Targeted Nanoparticles for Therapy

Abstract

The formulation comprises nanostructures formed from the self-assembly of a plurality of amphiphilic polymers comprising cationic groups. Each nanostructure comprises an application agent on the nanostructure or further added to the nanostructure. The application agent comprises a targeting agent having a negative charge.

Description

[Technical field]

[0001] [Government aid] This invention was made with government support under grant numbers R01CA223788 and CA219399 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 245,810, filed September 18, 2021, and U.S. Provisional Patent Application No. 63 / 299,431, filed January 14, 2022, the disclosures of which are incorporated herein by reference. [Background technology]

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

[0004] Nanoparticles / nanostructures are effective in delivering or co-delivering various types of therapeutic agents, including, for example, small molecule drugs and nucleic acids (e.g., siRNA), to tumors. Delivery of cancer therapeutic agents via nanocarriers is based on the concept that tumor vasculature is leaky with palisade vessels of a few to a few hundred nanometers, and long-circulating nanoparticles (NPs) can selectively accumulate in tumor tissues by passive targeting mechanisms. In addition, an impaired lymphatic system results in the inability to remove extravasated NPs from tumor tissues, a concept known as the Enhanced Permeation and Retention (EPR) effect. EPR is commonly observed in many pseudogene and human xenograft tumor models, but appears to be more heterogeneous in human cancer patients. Thus, there is a need to develop nanocarriers that can target tumors other than or in addition to EPR. Summary of the Invention

[0005] In one aspect, the formulation comprises a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers having cationic groups, and a coating, application, or layer applied to the nanostructure. The nanostructure may comprise an inner hydrophobic domain and an outer hydrophilic domain. The application comprises a negatively charged targeting agent that targets a target area (e.g., a tumor) within a patient's body. The targeting agent may be selected from the group consisting of, for example, a ligand for a cell receptor, a peptide, an aptamer, a polysaccharide, and an antibody. In many embodiments, the targeting agent is a ligand for a cell receptor (e.g., a CD44 ligand).

[0006] The application agent may further include a hydrophilic polymer. The hydrophilic polymer may have a negative charge. In many embodiments, the hydrophilic polymer includes a conjugate of a negatively charged molecule or compound and a hydrophilic polymer. The negatively charged molecule conjugated to the hydrophilic polymer may be, for example, the same compound as the targeting agent.

[0007] The formulation can further include a therapeutic compound associated with the nanostructure. The therapeutic compound can be, for example, a nucleic acid. The nucleic acid can be associated with the cationic groups of the nanostructure after its formation or form a conjugate with the nanostructure via charge-charge interactions.

[0008] In many embodiments, the therapeutic compound is a hydrophobic or lipophilic therapeutic compound. The hydrophobic or lipophilic therapeutic compound can be integrated with the hydrophobic domains inside the nanostructure. The therapeutic compound can be, for example, a small molecule therapeutic compound. The therapeutic compound can have, for example, a molecular weight of less than 1 kDa. In many embodiments, the therapeutic compound is a chemotherapeutic compound.

[0009] In many embodiments, the formulation further comprises a second therapeutic compound different from the therapeutic compound, the second therapeutic compound comprising or being a nucleic acid, which may be associated with the cationic group of the amphiphilic polymer of the nanostructure, as described above.

[0010] The nucleic acid may, for example, comprise or be RNA or DNA, hi many embodiments, the nucleic acid is a gene or an siRNA.

[0011] As used herein, the term "cationic group" refers to a group that is cationic in nature or that forms a cation in vivo. In many embodiments, the group that forms a cation in vivo is an amine group, wherein the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group. In many embodiments, the amine group is selected from the group consisting of metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

[0012] The hydrophilic polymer may be selected from the group consisting of, for example, polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides. In many embodiments, the hydrophilic polymer is polyethylene glycol.

[0013] The ratio of the negatively charged targeting agent to the hydrophilic polymer added to the nanostructure can be determined such that uptake of the nanostructure in one or more regions other than a region of interest is maintained at a sufficiently low level to allow interaction of the negatively charged targeting agent in the region of interest (e.g., a tumor).

[0014] In many embodiments, each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one of the cationic groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer (i.e., pendant hydrophilic polymer). The hydrophobic polymer backbone may further comprise, for example, a pendant lipid group.

[0015] In many embodiments, the hydrophobic polymer backbone is formed via free radical polymerization. The hydrophobic polymer backbone can be formed, for example, via controlled / living radical polymerization or reversible deactivation radical polymerization.

