The treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy. As used herein, the terms “prevent,” “preventing,” “prevention,” are art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount. As used herein, the terms “patient” and “subject” may be used interchangeably and mean animals, such as dogs, cats, cows, horses, and sheep (i.e., non-human animals) and humans. Particular patients are mammals (e.g., humans). The term patient includes males and females.

[0216] Specifically contemplated cancers that can be treated using the compounds and compositions described herein include, but are not limited to pancreatic cancers and brain cancers. In some cases, the cancer is pancreatic ductal adenocarcinoma or glioblastoma.

[0217] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.

[0218] It is to be understood that while the disclosure is read in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.Attorney Docket No.: 31134 / 70234 / PC EXAMPLES

[0219] The following examples are provided for illustration and are not intended to limit the scope of the disclosure in any way. Materials and Methods

[0220] N-vinyl caprolactam (NVCL), 1,4-dioxane, anhydrous dichloromethane (98 %), 4- Cyano-4- (phenyl carbono thioylthio) pentanoic acid, tris(hydroxymethyl)aminomethane hydrochloride buffer (TRIZMA, Sigma Aldrich, 99 %), 2-Hydroxyethyl acrylate, (97%, stabilized, Thermo Scientific) and t-Bu-OOC-PEG6-OH (Nano soft polymers), 2,20- Azobisisobutyronitrile (AIBN) (Sigma Aldrich) was recrystallized twice from methanol. NVCL was purified by recrystallization from hexane, dried under vacuum for 10 h at 35 °C and stored at 4 °C. (Sigma Aldrich 98%), Trimethylsilyldiazomethane (2 M solution in diethyl ether). Dialysis tubing of cellulose membrane (MW cut-off ¼ 10,000) was purchased from Sigma Aldrich. α-bromoisobutyrylbromide (α-BIBB), methacrylic acid, 1,1,4,7,10,10- hexamethyl triethylenetetramine (HMTETA), pentaerythritol, Cu(I)Cl,D-mannose anhydrous, H2O230 wt.% in H2O H2O2(Sigma- Aldrich), benzylamine, dry 1,4-dioxane, molecular sieves 4 Å, ethyl acetate, n-hexane and triethylamine (Et3N), dry dichloromethane (DCM) (Finar Chemicals limited), benzoyl chloride, sodium methoxide (NaOMe) and sodium acetate (Avra Synthesis Pvt Ltd) were used as received. Tetrahydrofuran (THF) (Finar Chemicals Ltd.) was distilled under a nitrogen atmosphere over sodium wire and benzophenone. (Meth)acryl chloride was prepared according to published methods. Buffer solution was of analytical grade preparation. Gemcitabine, Paclitaxel, were procured from LC Laboratories, MA, USA. DMEM, and Pen-Strep solution were purchased from Gibco, USA. Fetal bovine serum was purchased from Gemini Bioproducts, USA. Rabbit polyclonal Ki-67 and cleaved caspase-3 antibodies were purchased from Abcam, USA. Milli Q water was used for all the experiments.

[0221] Synthesized polymers were characterized by Fourier transform infrared (FT-IR) (Thermo 670 spectrometer) spectroscopy at a resolution of 4 cm-1using the KBr sampling method at room temperature (35 scans single averaged). The polymers proton nuclear magnetic resonance spectroscopy (1H NMR) was documented on AVANCE-400 or INOVA- 500 spectrometer in the CDCl3 (Aldrich) material solution with tetramethyl silane as an internal standard. Size, polydispersity index (PDI), and zeta potential of all the Gem and PTX-loaded. Empty PNPs were determined by the dynamic light scattering (DLS) method (Zetasizer Nano ZS, Malvern, UK) at 25 °C. All the samples were diluted to appropriate dilutions with water before the measurement to resolve these parameters. UV-Vis spectrometry (UV-2401 PC, USA) determined all the drugs' encapsulation efficiencies.Attorney Docket No.: 31134 / 70234 / PC Transmission electron microscopy (TEM) was performed using a Tecnai G220 TEM operated at an accelerating voltage of 120 kV. TEM samples were prepared by dropping 10 μL of 0.2 mg / mL PNP suspension on the copper grid and staining them with uranyl acetate (3 wt.%). Preparation of PEG6 based RAFT macro-initiator (PEG6-CTA).

[0222] 4-Cyano-4-(phenylcarbonothioylthio) pentanoic acid (0.25 g, 1.06 mmol) was added into a 250 mL round-bottom (RB) flask, followed by addition of 50 mL of Dry DCM. The reaction mixture was placed in an ice-water bath and purged with dry N2 for 30 min. A solution of DCC (0.4 g, 20.01 mmol) and DMAP (0.25 g, 2.07 mmol) in 20 mL of DCM was then poured into the RB flask, and the formation of a milky white precipitate was observed within 15 min. Next, Hydroxy-PEG6-t-butyl ester (0.5 g, 1.06 mmol) in 15 mL of DCM was added dropwise into the reaction mixture for 30 min. The reaction flask was kept under vigorous stirring at room temperature for 24 h. After N, N′-dicyclohexylurea (DCU) was removed by filtration, DCM was removed under reduced pressure. The yellow residue was dissolved in DCM, and the organic layer was washed first with 2 × 50 mL of 1.0 N HCl and then with 2 × 50 mL of brine solution. Next, the organic part was dried over anhydrous Na2SO4and concentrated under vacuum. Finally, the reaction residue was precipitated with n-Hexane to get a viscous yellow liquid, PEG6-CTA (65 % yield). Preparation of 6-O-methacryloyl-1,2,3,4-tetra-O-acetatyl-D-Manno pyranoside (MAcMn).

[0223] MAcMn was synthesized according to Scheme 1. A solution of 6-TAMnP weighing 30 g (0.086 mol) was mixed with 8.70 g (0.086 mol) of Et3N in 200 mL of dry THF and placed in a 500 mL two-necked round bottom flask equipped with a magnetic stirrer. The flask was then placed in an ice bath. In a separate additional funnel, Methacryloyl chloride weighing 9.90 g (0.094 mol) was mixed with 50 mL of dry THF. The solution was then added to the mixture in the round bottom flask with constant stirring, ensuring the temperature did not exceed 0°C. The reaction mixture was stirred for 5 h after removing the ice bath and then allowed to proceed for another 12 h at room temperature. A white precipitate was formed and filtered; the supernatant was washed with cold distilled water and dried with anhydrous Na2SO4. The MAcMn was isolated as a viscous liquid by evaporating the solvent and purified by column chromatography with a hexane-to-ethyl acetate ratio of 7:3 v / v. Yield: 70%.

[0224] MAcMn was characterized by FT-IR,1H NMR,13C NMR, and mass spectroscopy and these spectra are shown in Figures 1A, 1B, 2A, and 2B. FT-IR (KBr, cm-1): 2961 (-C-H- ), 1754 (-O-C=O-), 1637 (-C=C-), 1371, 1217 (-CH2- bending), 1217 (-C-O-C-).1H NMR (CDCl3, 400 MHz): 6.20–6.12 (m, 1H), 5.85 – 5.30 (m, 2H), 5.26 (m, j = 9.0, 4.5 Hz, 2H), 5.15 (dd, j = 9.9, 3.2 Hz, 1H), 4.39 – 4.20 (m, 1H), 4.20 – 4.02 (m, 1H), 3.87 (dd, j = 9.8, 5.2,Attorney Docket No.: 31134 / 70234 / PC 2.5 Hz, 1H), 2.42 – 1.66 (m, 12H), and 1.45 –1.24 (m, 3H) ppm.13C NMR (CDCl3, 101 MHz): 169.86-168.13, 167.15, 135.79, 126.21, 90.50, 73.32, 71.19, 69.13, 66.56, 62.42, 20.58 and 17.80 ppm. ESI MS: m / z: 439 (M+Na)+. Preparation of pentaerythritol tetra bis (2-bromoisobutyrate) initiator (4-armed / Star Initiator).

[0225] The synthesis of 4-armed, or star initiators was performed according to published methods. Briefly, pentaerythritol and α-bromoisobutyrylbromide were mixed in the presence of Et3N as a base in Dry THF for 12h while warming from 0 °C to room temperature. The cured star initiator was purified by column chromatography. Yield: 72%. Mp: 77-79 ºC. ESI- MS: m / z: 755 (M+Na)+.1H NMR (CDCl3, 400 MHz): 4.34-4.28 (s, 8H), and 2.01-1.90 (s, 24H) ppm.13C NMR (CDCl3, 100 MHz): 30.62, 44.93, 55.38, 63.11, and 171.46 ppm. FT-IR (KBr, cm-1): 1732 (-C=O-), 1488 and 1392 (-O=C-O-) and 1282 (-C-O-C-). Preparation and loading of PNPs

[0226] PTX-loaded polymeric nanoparticles P(PTX) (NPs) were prepared using the nanoprecipitation process. Briefly, 10 mg of the polymer [TTP-PEG6-b-P(NVCL-Co-HEA-g- LA)] and 1 mg of paclitaxel were dissolved in 200 µL of dichloromethane (DCM). Then, the DCM solution was slowly dripped into 2 mL of an aqueous solution containing a mixture of 0.5 % DSPE-PEG2000-OMe. The mixture was magnetically stirred at room temperature overnight at 500 rpm to evaporate the organic solvent. The resulting nanoparticle solution was centrifuged at 10000 rpm for 15 min using 3.5 K MWCO Amicon filter tubes and washed thrice with water to remove unencapsulated paclitaxel. Gemcitabine-loaded P(Gem) and dual drug-loaded P(Gem+PTX) PNPs were also prepared using the same procedure. Drug- free nanoparticles (empty polymeric NPs) were also prepared similarly without adding gemcitabine and paclitaxel. DLS and TEM are used to analyze the sizes and morphology of PNPs. Encapsulation efficiency and drug loading efficiency of PNPs

[0227] The drug loading content (DLE) was determined by analysis of UV-Vis spectrometry. First, the standard curves for gemcitabine and paclitaxel ranging from 10-100 μg / mL were determined using UV-Vis spectroscopy. The absorbance was measured at 275 and 230 nm, respectively. The Gem and PTX concentrations in PNPs were calculated by comparing the absorbance of Gem and PTX in the standard calibration curve. The measurement was repeated thrice.