[0016] In another aspect, a method of formulating a composition includes forming a nanostructure through self-assembly of a plurality of amphiphilic polymers having cationic groups in an aqueous medium, and providing a coating, targeting agent, or layer to the nanostructure by adding a negatively charged targeting agent to the nanostructure.

[0017] In another aspect, a method of delivering a therapeutic compound to a patient comprises administering a formulation comprising nanostructures formed from the self-assembly of a plurality of amphiphilic polymers having cationic groups, each of said nanostructures comprising a coating, application agent, or layer on said nanostructure, wherein said application agent comprises a targeting agent having a negative charge.

[0018] In many embodiments, the targeting agent is a CD44 ligand. In that regard, in one aspect, the formulation comprises a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers having cationic groups. The nanostructure comprises an application agent added to the nanostructure. The application agent comprises a negatively charged CD44 ligand and a hydrophilic polymer. The hydrophilic polymer may be negatively charged. In many embodiments, the hydrophilic polymer is a conjugate of a negatively charged molecule and a hydrophilic polymer. The negatively charged molecule bound to the hydrophilic polymer may be, for example, a CD44 ligand. In many embodiments, the hydrophilic polymer is a conjugate of a hydrophilic polymer present in the coating and the CD44 ligand.

[0019] The nanostructure may further comprise an inner hydrophobic domain and an outer hydrophilic domain. The nanostructure may be, for example, a micelle. The CD44 ligand herein may comprise, for example, osteopontin, collagen, matrix metalloprotease, chondroitin sulfate, hyaluronic acid, or derivatives of such ligands (which maintain activity as CD44 ligands). In many embodiments, the CD44 ligand is chondroitin sulfate or hyaluronic acid. In many embodiments, the CD44 ligand is chondroitin sulfate.

[0020] The formulation may further include a therapeutic compound associated with the nanostructure. The therapeutic compound may include, for example, a nucleic acid added to the nanostructure before the negatively charged CD44 ligand and the hydrophilic polymeric compound are applied, which may be applied as a mixture. The therapeutic compound may include, for example, a hydrophobic or lipophilic therapeutic compound. In many embodiments, the hydrophobic or lipophilic therapeutic compound is a small molecule therapeutic compound. The small molecule therapeutic compound may have, for example, a molecular weight of less than 1 kDa.

[0021] In many embodiments, the therapeutic compound (or first therapeutic compound) is a hydrophobic or lipophilic therapeutic compound, and the formulation further comprises a second therapeutic compound different from the therapeutic compound, the second therapeutic compound comprising a nucleic acid. As noted above, the therapeutic compound (or first therapeutic compound) may be a small molecule therapeutic compound.

[0022] The nucleic acid of the formulation herein may include, for example, RNA or DNA. In many embodiments, the nucleic acid is a gene or siRNA. In many embodiments, the nucleic acid is siRNA.

[0023] The cationic group may include an intrinsically cationic group or a group that forms a cation in vivo. In many embodiments, the group that forms a cation in vivo, the cationic group, is an amine group, where the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group. The amine group may be selected from the group consisting of, for example, metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine. In many embodiments, the amine group is biguanidine.

[0024] The hydrophilic polymer may be selected from the group consisting of, for example, polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides. In many embodiments, the hydrophilic polymer is polyethylene glycol.

[0025] In many embodiments, the ratio of the negatively charged CD44 ligand to the hydrophilic polymer is, or is determined to be, such that uptake of the nanostructure in the patient's liver is maintained at a sufficiently low level to allow interaction of CD44 with the negatively charged CD44 ligand in tumors distant from the liver.

[0026] In many embodiments, each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one of the cationic groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer. The pendant hydrophilic polymer may be selected from the group consisting of, for example, polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides. In many embodiments, the pendant hydrophilic polymer is polyethylene glycol. The hydrophobic polymer backbone may further comprise a pendant lipid group.

[0027] In many embodiments, the hydrophobic polymer backbone is formed via free radical polymerization. The hydrophobic polymer backbone can be formed, for example, via reversible deactivation radical polymerization.