[0228] The encapsulation efficiency (EE %) and drug loading, i.e. therapeutic agent content (DL %), were calculated as follows:Attorney Docket No.: 31134 / 70234 / PC

[0229] EE %= (amount of therapeutic agent encapsulated / amount of therapeutic agent used for incubation) X 100%

[0230] DL %= (amount of therapeutic agent encapsulated / weight of loaded nanoparticles) X 100% In vitro stability study

[0231] To evaluate the in vitro stability of NPs, empty PNPs and Gem and PTX-loaded P(Gem+PTX) NPs were stored at 4 °C in water for up to 30 days. The hydrodynamic size of the particles was measured at regular intervals to determine the stability of the formulation. In vitro drug release studies

[0232] A dialysis method was used to evaluate the drug release efficiency of polymer nanoparticles. It is known that the concentration of GSH in the blood is minimal (1-2 μM), but it is nearly 103times higher (2-10 mM) in the cellular cytoplasm. In this experiment, 10 mg of P(Gem+PTX) NPs (containing approx.440 μg of Gem and 820 μg of PTX) was suspended in 3 mL of PBS and placed in a dialysis bag (with a molecular weight cut-off of 3.5 kDa). The bag was then immersed in 10 mL of 0.5 % Tween in PBS with 10 mM GSH or without GSH, and an orbital shaker was set at 100 rpm, maintained at 37 ± 0.5 °C for 24 hrs. Every 1 h, 1 mL of dialysate was collected and replaced with an equal volume of pre-warmed PBS to maintain sink conditions. Finally, the PTX and Gem released from the NPs were analyzed using UV-Vis spectroscopy. The cumulative release % of the therapeutic agent can be calculated as follows: ^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^ (%) = (^^^^^^^^ℎ^^ ^^^^ ^^^^^^^^^^^^^^^^ ^^^^^^^^ ^^^^ ^^^^^^^^^^^^ (^^^^))(^^^^^^^^ℎ^^ ^^^^ ^^^^^^^^^^^^ ^^^^^^^^ ^^^^ ^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^ (^^^^)) × 100Cell culture

[0233] Pancreatic cancer cell lines PANC-1 and KPC were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). The cell lines were maintained at 37 °C with 5% CO2 in a humidified chamber. In vitro cellular uptake of polymer nanoparticles

[0234] Cellular uptake was investigated using a confocal microscope with fluorescent Rhodamine-B-loaded targeted or without TTP-PNPs prepared in the same manner described previously in the PNP preparation section. PANC-1 and KPC cells were seeded in a chamber slide at 2 × 104cells / well. After 24 h incubation, cells were treated with rhodamine-loaded targeted and without-TTP-PNPs formulations for two-time points, 2 and 4 h. After that, the cells were washed with PBS (pH 7.4) three times and then fixed using 4 % paraformaldehyde. The nuclei of the cells were counterstained with DAPI for the last 30 min.Attorney Docket No.: 31134 / 70234 / PC The cells were imaged in a Zeiss confocal microscope (LSM 880) using blue (Alexa 405), and red (Alexa 594) channels. In vitro cytotoxicity study

[0235] The in vitro cytotoxicities of free drugs and drug-load polymeric nanoparticles were evaluated at concentrations ranging from 600 nM to 4 μM and 300 nM to 2μM with receptive PTX and Gem, respectively, for 72 h using the MTS assay. Initially, PANC-1 and KPC (5 × 103) cells were seeded in 100 μL of DMEM medium in 96-well plates and cultured overnight. The medium was then replaced with fresh medium containing different concentrations of Gem and PTX and single and dual drug-loaded polymer nanoparticles. The control group did not receive any treatment. After 72 h, cell viability was measured by incubating cells with 10 μL MTS (10 mg / mL) for 1 h at 37 °C. The absorbance at 492 nm was measured using Spectra Max i3x. The experiment was performed in triplicate.

[0236] Percentage viability was calculated as follows: Viability (%) = 100 x (A Treated – A Blank) / (A Untreated – A Blank). Calculation of the Combination Index

[0237] The combination index (CI) of administering PTX and Gem together as a therapeutic approach was evaluated by using a formula based on the IC50values from the MTS assay. The CI is used to determine the overall outcome of the treatment. When a CI is greater than 1, it indicates antagonistic behavior, a CI of 1 corresponds to additive behavior, and a CI less than 1 represents synergistic behavior.where IC50(X)= IC50 of (Gem) / P(Gem) and IC50 (Y) = IC50of (PTX) / P(PTX) are the IC50 values obtained from each drug separately. IC50 (X + Y) =IC50 of (Gem+PTX) / P(Gem+PTX) is the IC50 value of both drugs in combination. In vivo tumor-targeting evaluation

[0238] Six- to eight-week-old male SCID mice were obtained from in-house breeding and housed in the institutional animal facilities. All animal work was performed following the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines under protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). The tumor-bearing models were established by orthotopic injection of GFP / Lucifer in 2× 106PANC-1 cells to evaluate the tumor-targeting efficiency of targeted polymer nanoformulations. After three weeks, when the tumors became palpable, mice were imaged using IVIS by bioluminescence imaging. Mice having similar luciferase signals wereAttorney Docket No.: 31134 / 70234 / PC randomly divided into four groups (n=3): i) control, ii) NIR dye, iii) without-targeted PNPs (CP), iv) targeted PNPs (TP), with 1mg / kg NIR dye administered via intravenous injection (i.v). After 24 h after administration, the mice were anesthetized and imaged using IVIS. Then mice were euthanized, and major organs were collected and imaged ex vivo to validate the increased targeting efficiency. In vivo antitumor efficacy and survival study

[0239] Anti-tumor efficacies of the various PNPs were evaluated in both PANC-1 xenografts and syngeneic KPC models. Briefly, GFP-Luciferase-labeled 2 × 106-PANC-1 and KPC cells were orthotopically injected into the head of the pancreas of 8-10 weeks old female SCID and C57BL / 6J mice, respectively. After three weeks of PANC-1 tumor cell and one week of KPC cell implantation, mice were imaged using IVIS by bioluminescence imaging. Mice having similar luciferase signals were randomly divided into five groups (n=5): i) control only PNPs, ii) free drugs gemcitabine and paclitaxel (Gem+PTX), iii) poly paclitaxel [P(PTX)], iv) poly gemcitabine, [P(Gem)], and v) poly gemcitabine plus paclitaxel [P(Gem+PTX)].

[0240] Treatments were administered twice a week for three weeks via intravenous and (i.v) injection. Two days after the final treatment, the mice were euthanized, and their tumors, hearts, livers, spleens, lungs, and kidneys were harvested. The tumors were weighed and measured in volume using calipers. The formula V = ½ (a × b2) was used to calculate the tumor volumes, where a and b represent the longest and shortest diameters, respectively. Additionally, a survival study was conducted on mice with PANC-1 and KPC tumors to determine the median survival enhancement. After administering the treatments mentioned above, the IACUC endpoint for each mouse was recorded as the survival termination date. Immunohistochemistry

[0241] Tumors and organs were fixed in 10% formalin buffer at room temperature for 24 h before embedding in paraffin for sectioning. Sections were deparaffinized and exposed to hematoxylin, eosin (H&E), and Ki67 immunochemistry according to the manufacturer’s instructions (DAB 150; Millipore). Stable diaminobenzidine and hematoxylin were used as the chromogen substrate and the counterstain, respectively. Photographs of cross sections were digitalized with the Aperio AT2 slide scanner (Leica). Images were analyzed using ImageScope software (Leica). Statistical Analysis

[0242] All experiments were performed independently in quadruplicates, with statistical analysis performed with the help of GraphPad Prism 10.0.2 (GraphPad et al., USA), and theAttorney Docket No.: 31134 / 70234 / PC results were expressed as the mean ± standard deviation. Differences between groups were studied using one-way analysis of variance (ANOVA), p< 0.05 was considered as a statistical significance. Example 1 – Synthesis and Characterization of a Triblock Copolymer of the Disclosure TTP-PEG6-b-P(NVCL-co-HEA-g-LA)

[0243] A TTP-conjugated amphiphilic triblock copolymer, such as TTP-PEG6-b-P(NVCL- co-HEA-g-LA), was successfully synthesized in three steps. The polymerization of NVCL and HEA was stopped before the total conversion of monomers to retain the RAFT end- group functionality. In the first step, t-Bu-OOC-PEG6-CTC, RAFT agent was used along with AIBN as an initiator to polymerize NVCL and HEA using the RAFT method at 90 °C for 24 h to obtain t-Bu-OOC-PEG6-b-P(NVCL-co-HEA). The obtained polymer was then post- modified with lipoic acid by esterification, followed by deprotection of the t-butyl group with 10% TFA to obtain HOOC-PEG6-b-P(NVCL-co-HEA-g-LA) in the second step. In the third step, the polymer obtained in step two was used for targeted peptide conjugation (TTP- PEG6-b-P(NVCL-co-HEA-g-LA), as shown in scheme 2.

[0244] Step 1: Preparation of PEG block copolymer (t-But-OOC-PEG6-b-P(NVCL-co- HEA). A mixture of N-vinyl caprolactam (NVCL) (3 g, 21.02 mmol), 2-Hydroxyethyl acrylate (HEA) (0.7 g, 15.2 mmol), AIBN (10 mg, 1.18 mmol) and PEG6-CTA RAFT agent (80 mg, 1. 58 mmol) in 10 mL of dry 1,4-dioxane were added in 50 mL round bottom flask. The polymerization reaction mixture was degassed using freeze-pump-thaw process cycles for 3 times under nitrogen atmosphere. Subsequently, the RB flask had been sealed and rest for 24 h in a pre-heated 90 °C with stirrer. The polymerization was terminated by adding 0.5 mL of methyl alcohol (MeOH) and air exposed and then precipitated in cold ether twice and finally, the resulting PEG block copolymer namely t-But-OOC-PEG6-b-P(NVCL-Co-HEA) The colorless solids were formed and dried out at 60 °C for 48 h beneath vacuum.

[0245] Step 2: Preparation of PEG block copolymer (t-But-OOC-PEG6-b-P(NVCL-co- HEA-g-LA). Lipoic acid conjugated t-But-OOC-PEG6-b-P(NVCL-Co-HEA-g-LA) (2.77 g, 1.34 × 10-2moles), PEG block copolymer (1.66 g, 1.27 × 10-2moles) and 4- (Dimethylamino)pyridine (DMAP) (1.02 g, 8.37 × 10-3moles) were dissolved in Dichloromethane (22 mL) in a round bottom flask and cooled under stirring over ice for 3 approx.10 min. N,N′-Dicyclohexylcarbodiimide (DCC) (3.36 g, 1.62 × 10-2moles) dissolved in Dichloromethane (22 mL) was added dropwise to the mixture. The reaction mixture was cooled with ice for 30 min and then left at room temperature overnight. The crude mixture was precipitated in diethyl ether dried under reduced pressure and stored at 40 C.Attorney Docket No.: 31134 / 70234 / PC

[0246] Step 3: Preparation of Targeted Peptide conjugated PEG block copolymer (PEG6-b-P(NVCL-co-HEA-g-LA). The TP-peptide conjugated PEG- block copolymer was prepared using the Fmoc strategy- based solid phase peptide synthesis method. T Peptide coupling with Polymer (2 equiv.) using HBTU (2 equiv.), HOBt (2 equiv.) and DIPEA (4 equiv.) using HBTU (2 equiv.), HOBt (2 equiv.), and DIPEA (4 equiv.) in DMF at room temperature for 6 h. Formation of the cyclic peptide, the two cystine amino acids were coupled to form a disulfide bond. The resin-bound peptide and thallium(iii) trifluoroacetate (2 equiv.) was dissolved in 5ml of DMF and reaction was performed for 1 h, followed by DMF washing three times. The resin-bound cyclic lipo peptide was carefully washed with DCM (5 x 10 mL) and thoroughly dried. To separate the peptide from the resin and to deprotect the protecting groups from the peptide, the dried resin was treated for 2 h at 0 °C with TFA: Tri isopropyl silane (TIS) (95:5, v / v, 4 mL) and then filtered the peptide with filter paper, the excess TFA: DCM was removed by using rotary evaporation. Finally, peptide conjugated polymer was dissolved in 2 mL methanol precipitated in cold ether dropwise to get pure TT- peptide conjugated polymer, final resulted TP-Polymer dried under reduced pressure stored at 4 °C.

[0247] The successful synthesis of the targeted polymer was characterized by using1H- NMR, gel permeation chromatography (GPC) and FT-IR spectroscopy techniques. The typical1H-NMR characterization in Fig.3 (A-C) showed that all the proton signals of NVCL, HEA, LA, and TTP correspond well to their molecular structures. PNVCL group characteristic peaks proton signals at δ 4.45 (1H, -N-CH), δ 3.65 (2H, −NCH2−), 3.16 (2H, −COCH2−), 2.33-2.17 (-CH2 – of the backbone from PNVCL) and 1.74-1.51 ppm (6H, −CH2− of the caprolactam ring). The PHEA block signal at δ 4.86-461 ppm comes from the resonance of the proton of the hydroxyl group, while the signals at δ 3.97-3.77 (4H -O-CH2) δ 3.24-3.18 (- CH2- of the backbone PHEA group’s 3.7 ppm from PEG block, in the triblock copolymer, and similar peaks were observed in1H NMR spectrum of step-2 and 3 (Fig.1 B and C) along with peaks corresponding to LA at δ 3.44-3.39 (-S-CH2), δ 2.60-250 (2H, −COCH2− ) and δ 1.2-1.8 ppm (6H-CH2- protons from long chain in LA group) and targeted peptide merged with polymer protons from δ 6.80 δ 5.90-5.87, δ 4.21-4.02, δ 3.4-3.22 ppm and δ 1.38-1.25 ppm corresponding to -C-H, -N-H, and -C=O-NH protons.

[0248] The GPC traces of Step-2 (Fig.4) showed that number average molecular weight (Mn) was 1.19 × 104, and the PDI was 1.34. The FT-IR spectrum of step-1 showed characteristic strong bands in signal, 2933.98 cm-1from -C-H, 2861.35 cm-1stretch from PEG, PNVCL block 3429.16 cm-1, and 1729.96 cm-1for the stretch OH and -O-C=O group from the PHEA block, and -N-C=O ester bond cyclic amide stretch from PNVCL block centered at ~1624.13 cm-1. While comparing the FT-IR spectrum of LA conjugated withAttorney Docket No.: 31134 / 70234 / PC polymer, showed in scheme Step-2 strong absorption of the carbonyl band -O-C=O 1736.90 cm–1was observed, confirming the peptide conjugation with polymer, amide carbonyl band - N-C=O 1646.11 cm-1which thus provides evidence of ester bond and amide conformation in TTP-PEG6-b-P(NVCL-co-HEA-g-LA) as shown in Fig-5 (A-C). Example 2 – Preparation and Characterization of Nanoparticles of the Disclosure

[0249] The single or dual drug-encapsulated tumor-targeted polymer formulations were prepared using a modified nanoprecipitation described in the methods. The hydrodynamic diameters, zeta potentials, and morphology of empty and drug-loaded polymer formulations were measured using dynamic light scattering (DLS) and transmission electron microscope (TEM) techniques. Fig.6A-6H shows the average hydrodynamic diameters (Intensity %) and the morphology of PNPs. These include empty polymer nanoparticles (P) -118.08 ±1.06 nm (Fig.6A), single drug-loaded polymer nanoparticles such as P(Gem)-131.16 ±0.89 nm (Fig. 6B) and P(PTX)-140.51 ± 0.72 nm (Fig.6C), and dual drug-loaded polymer nanoparticles P(Gem+PTX)-159.82 ±1.52 nm (Fig.6D). All the PNPs have a narrow range of PDI from 0.11-0.21 and zeta potentials ranging from +17.3 ± 2.06 mV to +30.2 ± 2.2 mV. The uniform spherical morphology of empty polymer (P) and single and dual drug-loaded PNPs (∼50 nm) was observed in transmission electron micrographs (Fig.6E-H). All physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values of respective polymer formulations, were summarized in Table 1. Table 1. Formulation Name Size (nm) PDI Zeta potential (mV) Polymer (P) 118.08 ± 1.06 0.191 ± 0.010 -17.3 ± 2.06 P(Gem) 131.16 ± 0.89 0.175 ± 0.007 -22.1 ± 1.16 P(PTX) 140.51 ± 0.72 0.180 ± 0.09 -26.7 ± 3.06 P(Gem+PTX) 169.82 ± 2.52 0.231 ± 0.012 -30.2 ± 2.2

[0250] The stability of empty PNPs and P(Gem+PTX) in deionized water was measured using the DLS technique (Fig.6E). Hydrodynamic diameters of empty and drug-loaded PNPs showed no significant changes. Moreover, the selected drug encapsulation did not influence the polymer nano formulation's hydrodynamic diameter, PDI, and zeta potentials. Table 2 summarizes the total polymer and drug amounts used to formulate single and dual drug loaded PNPs and their drug loading efficiency (DLE) and encapsulation efficiency (EE) values.Attorney Docket No.: 31134 / 70234 / PC Table 2. Formulation Total Initial Concentration DLE (%) EE (%) Name Polymer of Therapeutic Agent (mg / mL) Gem PTX Polymer (P) 10.0 - - - - P(Gem) 10.0 2.0 - 5.5 ± 0.01 20.2 ± 1 P(PTX) 10.0 - 0.5 3.5 ± 0.02 81.5 ± 2 P(Gem+PTX) 10.0 2.0 0.5 5.7 ± 0.1 (Gem)22 ± 1 (Gem) 3.45 ± 0.03 80.1 ± 0.03 (PTX) (PTX)

[0251] Hydrophobic drugs such as paclitaxel EE ranged between 81.5% and 82.1% for single- and dual-drug-loaded formulations, respectively. In contrast, the EE for the hydrophilic drug Gem was less than 25% in both cases. Water-insoluble hydrophobic drugs were naturally arranged within the polymersome, while water-soluble hydrophilic drugs tended to escape from the bilayer, resulting in lower EE. Therefore, higher EE was observed for hydrophobic drugs than for hydrophilic drugs. The DLE of both single and dual drugs was less than 8% to 10%. No significant variations were observed among the single and dual drug-loaded PNPs in EE and DLE. Example 3 –In vitro Drug Release and Stability Studies of Nanoparticles of the Disclosure

[0252] This study assessed the impact of GSH on the release of Gem and PTX from TTP- PNPs at pH 7.4 and 37 °C. Fig.6F shows that the release behavior depends on the presence of GSH. Notably, in 24 h at 37°C and pH 7.4, less than 20% of the Gem and 15 % PTX were released from P(Gem+PTX), indicating that PNPs are highly stable in circulation. However, the release of both Gem and PTX from the PNPs increased to more than 85% and 90%, respectively, when the GSH concentration was 10 mM. These findings suggest that P(Gem+PTX) is stable in circulation while quickly releasing drugs after entering the cell.

[0253] Next, in vitro cellular uptake studies were performed using a Rhodamine-B loaded polymer formulation with two different time points (2h, 4h) to evaluate the targeting efficacy of the targeted PNPs. As shown in Fig.7, cellular uptake was significantly higher at both time points for the TTP-PNPs (TP) in both KPC and PANC-1 cell lines. In contrast, minimalAttorney Docket No.: 31134 / 70234 / PC cellular uptake was observed for the PNPs without TTP (CP). These results suggest the excellent targeting efficiency of TTP-PNPs.

[0254] Following uptake studies, the cytotoxic effects of the polymer nanoparticles were investigated on PANC-1 and KPC cells while delivering single or combined drugs. The impact of different concentrations of free drugs and their combinations, as well as PNPs, single and dual drug-loaded PNPs such as P(Gem), P(PTX), and P(Gem+PTX) were analyzed on the two cell lines using the MTS assay. Gem / PTX combinations with various concentrations of Gem and PTX keeping their molar ratio 1:2 (i.e.2:4, 1:2, 0.5:1, 0.25:0.5, 0.125: 0.25, 0.06: 0.125 and 0.03:0.06) were used and the IC50 value of each drug and the combination index (CI) of combination chemotherapy was calculated and is presented in Table 3. Table 3. PANC-1 KPC Formulation Name IC50 (μM) ± SD Combination IC50 (μM) ± SD Combination Index (CI) Index (CI) Gem (alone) 0.777 ± 0.241 - 1.073 ± 0.073 - PTX (alone) 3.293 ± 0.299 - 3.981 ± 0.183 - Gem+PTX (no 0.185 ± 0.018 0.25 (Gem) 0.417 ± 0.035 0.50 (Gem) polymer) (Gem) 0.51 (PTX) (Gem) 1.01 (PTX) 0.374 ± 0.036 0.845 ± 0.069 (PTX) P(Gem) 0.708 ± 0.035 - 0.264 ± 0.025 - P(PTX) 0.575 ± 0.041 - 2.719 ± 0.280 - P(Gem+PTX) 0.085 ± 0.003 0.24 (Gem) 0.084 ± 0.006 0.34 (Gem) (Gem) 0.53 (PTX) (Gem) 0.69 (PTX) 0.173 ± 0.006 0.171 ± 0.011 (PTX) (PTX)

[0255] After 72 h, all the treatment groups demonstrated concentration-dependent cytotoxicity. The combination of Gem and PTX proved more effective against PANC-1 and KPC cancer cells than the individual drugs as shown in Fig.8A and 8B. For PANC-1 cells, the IC50values were 0.185± 0.018 for Gem+PTX and 0.777± 0.241 for free Gem alone. For KPC cells, the IC50values were 0.417±0.035 for Gem+PTX and 1.037±0.073 for free GemAttorney Docket No.: 31134 / 70234 / PC alone. Similarly, for PANC-1 cells, the IC50values were 0.374 ± 0.035 for Gem+PTX and 3.293±0.299 for free PTX alone. For KPC cells, the IC50values were 0.845 ± 0.069 for Gem+PTX and 3.981±0.183 for free PTX alone. The IC50values of the Gem+PTX combination were lower than those of individual drugs.