[0028] In another aspect, a method of formulating a composition includes forming nanostructures through self-assembly of a plurality of amphiphilic polymers containing cationic groups in an aqueous medium, and applying or forming a coating on the outside of each of the nanostructures by adding a negatively charged CD44 ligand and a hydrophilic polymer to the nanostructures. The hydrophilic polymer may be negatively charged. In many embodiments, the hydrophilic polymer comprises or is a conjugate of a negatively charged molecule and a hydrophilic polymer. The negatively charged molecule conjugated to the hydrophilic polymer may be, for example, a CD44 ligand. In many embodiments, the hydrophilic polymer is a conjugate of a hydrophilic polymer present in the coating and the CD44 ligand.

[0029] As described above, each of the nanostructures can include, for example, an inner hydrophobic domain and an outer hydrophilic domain. The nanostructures can be, for example, micelles. The CD44 ligands referred to herein can include, for example, osteopontin, collagen, matrix metalloproteases, chondroitin sulfate, hyaluronic acid, or derivatives of these ligands, where the derivatives retain activity as CD44 ligands. In many embodiments, the CD44 ligand is chondroitin sulfate or hyaluronic acid. In many embodiments, the CD44 ligand is chondroitin sulfate.

[0030] The method may further include associating a therapeutic compound with the nanostructure. The therapeutic compound may include, for example, a nucleic acid that is added to the nanostructure prior to applying the negatively charged CD44 ligand and the hydrophilic polymeric compound. The negatively charged CD44 ligand and the hydrophilic polymeric compound may be applied, for example, as a mixture. The therapeutic compound may include, for example, a hydrophobic or lipophilic therapeutic compound, or may be a hydrophobic or lipophilic therapeutic compound. In many embodiments, the hydrophobic or lipophilic therapeutic compound is a small molecule therapeutic compound. The small molecule therapeutic compound may have, for example, a molecular weight of less than 1 kDa.

[0031] In many embodiments, the therapeutic compound is a hydrophobic or lipophilic therapeutic compound and the formulation further comprises a second therapeutic compound different from the therapeutic compound, the second therapeutic compound comprising a nucleic acid. As noted above, the therapeutic compound may be a small molecule therapeutic compound.

[0032] The nucleic acid of the formulations herein may include, for example, RNA or DNA. In many embodiments, the nucleic acid is a gene or siRNA. In many embodiments, the nucleic acid is siRNA.

[0033] The cationic group may include an intrinsically cationic group or a group that forms a cation in vivo. In many embodiments, the group that forms a cation in vivo, the cationic group, is an amine group, where the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group. The amine group may be selected from the group consisting of, for example, metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine. In many embodiments, the amine group is biguanidine.

[0034] The hydrophilic polymer may be selected from the group consisting of, for example, polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides. In many embodiments, the hydrophilic polymer is polyethylene glycol.

[0035] In many embodiments, the ratio of the negatively charged CD44 ligand to the hydrophilic polymer is determined such that uptake of the nanostructure in the patient's liver is maintained at a sufficiently low level to allow interaction of CD44 with the negatively charged CD44 ligand in tumors distant from the liver.

[0036] In many embodiments, each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one of the cationic groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer. The pendant hydrophilic polymer may be selected from the group consisting of, for example, polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides. In many embodiments, the hydrophilic polymer is polyethylene glycol. The hydrophobic polymer backbone may further comprise a pendant lipid group.

[0037] In many embodiments, the hydrophobic polymer backbone is formed via free radical polymerization. The hydrophobic polymer backbone can be formed, for example, via reversible deactivation radical polymerization.

[0038] In a further embodiment, a method of delivering a therapeutic compound to a patient comprises administering a formulation comprising nanostructures formed from the self-assembly of a plurality of amphiphilic polymers comprising cationic groups and a coating on the exterior of each of the nanostructures. The coating comprises a CD44 ligand and a hydrophilic polymeric compound, and the therapeutic compound is integrated into the formulation. As described above, the nanostructures may further comprise an interior hydrophobic domain and an exterior hydrophilic domain. The nanostructures may be, for example, micelles. The nanostructures may be further characterized as described above and elsewhere herein.

[0039] The devices, systems, methods and compositions of the present invention, together with their attributes and attendant advantages, will be best understood in consideration of the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0040] [Figure 1] Figure 1 shows a representative example of co-delivery of TCF4 siRNA and 5-FU with OXP (FuOXP) prodrug conjugates in a mouse syngeneic CRC (CT26) model, N=5, ***P<0.01, ***P<0.001.

[0041] [Figure 2A] FIG. 2A illustrates generally one embodiment of a methodology for the formation of representative nanostructures or nanoparticles of the present nanocarriers for delivery of nucleic acids (siRNA) containing targeting agents such as the CD44 ligand chondroitin sulfate (CS) and / or small molecule therapeutic agents (FuOXP).