[0256] The CI of the Gem+PTX was less than 1 in PANC-1 and less than or equal to 1 in KPC cells, indicating a moderate synergistic effect between Gem and PTX. Fig.6C and 6D show single and dual-loaded PNPs and PNPs. PNPs did not cause any significant cytotoxicity at concentrations up to 100 µg / mL, which was higher than the maximum concentrations used in the drug-loading PNPs. P(Gem+ PTX) had considerably lowered IC50 (μM) of 0.085 ± 0.003 / 0.084 ± 0.006 with respect to Gem and 0.173±0.006 / 0.171±0.011 with respect to PTX in PANC-1 / KPC cell lines compared to IC50 (μM) of P(Gem) and P(PTX). The IC50 of P(Gem+ PTX) was 2.4-fold lower in PANC-1 and 5.2-fold lower in KPC cells compared to free Gem+ PTX in both cell lines. The CI of Gem and PTX in dual drug-loaded PNPs such as P(Gem+PTX) were 0.24 and 0.34 with respect to Gem and 0.53 and 0.69 with respect to PTX in PANC-1 and KPC cell lines respectively, it was pointing to a strong synergistic effect between Gem and PTX in P(Gem+PTX) treated cells.

[0257] Combination chemotherapy has been linked to reduced systemic toxicity when it shows synergistic effects. The results from the MTS assay indicated that P(Gem+PTX) demonstrated better cytotoxicity than all control groups, such as pristine drugs and their combinations, and single drug-loaded PNPs in both KPC and PANC-1 cell lines, as shown in Figs.8(A-D) and Table 3. Example 4 –In vivo Tumor Targeting Efficacy Studies of the Nanoparticles of the Disclosure

[0258] The in vivo tumor targeting efficacy of the TTP-PNPs was investigated using NIR dye-loaded PNPs instead of Rhodamine-B to avoid the interference of tissue autofluorescence signal usually seen with Rhodamine-B. NIRdye absorbs and emits in the IR region of the spectrum, which is less absorbed by living tissue, leading to minimal autofluorescence interfering with the actual signal intensity. NIR-dye labeled TTP- PNPs (TP) and PNPs without TTP (CP) and NIR dye solution were injected via the iv route in mice bearing orthotopic PANC-1 xenografts. As shown in Fig.9 (top), the TP showed a higher tumor-specific signal than the CP and NIR dye groups at 24 h after iv injection. Furthermore, the ex vivo imaging of the tumors and significant organs also confirmed that TP had a higher tumor-specific signal than CP as shown in Fig.9 (bottom). TP formulation shows less dye accumulation in the liver compared to the other groups, suggesting less hepatotoxicity. The findings summarized in Fig.9 are consistent with the supposition that TTP-PNPs efficiently deliver anticancer agents selectively to tumors.Attorney Docket No.: 31134 / 70234 / PC

[0259] The efficacy of P(Gem+PTX) in PANC-1 xenografts was evaluated and the results are summarized in Fig.10(A-D) and Table 4. Fig.10A shows schematically the experimental plan for the tumor growth inhibition study, Fig.10B shows tumor volume, and Fig.10C shows tumor weight, respectively. Therapeutic agents were administered at a concentration of 2mg / kg for PTX and 1mg / kg for Gem, twice a week for three weeks via the i.v route. Table 4. Formulation Name Median Survival (Days)Control 32 Gem+PTX 42 P(PTX) 44 P(Gem) 45 P(Gem+PTX) 57

[0260] Tumor volumes in P(Gem+PTX) treated group (727 ± 30 mm3) were significantly smaller in comparison with Control (2433.2 ± 209.7 mm3) or P(Gem) (1382 ± 88.09 mm3) or P(PTX) (1383 ± 86.09 mm3) and free Gem + PTX (1744.6 ± 83.0mm3) groups (Fig.10B). Similar patterns of results were observed from tumor weight measurements (Fig.10C). Another set of experiments was performed to estimate the median survival of various treatment groups. A similar in vivo experimental (Fig.10A) plan was used for survival study. The P(Gem+PTX) treated group showed a significant increase in median survival rate (57 days) compared with the control group (32 days), P(Gem) (45 days), P(PTX) (44 days), and Gem+PTX (42 days) as shown in Fig.10D and summarized in Table 4.

[0261] The tumor growth inhibition and survival studies were repeated in a syngeneic KPC model developed in C57BL6 to test whether this observed effect of P(Gem+PTX) holds in another pancreatic tumor model. These results are shown in Fig 11A-11D and Table 5. Here, the same drug concentrations were administered as for the PANC-1 tumor as described in Example 3.Attorney Docket No.: 31134 / 70234 / PC Table 5. Formulation Name Median Survival (Days)Control 25 Gem+PTX 38 P(PTX) 39 P(Gem) 39 P(Gem+PTX) 51

[0262] Similar patterns of results were observed in this model. The combination therapy decreased tumor volume and weight compared to control groups (Fig 11B, C). A significant improvement in survival was observed in the P(Gem+PTX) group compared to other treatment groups as shown in Fig.11D and Table 5.

[0263] Further, the PANC-1 and KPC tumor sections were stained with hematoxylin and eosin (H&E) to analyze the tumor growth (Fig.12A, first and third row) fewer nuclei staining in P(Gem+PTX) treatment group tumor sections than P(Gem), P(PTX) and (Gem+PTX). It indicates more tumor necrosis that happened due to dual drug treatment. The tumor sections were subjected to Ki67 staining to examine the proliferation state of the tumors, as shown in (Fig.12A, second and fourth row). P(Gem+PTX) showed less Ki-67 staining compared to other controls. Additionally, the quantification results for Ki67-positive nuclei (Fig.12B, C) corroborated with H&E. The endpoint mice seemed healthier in all polymer nanoformulations treated groups, indicating the non-toxic effect of the nanoformulations in major organs.

[0264] To evaluate the impact of P(Gem+PTX) nanoformulation on other major organs, tissue sections of major organs (lung, heart, kidney, spleen, and liver) of PANC-1 tumor- bearing SCID mice were analyzed in five groups using H&E staining. The results in Fig.13 indicate that fibrosis was not detected in heart and lung samples, inflammatory reactions were not found in liver sections, and glomerular and tubular structures in kidney samples were clearly displayed. Histological analysis of major organs revealed no pathological changes in histological specimens of the P(Gem+PTX) group.

[0265] The anti-tumor effects of combination of TTP-polymeric loaded paclitaxel(P) Gemcitabine(G) and cisplatin© treatments were investigated in a pancreatic orthotopic mouse model. Mice bearing KPC orthotopic pancreatic tumors were orally administered five deafferentations of treatment: (n = 5) Control, Pristine drugs (C+G+P), P(C), P(G+P) andAttorney Docket No.: 31134 / 70234 / PC P(C+G+P). The PNPS group was used as a control. As shown in Figures 14A and 14B, targeted polymeric-loaded cisplatin, paclitaxel, and gemcitabine-treated mice significantly inhibited tumor growth compared to individual drug treatments or control mice. Throughout the experiment, Cispatin 2mg / kg, paclitaxel 1 mg / kg, and gemcitabine 2 mg / kg were used. **** denotes p < 0.0001 compared to the indicated respective group. Example 5 – Synthesis and Characterization of Additional Polymers and Respective Nanoparticles of the Disclosure

[0266] The paclitaxel encapsulated tumor-targeted polymer formulations were prepared as described in Example 1. The PDI values of all the polymer formulations were less than 0.3, suggesting the nanoparticles are monodisperse. The drug encapsulation efficiency of hydrophobic drug Paclitaxel was found range 75 and 85 % in different polymerization formulations respectively. Encapsulation efficiency (EE) of the all the polymer formulation range from 5 to 10%. (PEG6-b-P(NVCL-co-VAc)

[0267] As shown in Scheme 3, (PEG6-b-P(NVCL-co-VAc) was synthesized and characterized by1H NMR spectroscopy (Fig.15).

[0268] The hydrodynamic diameters and zeta potentials of both empty and drug-loaded polymer formulations were measured by using the dynamic light scattering (DLS) technique. As shown in Fig.16A,B the average hydrodynamic diameters (intensity%) of the empty polymer (P) was 106 nm and PTX-loaded polymer (P-PTX), was 126 nm. All the other physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values of respective polymer formulations, were summarized in Table 6. Table 6. Formulation Name Size PDI Zeta potential DLE EE (nm) (mV) (%) (%) PEG6-b-P(NVCL-co-VAc) 106 ± 3 0.212 -29.6 ± 3 - -PEG6-b-P(NVCL-co-VAc) with126 ± 2 0.218 -31.8 ± 2 75 6.3PTX

[0269] The cytotoxic effects of the polymeric nanoparticles were also investigated on PANC-1 and KPC cells while delivering blank nanoparticles as well as nanoparticles loaded with PTX. As shown in Figs.17A-B, the cell viability for each cancer cell line decreased as the concentration of blank nanoparticles, PTX alone, or nanoparticles loaded with PTXAttorney Docket No.: 31134 / 70234 / PC increases. Additionally, the cell viability of the nanoparticles loaded with PTX was lower than the cell viability of the PTX or blank nanoparticles. PEG-b-P(NVCL-co- DMEAM-co-BA

[0270] As shown in Scheme 4, PEG-b-P(NVCL-co- DMEAM-co-BA was synthesized and characterized by1H NMR spectroscopy (Fig.18).

[0271] The hydrodynamic diameters and zeta potentials of both empty and drug-loaded polymer formulations were measured by using the dynamic light scattering (DLS) technique. As shown in Figs.19A-B, the average hydrodynamic diameters (intensity%) of the empty polymer (P) was 116 nm and PTX-loaded polymer (P-PTX) was 147 nm. All the other physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values of respective polymer formulations, were summarized in Table 7. Table 7. Formulation Name Size (nm) PDI Zeta potential (mV) PEG-b-P(VCL-co-DMEAM) 116 ± 3 0.185 -0.657 ± 7 PEG-b-P(VCL-co-DMEAM-co-BA) 147 ± 4 0.202 +35.9 ± 2 PEG-b-P(AHAM-co-DMEAM)

[0272] As shown in Scheme 5, PEG-b-P(AHAM-co- DMEAM) was synthesized. The hydrodynamic diameters and zeta potentials of both empty and drug-loaded polymer formulations were measured by using the dynamic light scattering (DLS) technique. As shown in Figs.19A-B, the average hydrodynamic diameters (intensity%) of the empty polymer (P) was 116 nm and PTX-loaded polymer (P-PTX), was 165 nm. All the other physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values of respective polymer formulations, were summarized in Table 8. Table 8. Formulation Name Size (nm) PDI Zeta potential (mV) PEG-b-P(AHAM-co- 116 ± 3 0.198 -26.6 ± 1 DMEAM) PEG-b-P(AHAM-co- 165 ± 3 0.275 -28.8 ± 3 DMEAM) with PTXAttorney Docket No.: 31134 / 70234 / PC

[0273] The cytotoxic effects of the polymeric nanoparticles were also investigated on PANC-1 and KPC cells while delivering blank nanoparticles as well as nanoparticles loaded with PTX. As shown in Figs.20A-B, the cell viability for each cancer cell line decreased as the concentration of blank nanoparticles, PTX alone, or nanoparticles loaded with PTX increases. Additionally, the cell viability of the nanoparticles loaded with PTX was lower than the cell viability of the PTX or blank nanoparticles (Figs.20A-B). (PEG-S-S-PLA)

[0274] As shown in Scheme 6, (PEG-S-S-PLA) was synthesized. The hydrodynamic diameters and zeta potentials of both empty and drug-loaded polymer formulations were measured by using the dynamic light scattering (DLS) technique. As shown in Figs.22A-B, the average hydrodynamic diameters (intensity%) of the empty polymer (P) was 116 nm and PTX-loaded polymer (P-PTX), was 185 nm. All the other physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values of respective polymer formulations, were summarized in Table 9. Table 9. Formulation Name Size (nm) PDI Zeta potential (mV) DLE (%) EE (%) PEG-S-S-PLA 116 ± 2 0.197 -24.6 ± 4 - - PEG-S-S-PLA with PTX 185 ± 2 0.213 -28.8 ± 2 70 5.4

[0275] The cytotoxic effects of the polymeric nanoparticles were also investigated on PANC-1 and KPC cells while delivering blank nanoparticles as well as nanoparticles loaded with PTX. As shown in Figs.23A-B, the cell viability for each cancer cell line decreased as the concentration of blank nanoparticles, PTX alone, or nanoparticles loaded with PTX increases. Additionally, the cell viability of the nanoparticles loaded with PTX was lower than the cell viability of the PTX or blank nanoparticles (Figs.23A-B). (TP-PEG6-b-P( NVCL-co-GMA)

[0276] As shown in Scheme 7, polymer 6 was synthesized. The hydrodynamic diameters and zeta potentials of both empty and drug-loaded polymer formulations were measured by using the dynamic light scattering (DLS) technique. As shown in Figs.24A-B, the average hydrodynamic diameters (intensity%) of the empty polymer (P) was 106 nm and PTX-loaded polymer (P-PTX), was 155 nm. All the other physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values of respective polymer formulations, were summarized in Table 10.Attorney Docket No.: 31134 / 70234 / PC Table 10. Formulation Name Size PDI Zeta potential DLE EE (nm) (mV) (%) (%)106 ± 1 0.192 -4.6 ± 4 - -PEG6-b-P(NVCL-co-155 ± 4 0.210 -8.8 ± 2 80 5.1GMA)+PTX Example 6 – Synthesis and Characterization of Additional Polymers of the Disclosure

[0277] The ROS-responsive S-MnP was synthesized in three steps, as shown in Scheme 8. First, MAcMn was polymerized independently using ATRP star initiator and copper(I) chloride-HMTETA catalyst at 90°C for 30 h to obtain star-P(MAcMn). Then, the resulting acetylated star mannose-based glycopolymer was deacetylated using NaOMe in a chloroform-methanol mixture to get the hydroxyl group free star-P(MHMn). Next, the free star-P(MHMn) was conjugated with dithioketal dioic acid with a 10:1 w / w ratio to produce the final ROS-responsive star mannose-based glycopolymer (ROS-S-MnP).

[0278] Step 1: Synthesis of 4-armed or star poly(1-O-methacryl-2,3,4,6-tetra-O- acetyl-D-mannopyranoside) [Star-P(MAcMn)]. A 1.0 g (0.0024 mol) of MMn and 5.0 mL of 1,4-dioxane as a solvent were placed into a 50 mL schlock flask fitted with rubber septa and purged with nitrogen for 20 min. A 55 mg (0.00024mol) of HMTETA and 87 mg (0.00012 mol) of pentaerythritol-based tetra functional Star ATRP initiator were added to the solution through a micro-syringe. Then the mixture was degassed with nitrogen for 10 min to remove air. At last, 24 mg (0.00024 mol) of Cu(I)Cl was placed into the reaction mixture. The mixture was degassed four times using the freeze-pump-thaw procedure cycles and sealed. The reaction mixture was placed in a preheated 90 °C oil bath for 30 h with magnetic stirring. The polymerization was terminated by the addition of 0.2 mL of methanol. To remove the copper, the polymerization mixture was passed through a neutral alumina column with 1,4-dioxane as eluent. The filtrate was concentrated under reduced pressure and precipitated into methanol. The re-precipitating further purified the Star-P(MAcMn) of 1,4-dioxane polymer solution into methanol. Finally, the polymer was dried under vacuum and stored at 4°C until further use. Yield: 65%.1H NMR (CDCl3, 500 MHz): 6.06, 5.89, 5.47, 5.35, 5.25, 4.23, 4.08, 3.86, 2.19, 2.17, 2.07, 2.01, 1.77, 1.15, 1.00 and 0.86 ppm.13C NMR (CDCl3, 126 MHz): 169.61, 167.80, 89.21, 72.85, 70.17, 68.66, 68.36, 66.11, 45.15 and 20.66 ppm. FT-IR (KBr, cm-1): 2988.32 (-CH-), 1752.54 (-O-C=O-), 1438.24 and 1372.06 (-CH2- bending) and 1225.48 (-C-O-C-).Attorney Docket No.: 31134 / 70234 / PC

[0279] Step 2. Formation of Star poly(methacryl-O-Hydroxy-D-manopyranoside) [Star-P(MHMn)] via Deacetylation of Star-P(MAcMn). The acetylated Star-P(MAcMn) (600 mg) was dissolved in anhydrous methanol and chloroform (1:2) (total volume: 30 mL) and left stirring for 10 min. under a nitrogen atmosphere. A solution of NaOMe (510 mg) in anhydrous methanol (10 mL) was taken through a syringe, and the reaction mixture was stirred for 90 min. Then, methanol and chloroform were removed by vacuum evaporation and neutralized with 2M HCl solution by stirring overnight. Subsequently, the solution was passed through a basic alumina to neutralize the solution and dried by lyophilization, toprovide Star poly(methacryl-O-Hydroxy-D-manopyranoside) [Star-P(MHMn)]. Yield: 90%. 1H-NMR (D2O, 500 MHz, d): 5.96, 5.83, 5.09, 3.97, 3.84, 3.74, 3.54, 3.33, 2.01, 1.89, 1.30, and0.76 ppm. FT -IR (KBr, cm-1, n): 3416.88 (-OH-), 2930.30 (-CH-), 1715.04 (-O-C=O-), and1256 (-C-O-C-).

[0280] Step 3: Synthesis of ROS-responsive mannose-based star Glycopolymer (S- MP). A DCC / DMAP esterification method commonly known in the art was used to conjugate Dithio ketal dioic acid (ROS Responsive Group) with Star-P(MHMn) (1: 10 w / w) to form S- MP . Yield: 90%.1H-NMR (D2O, 500 MHz, d): 5.96, 5.83, 5.09, 3.97, 3.84, 3.74, 3.54, 3.33,2.01, 1.89, 1.30, and 0.76 ppm. FT -IR (KBr, cm-1): 3419.89 (-OH-), 2929.22 (-CH-), 1731.30(-O-C=O-), and 1239 (-C-O-C-).

[0281] NMR and FT-IR spectroscopy confirmed the chemical structures resulting from each polymerization step. In the1H NMR spectra of the star-P(MAcMn) monomer methacrylate double bond chemical shifts at δ 6.20 and 5.88 ppm disappeared, and new peaks appeared at δ 0.93-0.71 ppm, which correspond to the methacrylate polymer backbone. The acetylated polymers with pendant 2,3,4,6-tetra-O-acetyl signals showed integrations at δ 2.6-1.7 ppm, and the anomeric proton of the mannose appeared at δ 6.02 ppm. The remaining mannose protons appeared at δ 5.5-3.8 ppm, and small low-intensity peaks were noticed centered at δ 4.1-3.95 ppm, which corresponded to –O-CH2- groups of the star initiators as shown in Fig.25 (top). After deacetylation, the acetyl signals vanished, and fresh peaks related to -OH groups of the mannose appeared at δ 3.78–3.9 ppm, along with the remaining peaks from star-P(MAcMn) as shown in Fig.25 (middle). After the conjugation of the ROS group, the dithiol methyl protons appeared at 1.75-1.86 ppm, along with all the proton peaks from deacetylated star-P(MHMn), as shown in Fig.25 (bottom).

[0282] The13C NMR spectra shown in Figure 26 of the star-P(MAcMn) polymer was consistent with the structure as signals at 126.21 ppm corresponding to double bond carbons from monomer disappeared. The carbon peaks of the -O-C=O- group were observed at δ 168-170 ppm, and the pendant acetyl mannose anomic carbon was exhibited at δ 90.5-91.8 ppm. (Fig.26) The -CH2- (methylene) carbon signals appeared at δ 70.7 ppm.Attorney Docket No.: 31134 / 70234 / PC From1H and13C NMR spectra, the monomer conversion and the purity of the polymer was confirmed.

[0283] The acetylated Star-P(MAcMn) polymer FT-IR spectra manifested the signals for ester -O-C=O stretching at ~1752.54 cm-1, while the deacetyl polymer exhibited the -C=O signal at about 1715 cm-1. The OH stretch of deacetyl S-P(MHMn) showed the characteristic strong band at 3416.88 cm-1. Notably, the final ROS responsive star glycopolymer (ROS-S- MnP) signals are the same as S-P(MHMn) with –O-C=O- ester symmetric bond at 1731.30 cm-1shown in Figure 27A.

[0284] The average molecular Weight of S-P(MACMn) was determined using GPC in THF eluent. The average molecular Weight of S-P(MACMn) was 13597 amu with a PDI of 1.24. Additionally, the molecular weights and the percentage of pendant acetylated / deacetylated mannose of Star-P(MAcMn), Star-P(MHMn), and S-MP were calculated using1H NMR spectra with 11212 amu, 8172 amu, and 8612 amu, and 75.0%, 64%, and 52.1%, respectively, as summarized in Table 11. The molecular weights obtained from1H NMR data were consistent with those obtained from GPC. Table 11. aCal.bGPCdTGA eDSCPolymer molecularcMannoseT5 / °C T10Tm weight by Mn Đ content % / °C / oCspectrum S-P(MAcMn) 11232 13597 1.24 - 276 288 149 S-P(MHMn) 7032 - - 63.9 162 227 105 S-MP 8792 - - 51.2 201 244 117a[MTAcMn)(Fw)×(I6.2-6.0compared to I1.2-1.0)]+X(Fw); where,): MTAcMn)(Fw) methacrylate mannose monomer molecular weight, I6.2-6.0, and I1.2-1.0: intensities in1H NMR spectra at d 6.2- 6.0 (Mannose anomeric proton) and d 1.2-1.0 ppm (terminal methyl proton) and X: 731 molecular weight of terminal ends of ATRP star initiator;bTHF used as an eluent at room temperature,c=100×Dp × [M(Fw) / SP(Mn)]. Where M(Fw) is the mannose formula weight, SP Mn is the star glycopolymer molecular weight and DP is the degree of polymerization;dthermal degradation studies;efrom DSC curve on second heating.