[0042] [Figure 2B] FIG. 2B is a table showing the size, zeta potential, drug loading content (DLC), and drug loading efficiency (DLE) of PMAOB / FuOXP mixed micelles at various carrier / drug ratios (w / w).

[0043] [Figure 2C] FIG. 2C shows a gel retardation assay demonstrating that stable conjugates were formed at nitrogen (N) / phosphate (P) ratios (ratio of positively charged polymer amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups) of 1 or greater.

[0044] [Figure 2D] FIG. 2D shows the size and zeta potential of FuOXP / siRNA-colloidal micelles at different NP ratios.

[0045] [Figure 2E] FIG. 2E shows the size and zeta potential of FuOXP / siRNA-colloidal micelles at different NP / sulfate (S) ratios.

[0046] [Figure 2F] FIG. 2F shows the size and zeta potential of FuOXP / siRNA-colloidal micelles at different NP / S(CS) / S(CS-PEG) ratios.

[0047] [Figure 2G] FIG. 2G shows a study of the size change of NPs after 2 weeks in PBS, at room temperature, and after 24 hours in mouse serum (mouseserum:MS).

[0048] [Figure 2H]Figure 2H shows gel retardation studies indicating that siRNA loaded onto PMAOB-CP ​​NPs was well protected from degradation by RNase III, whereas free siRNA was completely degraded after treatment with RNase III at 37 °C for 1 h.

[0049] [Figure 3A] FIG. 3A shows one embodiment of a synthetic route to polymer PMAOB.

[0050] [Figure 3B] FIG. 3B shows the size and zeta potential study of micelle / siRNA complexes (w / o FuOXP) at different NP ratios.

[0051] [Figure 3C] FIG. 3C shows tissue distribution studies of Cy5-labeled siRNA in the liver and tumor for various CS / CS-PEG ratios.

[0052] [Figure 4A] FIG. 4A shows whole-body NIR imaging of a tumor-bearing mouse showing that the Cy5 signal was concentrated in the tumor area (subcutaneous (sc) CT26 model).

[0053] [Figure 4B] FIG. 4B shows ex vivo images of the heart, kidney, spleen, lung, liver, and tumor.

[0054] [Figure 4C] FIG. 4C shows the quantitative fluorescence intensity in the liver and tumor over a 48 hour period.

[0055] [Figure 4D] FIG. 4D shows images of Cy5-labeled siRNA in NPs and free Cy5-labeled siRNA in blood over time.

[0056] [Figure 4E]FIG. 4E shows NIR images of the heart, kidney, spleen, lung, liver, and tumor in human colon cancer (WiDr), human breast cancer (BT-474), mouse pancreatic cancer (Panc02), and mouse breast cancer (4T1.2) models.

[0057] [Figure 4F] FIG. 4F shows tissue distribution studies in an orthotopic mouse colon cancer model.

[0058] [Figure 4G] FIG. 4G shows the distribution of Cy5 siRNA in tumor sections 24 hours after iv administration of siRNA NPs.

[0059] [Figure 5A-5C] FIG. 5A shows whole-body NIR imaging showing the distribution of Cy5-labeled siRNA in tumors in wild-type (WT) and CD44 − / − mice.

[0060] Figure 5B shows WT mice and CD44 - / - Ex vivo imaging of liver and tumors in mice.

[0061] Figure 5C shows WT mice and CD44 - / - Images of Cy5-labeled siRNA in serum from mice are shown.

[0062] [Figure 5D] FIG. 5D shows the quantitative intensity of Cy5 signals in tumor and liver tissues in wild-type (WT) CD44 and CD44− / − mice.

[0063] [Figure 5E] FIG. 5E shows the quantitative intensity of Cy5 signals in blood in wild-type (WT) CD44 mice and CD44− / − mice.

[0064] [Figure 5F]FIG. 5F shows whole-body NIR imaging of WT and zombie mice, in which active transendothelial transport mechanisms are inhibited while passive targeting mechanisms such as EPR remain active.

[0065] [Figure 5G] FIG. 5G shows the quantitative intensity of Cy5 signals in tumor and liver tissues in wild-type (WT) and zombie mice.

[0066] [Figure 5H] FIG. 5H shows a study of NP uptake as a function of CS / PEG-CS ratio in mouse liver sinusoidal endothelial cells (LSECs) and human umbilical vein endothelial cells (HUVECs).