[0285] The thermal stability of the S-P(MAcMn), S-P(MHMn), and ROS-responsive Star mannose polymer (ROS-S-MnP) were investigated by TGA and DSC under nitrogen atmosphere. The TG curves of the glycopolymers are shown in Fig.27B. Most of the threeAttorney Docket No.: 31134 / 70234 / PC star polymers exhibited three stages of degradation. S-P(MAcMn), S-P(MHMn), and S-MnP of 5% weight losses (Td5) (162-277 ºC) and 10% (Td10) (227-288 ºC) weight losses the temperatures summarized in Table 11. Among the three polymers, the S-P(MAcMn) polymer resulted in relatively more thermal stability.

[0286] Fig.24C shows the DSC curves of protected S-P(MAcMn), S-P(MHMn), andROS- S-MnP on second heating cycles under the nitrogen atmosphere. All three steps of star- mannose glycopolymer architectures exhibited melting temperatures (Tm) in the range of 97- 148 ºC as given in Table 11. All these steps resulted in star mannose polymer exhibiting only Tm values, which indicated that these star mannose polymers were in the solid state. On the other hand, the acetyl-protected S-P(MAcMn) polymer exhibited higher Tm values than S- P(MHMn) and ROS-S-MnP polymer architectures. Example 7 – Preparation and Characterization of Additional Nanoparticles of the Disclosure

[0287] The PTX-encapsulated S-MP nanoformulation was prepared using procedure described above. Through controlled polymerization of mannose moieties, nanoformulations were prepared with a composition of 35.8% hydrophobic polymer backbone, 51.2% hydrophilic and target mannose moiety, and 13% ROS responsive group conjugation through the anomeric carbon of mannose to mannose.

[0288] The hydrodynamic diameters, zeta potentials, and morphology of empty and PTX- loaded S-MP formulations were measured using dynamic light scattering (DLS) and TEM techniques as shown in Fig.28A, 28B, and 28E, and Fig.28C, 28D, respectively. and the morphology of S-MP micelles. The average hydrodynamic diameters (as Intensity %) for the S-MP nanoparticles - 98.8 ± 0.02 nm with 0.174 PDI (Fig.3A), PTX-loaded S-MP nanoparticles - 126.7 ± 0.14 nm with narrow 0.136 PDI (Fig.28B). The uniform spherical morphology of S-MP and S-MP(PTX) (∼50 nm) was observed in TEM images (Fig.28C, 28D). S-MP(PTX) in 1mM H2O2after 24h, the hydrodynamic diameter was- 507 ± 16.49 nm with 0.648 ± 0.02 broad PDI, and these micelles zeta potentials ranging from -23 to -27 ± 0.06 mV (Fig.28E,28F). All physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values with and without 1mM H2O2S-MP micelles, were summarized in Table 12.Attorney Docket No.: 31134 / 70234 / PC Table 12. Formulation Hydrodynamic Zeta Potential EE DLE PDI Name Diameter(nm) (mV) (%) (%) S-MP 98.8± 0.02 0.17± -26± 0.3 - - 0.04 S-MP(PTX) 126.7± 0.14 0.13 ±-27± 0.6 78.2±1 7.2 ±0.07 0.5 S-MP+1mM 507± 16.49 0.64 ± -23± 1 - - H2O20.08 PDI: Polydispersity Index; DLC: Drug loading content; EE: Encapsulation efficiency. Example 8 – In vitro Drug Release and Stability Studies of Nanoparticles of the Disclosure

[0289] To evaluate the in vitro stability of the PTX-encapsulated S-MP nanoformulation PTX-encapsulated S-MP nanoformulation, PTX-loaded S-MP(PTX) micelles and 1mM H2O2 containing S-MP(PTX) were stored at room temperature in PBS for up to 24h. The hydrodynamic size of the particles was measured at regular intervals to determine the formulation's stability. As shown in Figure 28G, the hydrodynamic diameters of micelles without H2O2did not show any significant changes in size and PDI. However, when 1nM H2O2was added, there was a considerable change in the hydrodynamic diameters. Without being bound by theory, this was believed to result from reactions between the ROS- responsive group and the peroxide ion, causing the micelles to break. As a result, PTX was slowly released from the micelles.

[0290] The study assessed the impact of H2O2on the release of PTX from S-MP(PTX) at pH 7.4 and 37°C. Fig.28H shows that the release behavior depends on the presence of 1mM H2O2. Notably, in 24 hours at 37°C and pH 7.4, less than 21.2% of the PTX was released from S-MP(PTX), indicating that S-MP Micelles are highly stable in circulation. However, the release of PTX from the S-MP increased to more than 90.3% when the H2O2concentration was 1 mM. These findings suggest that S-MP(PTX) may be stable in circulation while quickly releasing drugs after entering the cell. Example 9 – In vitro Tumor Targeting Efficacy Studies of the Nanoparticles of the Disclosure

[0291] The cytotoxic effects of the S-MP micellar system on PANC-1 and AsPC cells when delivering PTX were also evaluated. In vitro cellular uptake studies were performed using Rhodamine-B loaded S-GP (Rhd) micelles with three different time points (2h, 4h, and 6h) to evaluate the targeting efficacy of the star polymeric micelles. As shown in Fig 29,Attorney Docket No.: 31134 / 70234 / PC cellular uptake was significantly higher at three-time points for the S-MP(Rhd) in PANC-1 and AsPC cell lines than only free Rhd in 6h. These results indicate that because of the mannose-binding receptors, the S-MP micelles have a highly effective uptake, and this result suggests that the synthesized star micelles have a selective targeting nature. Thus, the time- dependent internalization of these micelles leads to the accumulation of antitumor drugs inside the cells, thereby improving the efficiency of chemotherapy.

[0292] The impact of different concentrations of free PTX and S-MP(PTX) micelles was investigated on both cell lines using the MTS assay at various concentrations of PTX (i.e., 20, 10, 5, 2.5, 1.25, 0.6, and 0.3 uM). After 72 h, all the treatment groups demonstrated concentration-dependent cytotoxicity. The S-MP(PTX) micelles proved more effective against PANC-1 and AsPC cancer cells than the free PTX, as shown in Fig.30A and 30B. The IC50 values of the S-GP(PTX) are lower than Free PTX. In PANC-1 cells, the IC50 value of S-MP(PTX) is 1.36 ± 0.001 and 4.83 ± 0.008 for free PTX alone. AsPC cells, the IC50 values were S-MP(PTX) 1.55 ± 0.006 and 4.77 ± 0.004 for free PTX alone. S-MP micelles did not cause significant cytotoxicity at 1mg / mL concentrations, higher than the maximum concentrations used in the PTX S-MP micelles. The IC50 of S-MP(PTX) was ~3.6 fold lower in PANC-1 and ~3.1 fold lower in AsPC cells than free PTX in both cell lines. The results from the MTS assay indicated that S-MP(PTX) demonstrated better cytotoxicity than PTX alone in both PANC-1 and AsPC cell lines, as shown in Table 13. Table 13.

[0293] A 3.1 to 3.6-fold decrease in the IC50value in both the cells were mainly attributed to the higher intracellular uptake of S-MP in the mannose receptor-overexpressed cancer cells. Without being bound by theory, a higher delivery potential of S-MP micelles and stimulative drug release to the nuclear target is understood be the main reason for the higher cytotoxic effect observed in cancer cells. Example 10 – Reactive Oxygen Species Generation and Colony Formation Assay Studies of Nanoparticles of the Disclosure

[0294] In order to test whether S-MP(PTX) micelles work against tumors by generating reactive oxygen species (ROS)PANC-1 and AsPC pancreatic cancer cells were treated with PBS, 50 μM Nac, PTX, and S-GP(PTX) using the IC50concentration for 2h. After, it was found that S-MP(PTX) significantly (p < 0.05) increased the production of ROS in both cell lines compared to the PTX-only and other control groups, as shown in Fig.30D,30E.Attorney Docket No.: 31134 / 70234 / PC

[0295] The ability of PTX and S-MP(PTX) micelles to form colonies was tested in PANC-1 and AsPC cell lines, as shown in Fig.31A, 31B. The results indicated that PTX had a slight potential to inhibit colony formations compared to the control. However, S-MP(PTX) showed a remarkable ability to control the colony formation ability of the cancer cells. The colony formation rate for S-MP(PTX) was only ~20% compared to ~55 % for PTX, indicating the superior anticancer effect of S-MP(PTX) in both cell lines shown in Fig.31C, 31D.

[0296] The effectiveness of various formulations in inducing apoptosis was analyzed quantitatively. The Panc-1 and AsPC cells were treated with the IC50concentration PTX in S-MP(PTX) and PTX formulations and incubated for 48 hours prior to staining with Annexin V and PI. Untreated control cells had more than 99% viable cells. However, treatment with PTX decreased viable cells to 90% and induced about 9% apoptosis. On the other hand, S- MP (PTX) micelles showed a significant (p < 0.05) reduction in viable cells to about 65%, with approximately 30% of the cells undergoing early and late apoptosis. The increased potential for apoptosis in S-MP micelles was attributed to the mannose receptor-mediated cellular uptake and higher accumulation of anticancer drugs in the cancer cells, which could enhance their anticancer effects in tumor cells. Example 11 – Oral co-drug delivery Studies of Hydrogel-coated Tumor-targeted polymer nanoparticles (PNPs) of the disclosure

[0297] The in vivo tumor targeting efficacy of the TTP-PNPs was investigated in an KPC orthotopic pancreatic cancer model using NIR dye-loaded hydrogel coated tumor-targeted peptide PNPs. As shown in Figs.32A–C, the TP showed a higher tumor-specific signal than the NIR dye formulation without PNPs (Con) and the NIR dye-loaded PNPs without tumor- targeted peptide (NTP) groups at 24 h after iv injection and emitted IR radiation for up to 72 h after administration.

[0298] Additional tumor growth inhibition and survival studies were conducted in an orthotopic pancreatic cancer model to test whether the effect observed for P(Gem+PTX) holds for orally administered hydrogel coated tumor-targeted peptide PNPs. Five different treatment groups of healthy SCID mice with three animals in each group, were orally administered PNPs loaded with cisplatin+Gem+PTX, Gem+PTX, and cisplatin as well as free cisplatin+Gem+PTX. These results are shown in Figs.33 and 34A–D and Table 14. Here, cispatin was administered at 1mg / Kg, Gemcitabine was administered at 4mg / kg and Paclitaxel was administered at 2mg / kg.Attorney Docket No.: 31134 / 70234 / PC Table 14. Formulation Name Median Survival (Days) Control 24 Cisplatin+Gem+PTX 32 P(Gem+PTX) 33 P(Cisplatin+Gem+PTX) – Oral 39P(Cisplatin+Gem+PTX) – I.V. 47

[0299] Similar patterns of results were observed in this model. The combination therapy decreased tumor volume and weight compared to control groups (Fig 34B, C). A significant improvement in survival was observed in both P(cisplatin+Gem+PTX) groups compared to other treatment groups as shown in Fig.34D and Table 5, with I.V. administered cisplatin+Gem+PTX demonstrating the highest median survival of 47 days.

[0300] Further studies and ex vivo imaging of the tumors were performed in an orthotopic PANC-1 mouse model and these results are shown in Fig 35A-35C. Similar patterns of results were observed in this model. In particular, the combination therapy of cisplatin+Gem+PTX decreased tumor volume and weight compared to control groups in PANC-1 mouse models (Fig 35B, C). Here, cispatin was administered at 1mg / Kg, Gemcitabine was administered at 4mg / kg and Paclitaxel was administered at 2mg / kg. Example 12 – In vivo Renca Targeting Efficacy Studies of the Nanoparticles of the Disclosure

[0301] To evaluate the in vivo tumor targeting efficacy of the TTP-PNPs with the Renca cancer cell model using rapamycin and vinorelbine. The impact of different concentrations of free drugs and their combinations, as well as PNPs, single and dual drug-loaded PNPs such as P(RV) were analyzed. Therapeutic agents were administered at a concentration of 2.5 mg / kg and 5.0 mg / kg. As shown in Figs.36A–C, tumor volumes were recorded over 14 days. As shown in Fig, 36D, the groups treated with P(RV) were significantly smaller in comparison with Control, and the group treated with PNPs at a higher concentration was shown to have the largest reduction in tumor volume. Example 12 –Neuropilin-1 (#9) and Tyrosine Kinase (Cabozantinib) Inhibitor Delivery for Targeted Glioblastoma Therapy Studies of Nanoparticles of the Disclosure

[0302] Tumor-targeted polymer formulations encapsulating Neuropilin-1 (#9) and Tyrosine Kinase (Cabozantinib) were prepared as described in Example 1. The hydrodynamicAttorney Docket No.: 31134 / 70234 / PC diameters and zeta potentials of both empty and drug-loaded polymer formulations were measured by using the dynamic light scattering (DLS) technique. As shown in Fig.37A–D the average hydrodynamic diameters (intensity%) of the empty polymer (P) was 126 nm, the Neuropilin-1 -loaded polymer P(#9), was 154 nm, the cabozantinib-loaded polymer P(Cabo), was 135 nm, and the polymer loaded with Neuropilin-1 and cabozantinib P(#9+Cabo) was 165 nm. All the other physicochemical characteristics, including size, zeta potentials, and polydispersity indices (PDI) values of respective polymer formulations, were summarized in Table 15. Table 15. Formulation Name Size (nm) PDI Zeta potential (mV) DLE (%) EE (%)TP-Polymer0.151 -1.20 ± 4.44 - -TP-P(#9) 154.3 0.172 -5.64 ± 2.28 8.2 70.2TP-P(Cabo) 135.3 0.181 -2.63 ± 2.28 3.7 60.3TP-P(#9+Cabo) 164.5 0.200 -3.14 ± 10.2 5.8 (#9)60.5 (#9) 3.0 (Cabo) 62 (Cabo)

[0303] The PDI values of all the polymer formulations were less than 0.3, suggesting the nanoparticles are monodisperse. The drug encapsulation efficiency of Neuropilin-1 was found to be 8% and 6% in different polymerization formulations respectively. The drug encapsulation efficiency of cabozantinib was found to be 4% and 3% in different polymerization formulations respectively. Encapsulation efficiency (EE) of the all the polymer formulations ranged from 60 to 70%.

[0304] The cytotoxic effects of the polymeric nanoparticles were also investigated on GBM-22 and GBM-1A cells while delivering free drugs as well as nanoparticles loaded with Neuropilin-1 P(#9), cabozantinib P(Cabo), and Neuropilin-1 with cabozantinib P(#9+Cabo). As shown in Figs.38A,B, the cell viability for each cancer cell line decreased as the concentration of free Neuropilin-1 (#9), cabozantinib P(Cabo), or Neuropilin-1 with cabozantinib P(#9+Cabo)alone. A similar pattern was observed for nanoparticles loaded with Neuropilin-1 P(#9), cabozantinib P(Cabo), and Neuropilin-1 with cabozantinib P(#9+Cabo). Further, the cell viability of the nanoparticles loaded with Neuropilin-1 and cabozantinib P(#9+Cabo) was lower than the cell viability of the free Neuropilin-1 with cabozantinib or nanoparticles loaded with Neuropilin-1 or cabozantinib alone.Attorney Docket No.: 31134 / 70234 / PC

[0305] The ability of blank nanoparticles, free Neuropilin-1 with cabozantinib, nanoparticles loaded with Neuropilin-1 or cabozantinib, and nanoparticles loaded with Neuropilin-1 and cabozantinib micelles to form colonies was tested in a GBM-22 cell line, as shown in Fig.39A. The results indicated that P(#9) and cabozantinib P(Cabo) had a slight potential to inhibit colony formations compared to the control. However, P(#9+Cabo) showed a remarkable ability to control the colony formation ability of the cancer cells. The colony formation rate for P(#9+Cabo) was only ~20% compared to ~45 % for P(#9) and P(Cabo), indicating P(#9+Cabo) exhibited a superior anticancer effect of in a GBM-22 cell line (Fig. 39B).

[0306] The in vivo tumor targeting efficacy of the TTP-PNPs was investigated using NIR dye-loaded PNPs. NIR-dye labeled TTP- PNPs (With Targeted P(Dye)) and PNPs without tumor-targeted peptide (With out Targeted P(Dye)), NIR dye solution, and blank nanoparticles were injected via the iv route in mice bearing orthotopic GBM xenografts. As shown in Fig.40 (top), the With Targeted P(Dye) group showed a higher tumor-specific signal than the With out Targeted P(Dye), NIR dye solution, or blank nanoparticle groups at 24 h after iv injection. Furthermore, the ex vivo imaging of the tumors and significant organs also confirmed that the With Targeted P(Dye) group had a higher tumor-specific signal than the With out Targeted P(Dye) group as shown in Fig.40 (bottom). The With Targeted P(Dye) group formulation shows less dye accumulation in the liver compared to the other groups, suggesting less hepatotoxicity. The findings summarized in Fig.40 are consistent with the supposition that TTP-PNPs efficiently deliver anticancer agents selectively to tumors.

[0307] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the disclosure may be apparent to those having ordinary skill in the art.

[0308] All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In the case of conflict, the present description, including definitions, will control.

[0309] Throughout the specification, where the compounds, compositions, methods, and / or processes are described as including components, steps, or materials, it is contemplated that the compounds, compositions, methods, and / or processes can also comprise, consist essentially of, or consist of any combination of the recited components or materials, unless described otherwise. Component concentrations can be expressed in terms of weight concentrations, unless specifically indicated otherwise. Combinations of components are contemplated to include homogeneous and / or heterogeneous mixtures, asAttorney Docket No.: 31134 / 70234 / PC would be understood by a person of ordinary skill in the art in view of the foregoing disclosure.

Claims

Attorney Docket No.: 31134 / 70234 / PC What is claimed is:

1. A polymeric nanoparticle for targeted delivery of one or more therapeutic agents, comprising: a polymeric material comprising a triblock copolymer having a structure according to Formula (I) or Formula (II):wherein: t, v, w, and z are each independently 6 to 500; y is 0 to 500; A is OC1-6alkylene; RBis C(O)NH–C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N(RN)2, C(O)NH– C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RBis substituted with 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O; each of RCand RC′ are C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2, C(O)NH–C1- 6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and RCis substituted with 0 to 2 substituents selected from C1-3alkyl and =O; D is –C(O)C1-6alkylene–O substituted with 0 to 2 C1-3alkyl substituents; X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then RBis the targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; each RNis independently H or C1-3alkyl; the nanoparticle has an average particle diameter in the range of 100 nm to 500 nm;Attorney Docket No.: 31134 / 70234 / PC the one or more therapeutic agents are encapsulated by the polymeric material; each therapeutic agent has a content in the range of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle; the nanoparticle has a Zeta potential in the range of -35 mV to +40 mV; and the nanoparticle has a polydispersity index in the range of 0.150 to 0.

300.

2. The nanoparticle of claim 1, wherein each therapeutic agent is independently selected from an snRNA, a peptide, a nucleic acid, a nucleotide, and a pharmaceutically active compound.

3. The nanoparticle of claims 1 or 2, wherein at least one therapeutic agent is a peptide.

4. The nanoparticle of claim 3, wherein at least one peptide is an antibody.

5. The nanoparticle of any one of the preceding claims, wherein at least one therapeutic agent is a pharmaceutically active compound.

6. The nanoparticle of claim 5, wherein the pharmaceutically active compound comprises gemcitabine , paclitaxel, cisplatin, cabozantinib, or neuropilin-1.

7. The nanoparticle of any one of the preceding claims, wherein A is OC3alkylene.

8. The nanoparticle of any one of the preceding claims, wherein each X is NRN.

9. The nanoparticle of claim 7, wherein each CAP group is a targeting moiety.

10. The nanoparticle of any one of the preceding claims, wherein each X is O.

11. The nanoparticle of claim 9, wherein each CAP group is C1-6alkyl.

12. The nanoparticle of any one of the preceding claims, wherein the END group is C6aryl.

13. The nanoparticle of any one of the preceding claims, wherein RBis C(O)NH– C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O.

14. The nanoparticle of any one of the preceding claims, wherein RBis C(O)NH– C1-6alkylene–COOH, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and substituted with 0 to 2 substituents selected from C1-3alkyl and =O.Attorney Docket No.: 31134 / 70234 / PC 15. The nanoparticle of any one of claims 1-12, wherein RBis C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl, substituted with 0 to 2 B(OH)2.

16. The nanoparticle of any one of the preceding claims, wherein RCis C(O)O– C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1- 6alkylene–N(RN)2 or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and RCis substituted with 0 to 2 substituents selected from C1-3alkyl and =O.

17. The nanoparticle of any one of the preceding claims, wherein RCis C(O)O– C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, or C(O)O–C1-6alkyl, wherein the heterocycloalkyl has 1-2 ring heteroatoms selected from N, O, and S and is substituted with 0 to 2 C1-3alkyl substituents.

18. The nanoparticle of claims 1-15, wherein RCis NHC(O)C1-6alkylene–N(RN)2 or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S and is substituted with 0 to 2 =O substituents.

19. The nanoparticle of any one of the preceding claims, wherein RC′ is C(O)O– C1-6alkylene–OC(O)–C1-6alkylene–C3-8heterocycloalkyl, having 1-2 ring heteroatoms selected from N, O, and S and is substituted with 0 to 2 =O substituents.