[0067] [Figure 5I] FIG. 5I shows an investigation of the role of transcytosis in tumor targeting by the NPs herein, where significant transfection was observed in CT26 plated in the lower chamber of the transwell when Cy5 siRNA NPs were applied to HUVECs in the upper chamber, and transfection of CT26 cells was significantly inhibited by the endocytosis inhibitor Dynasore (NP+I), indicating the effectiveness of the NPs herein in mediating transcytosis through vascular ECs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] It will be readily understood that the components of the embodiments as generally described and illustrated in the Figures herein could be arranged and designed in a wide variety of different configurations in addition to the representative embodiments described. Thus, the following more detailed description of the representative embodiments as illustrated in the Figures is not intended to limit the scope of the embodiments as claimed, but merely to illustrate representative embodiments.

[0069] References throughout this specification to "one embodiment" or "an embodiment" (or the like) mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, although "in one embodiment" or "in an embodiment" or similar phrases appear in various places throughout this specification, they are not necessarily all referring to the same embodiment.

[0070] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to provide a thorough understanding of the embodiments. However, one of ordinary skill in the relevant art will recognize that various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obfuscation.

[0071] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "therapeutic compound" includes a plurality of such therapeutic compounds known to those of skill in the art, and equivalents thereof, and a reference to a "therapeutic compound" is a reference to one or more such therapeutic compounds known to those of skill in the art, and equivalents thereof, and the like. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value and intermediate range is incorporated herein as if it were individually set forth herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contraindicated by the text.

[0072] As used herein, the term "polymer" refers to a chemical compound consisting of multiple small molecules or monomers arranged in a repeating structure to form a larger molecule. Polymers can occur naturally or can be synthetically formed. The term "polymer" includes homopolymers and copolymers. The term "copolymer" is used herein to include any polymer having two or more different monomers. Copolymers can include, for example, alternating copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, graft copolymers, and the like. Examples of polymers include, for example, polyalkylene oxides.

[0073] As used herein, the term "pendant" refers to a group or moiety attached to the backbone of a longer molecule, such as a polymer as described above. A pendant group is either (1) a short chain or low molecular weight group, or (2) a long chain or high molecular weight group, such as a polymer. A pendant group may also be referred to as a side group. A long chain or high molecular weight pendant group may also be referred to as a "pendant chain" or "side chain."

[0074] In many embodiments, the systems, formulations, methods, and compositions herein are used in the delivery and / or co-delivery of small molecule therapeutics or agents (e.g., chemotherapeutic therapeutics or agents) and / or nucleic acid-based therapeutics or agents. The amphiphilic polymers can be formed, for example, via radical polymerization to have a hydrophobic polymer backbone. The hydrophobic polymer backbone can be formed, for example, via free radical polymerization or reversible deactivation radical polymerization or RDRP (previously called controlled radical polymerization or CRP).

[0075] Reversible-Deactivation Radical Polymerization (RDRP) procedures include, for example, nitroxide-mediated polymerization (NMP), atom transfer radical polymerization (ATRP), and reversible addition-fragmentation transfer (RAFT), as well as others that have developed in the past two decades (including cobalt-mediated transfer). RDRP provides access to polymers and copolymers containing radically polymerizable / copolymerizable monomers with predefined molecular weight, composition, architecture, and narrow / controlled molecular weight distribution. Because RDRP processes can provide compositionally homogeneous and well-defined polymers with predicted molecular weight, narrow / designed molecular weight distribution, and a high degree of α- and ω-chain end functionalization, they have been the subject of much research, as reported in several reviews and ACS symposia. For example, Qiu, J.; Charleux, B.;Matyjaszewski, K., Prog. Polym. Sci. 2001, 26, 2083; Davis, KA;Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1; Matyjaszewski, K., Ed.Controlled Radical Polymerization; ACS: Washington, DC, 1998; ACS SymposiumSeries 685. Matyjaszewski, K., Ed.; Controlled / Living Radical Polymerization.Progress in ATRP, NMP, and RAFT; ACS: Washington, DC, 2000; ACS SymposiumSeries 768; and Matyjaszewski, K., Davis, TP, Eds. Handbook of Radical Polymerization; Wiley: Hoboken, The disclosure of 2002 is incorporated herein by reference.

[0076] The hydrophobic polymer backbone may be formed via radical polymerization of radically polymerizable monomers (including conventional or free radical polymerization, as well as RDRP). Such monomers may contain pendant groups prior to polymerization. Alternatively, such pendant groups may be attached after polymerization. Representative monomers for use herein include styrene, acrylic acid, methacrylic acid, acrylonitrile, vinyl monomers and their derivatives. In many embodiments, the degree of polymerization of the hydrophobic polymers herein is, for example, less than 500.

[0077] In many embodiments, the polymer further comprises a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one cationic group, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer (as described above). The pendant groups may also comprise both at least one cationic group and at least one hydrophilic polymer. In many embodiments, at least one of the first plurality of pendant groups and the second plurality of pendant groups is attached to the hydrophobic polymer backbone via a linking moiety.

[0078] As described above, the at least one cationic group may, for example, comprise an inherently cationic group or a group that forms a cation in the formulation herein and / or in vivo (e.g., an amine group that forms a cation in vivo). The amine group may be an acyclic amine group, a cyclic amine group, or a heterocyclic amine group. The at least one cationic group may, for example, be selected from the group consisting of biguanidine group, metformin group, morpholine group, piperazine group, pyrrolidine group, piperidine group, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, or creatine. In many embodiments, the at least one cationic group is selected from the group consisting of metformin group, morpholine group, piperazine group, or creatine. The cationic amine group described herein may be substituted or unsubstituted.

[0079] The pendant groups herein can be attached to the hydrophobic polymer backbone via a linking moiety that is labile, for example, under in vivo conditions (e.g., under acidic pH conditions). The labile bond can be sensitive, for example, to conditions in the target region (e.g., sensitive to acidic conditions in the region of a tumor or unstable under acidic conditions). Acid-labile bonds include, for example, carboxydimethyl maleate, hydrazine, imine, acetal, oxime, silyl ether, cis-sonityl, or other acid-labile bonds or linkages. The use of labile bonds that are sensitive to acidic conditions can be used, for example, to cleave hydrophilic polymers / oligomers in, for example, the acidic tumor environment. Examples of other suitable labile bonds include disulfide bonds, hypoxia-sensitive bonds, and glucose-sensitive bonds.

[0080] The hydrophilic oligomer or hydrophilic polymer may be selected from the group consisting of, for example, polyalkylene oxide, polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyoxazoline, polysaccharide, and polypeptide. In many embodiments, at least one hydrophilic polymer is a polyalkylene oxide. The polyalkylene oxide may be, for example, polyethylene glycol. The polyethylene glycol or other hydrophilic polymer may have, for example, a molecular weight of at least 500 Da. In many embodiments, the polyethylene glycol of these other hydrophilic polymers has a molecular weight in the range of 200 Da to 10 kDa, or in the range of 500 Da to 5 kDa.

[0081] In many embodiments, the formulation or nanocarrier formulation comprises nanostructures or nanoparticles formed from the self-assembly (in an aqueous medium) of a plurality of amphiphilic polymers comprising cationic groups. The nanostructures can, for example, comprise an inner hydrophobic domain and an outer hydrophilic domain. The nanostructures may also comprise a coating, application, or layer on the outer region or on the outer region of the nanostructure. The coating, application, or layer need not be continuous. In many embodiments, the coating, application, or layer comprises a negatively charged targeting agent. As used herein, the term "targeting agent" generally refers to an agent that actively targets an area of ​​interest, such as a tumor. The negative charge of the targeting agent provides fixation via charge-charge interactions with the cationic groups of the amphiphilic polymers forming the nanostructure. In addition to providing targeting, such negatively charged agents aid in charge neutralization / shielding of positive charges to achieve nanostructures / nanocarriers that exhibit near charge neutrality. The coating, application, or layer may further comprise a hydrophilic polymeric compound, which may comprise, for example, a negative charge to anchor the hydrophilic polymeric compound to the cationic group via charge-charge interactions. The hydrophilic polymeric compound may further provide charge neutralization and, in some embodiments, may provide a degree of shielding for targeting agents, as discussed further below.