20. The nanoparticle of any one of the preceding claims, wherein D is –C(O)C1-3alkylene–O.

21. The nanoparticle of claim 18, wherein D is substituted with C1alkyl.

22. The nanoparticle of any one of the preceding claims, wherein each RNis independently H or C2alkyl.

23. The nanoparticle of any one of the preceding claims, wherein the polymeric material has a structure according to formula (I), wherein: t is 6; v and w are each 100 to 500; y is 100 to 500; A is OC3alkylene;Attorney Docket No.: 31134 / 70234 / PCeach CAP group is a targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl, and C0-3alkylene–CN; and each RNis H.

24. The nanoparticle of claim 22, wherein: the one or more therapeutic agents are Gemcitabine and Paclitaxel; the average particle diameter is in the range of 100 nm to 200 nm; and each therapeutic agent has a content in the range of 3 wt.% to 6 wt.%, based on the entire weight of the particle.

25. The nanoparticle of any one of the preceding claims, wherein the polymeric material comprises: t is 6; v and w are each 100 to 500; A is OC3alkylene;each CAP group is a targeting moiety; the END group is C6aryl; and each RNis H; 26. The nanoparticle of any one of the preceding claims, wherein the polymeric material comprises: t is 6; v and w are each 100 to 500; A is OC3alkylene;Attorney Docket No.: 31134 / 70234 / PCX is O; each CAP group is t-butyl; the END group is C6aryl; and each RNis H.

27. The nanoparticle of claim 25, wherein the targeting moiety is RB.

28. The nanoparticle of claim 26, wherein the targeting moiety is targeted to reactive oxygen species.

29. The nanoparticle of any one of the preceding claims, wherein the polymeric material comprises: t is 6; v and w are each 100 to 500; A is OC3alkylene;X is NRN; each CAP group is a targeting moiety; the END group is C3alkyl substituted with C1alkyl and CN; and each RNis H or C1-3alkyl.

30. The nanoparticle of any one of the preceding claims, wherein the polymeric material comprises: t is 6; z is 100 to 500; A is OC3alkylene;Attorney Docket No.: 31134 / 70234 / PCeach X is NRN; each CAP group is a targeting moiety; and each RNis H.

31. The nanoparticle of any one of the preceding claims, wherein the polymeric material comprises: v is 6; w and y are each 100 to 500; A is OC3alkylene;the CAP group is a targeting moiety; the END group is C6aryl; and each RNis H.

32. The nanoparticle of any one of the preceding claims, wherein the targeting moiety is a tumor-targeted peptide.

33. The nanoparticle of any one of the preceding claims, wherein the nanoparticle has an average particle diameter in the range of 100 nm to 300 nm.

34. The nanoparticle of any one of the preceding claims, wherein each therapeutic agent has a content in the range of 2 wt.% to 8 wt.%, based on the entire weight of the particle.

35. The nanoparticle of any one of the preceding claims, wherein the nanoparticle has a Zeta potential in the range of -35 mV to + 35 mV.

36. The nanoparticle of any one of the preceding claims, wherein the nanoparticle has a polydispersity index in the range of 0.150 to 0.

250.

37. A polymeric nanoparticle for targeted delivery of one or more therapeutic agents, comprising:Attorney Docket No.: 31134 / 70234 / PC a polymeric material comprising a triblock copolymer having a structure according to Formula (III):one or more therapeutic agents encapsulated by the polymeric material; wherein: t, v, w, and z are each independently 6 to 500; y is 0 to 500; E is a hydrophilic polymer; each of F, G and G′ are a targeting moiety, a hydrophobic polymer, or alipophilic polymer, provided that at least one hydrophobic polymer and lipophilic polymer are each present, X is NRNor O; each CAP group is a targeting moiety or C1-6alkyl, and if each CAP group is C1-6alkyl, then F is the targeting moiety; the END group is C1-6alkyl or C6-12aryl and is substituted with 0 to 2 substituents selected from C1-3alkyl and C0-3alkylene–CN; each RNis independently H or C1-3alkyl; the nanoparticle has an average particle diameter in the range of 100 nm to 500 nm; each therapeutic agent has a content in the range of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle; the nanoparticle has a Zeta potential in the range of -35 mV to +40 mV; and the nanoparticle has a polydispersity index in the range of 0.150 to 0.

300.

38. The polymeric nanoparticle of claim 39, wherein F is hydrophobic polymer and each of G and G′ are a lipophilic polymer.

39. The polymeric nanoparticle of any one of claims 39 or 40, wherein F is a targeting moiety.

40. The polymeric nanoparticle of claim 41, wherein the targeting moiety is C(O)NH–C1-6alkylene–N+(RN)2–C1-6alkylene–C6-12aryl.

41. The polymeric nanoparticle of any one of claims 39 to 42, wherein the hydrophilic polymer is a PEG polymer.

42. The polymeric nanoparticle of claim 40, wherein the hydrophilic polymer is OC1-6alkylene.Attorney Docket No.: 31134 / 70234 / PC 43. The polymeric nanoparticle of any one of claims 39 to 43, wherein the hydrophilic polymer is C(O)NH–C1-6alkylene–COOH, C(O)NH–C1-6alkylene–N(RN)2, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and F is substituted with 0 to 2 substituents selected from C1-3alkyl, B(OH)2, and =O.

44. The polymeric nanoparticle of any one of claims 39 to 44, wherein each lipophilic polymer unit is C(O)O–C1-6alkylene–[OC(O)–C1-6alkylene]0-1–C3-8heterocycloalkyl, C(O)O–C1-6alkyl, NHC(O)C1-6alkylene–N(RN)2, or C3-8heterocycloalkyl having 1-2 ring heteroatoms selected from N, O, and S, and G or G′ is substituted with 0 to 2 substituents selected from C1-3alkyl and =O.

45. A polymeric nanoparticle for targeted delivery of one or more therapeutic agents, comprising: a polymeric material comprising a triblock copolymer having a structure according to Formula (IV):one or more therapeutic agents encapsulated by the polymeric material; wherein: J is a targeting moiety, said moiety targeted to reactive oxygen species; each Q is a hydrophilic polymer and optionally comprises a targeting moiety; and M is a hydrophobic polymer having a structure according to Formula (IV’):wherein: X is halo; and each of n, m, p, and q, are independently selected from 1 to 20; and the nanoparticle has an average particle diameter in the range of 100 nm to 500 nm; each therapeutic agent has a content in the range of 1 wt.% to 10 wt.%, based on the entire weight of the nanoparticle;Attorney Docket No.: 31134 / 70234 / PC the nanoparticle has a Zeta potential in the range of -35 mV to +40 mV; the nanoparticle has a polydispersity index in the range of 0.150 to 0.300; and the hydrophilic polymer has a content in the range of 40 wt.% to 60 wt.%, based on the entire weight of the nanoparticle.

46. The nanoparticle of claim 44, wherein J is.

47. The nanoparticle of claim 44 or 45, wherein each Q is a hydrophilic polymer comprising a targeting moiety.

48. The nanoparticle of any one of claims 44-46, wherein each Q is a saccharide.

49. The nanoparticle of claim 47, wherein each Q is mannose or glucose.

50. The nanoparticle of any one of claims 44-48, wherein each of m, n, p, and q are the same.

51. The nanoparticle of any one of claims 44-48, wherein at least one of m, n, p, and q is not the same.

52. The nanoparticle of any one of claims 44-50, wherein each of m, n, p, and q are independently selected from 1-10.

53. The nanoparticle of any one of claims 44-50, wherein each of m, n, p, and q are independently selected from 11-20.

54. The nanoparticle of any one of claims 44-52, wherein X is Cl or Br.

55. The nanoparticle of any one of claims 44-53, wherein the nanoparticle has an average particle diameter in the range of 100 nm to 300 nm.

56. The nanoparticle of any one of claims 44-54, wherein each therapeutic agent has a content in the range of 2 wt.% to 8 wt.%, based on the entire weight of the particle.

57. The nanoparticle of any one of claims 44-55, wherein the nanoparticle has a Zeta potential in the range of -35 mV to + 35 mV.

58. The nanoparticle of any one of claims 44-56, wherein the nanoparticle has a polydispersity index in the range of 0.150 to 0.

250.

59. The nanoparticle of any one of claims 44-57, wherein the saccharide content in the range of 45 wt.% to 55 wt.%, based on the entire weight of the nanoparticle.

60. The nanoparticle of any one of claims 44-59, further comprising a hydrogel coating surrounding the nanoparticle.Attorney Docket No.: 31134 / 70234 / PC 61. The nanoparticle of claim 60, wherein the hydrogel comprises a functionalized sodium alginate, a modified hyaluronic acid, or an ROS responsive PEGylated hydrogel.

62. A formulation comprising the nanoparticle of any one of the preceding claims and one or more pharmaceutically acceptable excipients.

63. The formulation of claim 62, wherein the formulation is administered orally.

64. The formulation of claim 62, wherein the formulation is administered intravenously.

65. The formulation of claim 62 or 63, further comprising a hydrogel.

66. The formulation of claim 65, wherein the hydrogel comprises a functionalized sodium alginate, a modified hyaluronic acid, or an ROS responsive PEGylated hydrogel.

67. A layered drug delivery system comprising alternating ones of one or more therapeutic agent layers and one or more hydrogel layers, each therapeutic agent layer comprising a plurality of the nanoparticle of any one of claims 1 to 61.

68. The layered drug delivery system of claim 67, wherein the one or more therapeutic agent layers comprises the same one or more therapeutic agents.

69. The layered drug delivery system of claim 67, wherein the one or more therapeutic agent layers comprises different therapeutic agents.

70. The layered drug delivery system of any one of claims 67 to 69, wherein the one or more hydrogel layers comprises a functionalized sodium alginate, a modified hyaluronic acid, or an ROS responsive PEGylated hydrogel.

71. The layered drug delivery system of any one or claims 67 to 70, wherein an outermost layer of the drug delivery system is a hydrogel layer.

72. A method of treating cancer comprising administering the nanoparticle of any one of claims 1 to 61.

73. A method of treating cancer, comprising administering the formulation of any one of claims 62 to 66.

74. A method of treating cancer, comprising administering the layered drug delivery system of any one of claims 67 to 71.

75. The method of any one of claims 72 to 74, wherein the cancer is pancreatic cancer or brain cancer.

76. The method of claim 78, wherein the cancer is pancreatic ductal adenocarcinoma or glioblastoma.Attorney Docket No.: 31134 / 70234 / PC 77. Use of the nanoparticle in accordance with any one of claims 1 to 61 as a medicament for treating cancer.

78. Use of the formulation in accordance with any one of claims 62 to 66 as a medicament for treating cancer.

79. Use of layered drug delivery system in accordance with any one of claims 67 to 71 as a medicament for treating cancer.

80. The use of any one of claims 77 to 79, wherein the cancer is pancreatic cancer or brain cancer.

81. The use of claim 80, wherein the cancer is pancreatic ductal adenocarcinoma or glioblastoma.

Images