[0082] In many embodiments, the negatively charged targeting agent includes or is a ligand for a cell receptor, a peptide, an aptamer, a polysaccharide, and an antibody. The negative charge may be intrinsic to the targeting agent or may be added thereto (e.g., via binding to a negatively charged molecule). In many representative studies, the targeting agent is a negatively charged CD44 ligand. The application may include, for example, a negatively charged CD44 ligand and a hydrophilic polymeric compound as described above. Examples of suitable CD44 ligands include osteopontin, collagen, matrix metalloproteases, chondroitin sulfate (CS), hyaluronic acid (HA), or derivatives of such ligands that retain targeting activity. As described above, the hydrophilic polymeric compound may include a negative charge. The hydrophilic polymeric compound may be formed by binding a negatively charged molecule, such as chondroitin sulfate or CS, to a hydrophilic polymer as described above. For example, in a CS-PEG conjugate, CS provides a negative charge that interacts with the positive charge associated with the cation of the amphiphilic polymer of the nanostructure via charge-charge interaction. In general, a compound with a suitable negative charge can be conjugated with a hydrophilic polymer such as PEG to immobilize the hydrophilic polymer conjugate on the nanostructure. Such compounds can be, for example, a CD44 ligand, a biological compound, a synthetic compound, etc., that has another negative charge. Alternatively, a portion of the hydrophilic polymer can be modified to include a negative charge. In many embodiments, the hydrophilic polymer compound is a conjugate of a hydrophilic polymer and a CD44 ligand present in the application.

[0083] The nanocarrier formulations herein can be used, for example, to deliver therapeutic compounds that associate or interact with the hydrophobic domains and / or cationic groups of the nanocarrier. The nanocarrier formulations of the present invention can deliver or co-deliver, for example, small molecule, hydrophobic or lipophilic therapeutic compounds or drugs and / or nucleic acids (e.g., siRNA, genes, plasmids, etc.). The incorporation of nucleic acids into nanostructures or nanoparticles formed from polymers containing cationic groups is described, for example, in published U.S. Patent Application No. 2021 / 0236645, the disclosure of which is incorporated herein by reference. The multivalent charge-charge interactions between cationic groups of amphiphilic polymer molecules and nucleic acids can serve as a simple approach to form interactive non-covalent crosslinks between the amphiphilic polymer molecules of the micelles herein.

[0084] The present nanocarrier formulations coated with CD44 ligands are highly effective in tumor targeting by both EPR and transcytosis through tumor endothelial cells. In many embodiments, such negatively charged ligands can help stabilize the micelles. Such nanocarriers have been characterized for both their biophysical properties and tumor targeting efficiency.

[0085] In addition, non-covalent interactions such as π-π stacking between amphiphiles (e.g., via inclusion of aromatic groups), hydrogen bonds, etc. π-π and hydrophobic interactions or stacking and other interactions between amphiphilic polymer groups forming nanostructures / micelles and many compounds such as drugs are disclosed, for example, in U.S. Pat. Nos. 10,172,795 and 9,855,341, and U.S. Patent Publication Nos. 2018 / 0214563 and 2021 / 0236645, the disclosures of which are incorporated herein by reference.

[0086] In many studies, the nanocarriers herein have been demonstrated to be highly effective in co-delivery of nucleic acids, such as siRNA, and drugs, such as chemotherapy drugs. Immunotherapy is one of the most rapidly developing strategies in cancer treatment. In particular, immune checkpoint blockade (ICB) using inhibitors of PD-1 and / or CTLA-4 has clearly demonstrated its therapeutic potential in the clinic. However, only a small number of patients benefit from this therapy. There is an urgent need to develop novel therapies targeting other immune checkpoints to benefit more cancer patients.

[0087] The therapeutic effect and underlying mechanism of co-delivery of a representative siRNA and a representative chemotherapeutic drug, 5-Fu / cisplatin, were investigated using this nanocarrier in various cancer models. 5-Fu and OXP are the first-line therapeutic agents for colorectal cancer (CRC) and the main therapeutic agents for pancreatic cancer (PCa) patients at various stages, including advanced or metastatic PCa, but they are accompanied by limited efficacy and systemic toxicity issues. Previously, lipid-derivatized prodrug conjugates of 5-FU and cisplatin (Fuplatin) were reported to improve antitumor activity and reduce cytotoxicity against normal cells. Since OXP is superior to cisplatin in clinical trials, a prodrug conjugate of 5-Fu and OXP (FuOXP) was also synthesized. CT26 is an isogenic CRC model that responds poorly to 5-FuOXP as well as moderately to poorly to FuOXP (data not shown). In many studies, RNA-seq of CT26 tumors was performed after treatment with FuOXP three times every 5 days, and TCF4 was one of the significantly induced genes (data not shown). For example, Figure 1 shows a representative example of co-transfection of TCF4 siRNA and FuOXP, which significantly suppressed tumor growth in a mouse pseudo-CRC (CT26) model. Transcription factor 4 (TCF4) is an oncoprotein and is involved in the oncogenesis and drug resistance of CRC. As mentioned above, mice bearing CT26 tumors received various treatments three times, once every 5 days, with siRNA at a dose of 1 mg / kg and FuOXP at a dose of 5 mg / kg. FuOXP nanoparticles (NPs) alone slightly inhibited the growth of CT26 tumors. TCF4 siRNA NPs alone had a small effect on suppressing tumor growth. However, the combination of the two significantly improved the antitumor activity. Tumor volumes were tracked once every 2 days.

[0088] In numerous embodiments herein, nanocarriers formed from a representative poly(maleic anhydride-alt-1-octadecene) or PMAO polymer (PMAOB-CP) were developed to achieve codelivery of siRNA and FuOXP (see, e.g., Figures 2A to 2H). Figure 2A illustrates the main components and steps in the development of PMAOB-CP ​​nanocarriers. PMAOB is an amphiphilic polymer that self-assembles to form micelles in aqueous solution. The lipid motif can promote interaction with cell membranes and improve transfection. It also helps to improve loading of FuOXP into the hydrophobic / lipophilic core. The biguanidine motif was designed to enhance the interaction with siRNA as a result of its highly positive nature. The synthetic route of PMAOB is shown in Scheme 1 of Figure 3A. Poly(maleic anhydride-alt-1-octadecene) or PMAO (compound 1: a commercially available polymer) was first reacted with ethylenediamine to introduce amine groups. The amine-containing PMAO polymer (compound 2) was then coupled to PEG-20 ... 2K The PMAOB (compound 3) was reacted with -NHS and dicyandiamide in sequence to introduce PEG and biguanide pendant groups, respectively. 1 The H NMR spectrum showed the respective PMAO methyl peaks (1.0 ppm-1.2 ppm), PEG methyl peaks (3.28 ppm), and methylene peaks (3.3 ppm-3.6 ppm). The PEG substitution was approximately 10%, and all other amine groups in compound 2 were derivatized with biguanide groups based on the ninhydrin assay.

[0089] PMAOB polymers readily formed micelles in PBS, with a size of 173.2 nm. FuOXP could be loaded into PMAOB micelles at a carrier / drug ratio as low as 2 / 1. Figure 2B shows the size of PMAOB / FuOXP mixed micelles with various carrier / drug ratios (w / w). PMAOB / FuOXP mixed micelles with a carrier / drug ratio of 10 / 1 were used for further complexation with siRNA.

[0090] Both drug-free and FuOXP-loaded PMAOB micelles readily formed complexes with siRNA in aqueous solution. Gel retardation assays showed that stable complexes were formed at nitrogen (N) / phosphate (P) ratios of 1 or higher (Figure 2C). Figure 2D shows the size and zeta potential of FuOXP / siRNA conjugated micelles at different NP ratios. Increasing the NP ratio from 1 / 1 to 2.5 / 1 decreased the size of the conjugates and made the zeta potential of the conjugates negative. Increasing the NP ratio to 5 / 1 increased the size of the complexes. This increase was likely due to the charge becoming neutral at this ratio, making the complexes more susceptible to aggregation. Further increase in the NP ratio led to the formation of complexes with gradually increasing zeta potential and decreasing size. At a NP ratio of 10 / 1, the resulting FuOXP / siRNA / PMAOB complexes were positively charged (17.5 mV) and had a size of 107.5 nm, smaller than the size of PMAOB micelles loaded with FuOXP alone (179.1 nm) (Figure 2D). Similar results were observed when siRNA was complexed with drug-free PMAOB micelles (see Figure 3B), indicating that the PMAOB-based carriers described herein can be used to deliver siRNA alone or co-deliver siRNA with FuOXP.

[0091] Despite the potential for delivery to the pulmonary vasculature, including lung metastases, it is well known that carriers with cationic surfaces are not suitable for systemic delivery to distant solid tumors. Therefore, in a representative study, stable FuOXP / siRNA-colloidal micelles formed at a NP / sulfate (S) ratio of 10 / 1 were surface-coated / applicationed with a mixture of representative hydrophilic compounds in the form of conjugates of chondroitin sulfate (CS) and CS-PEG. CS is a highly charged molecule, and CS / CS-PEG was used to reduce the positive charge on the surface of the resulting NPs (PMAOB-CP ​​NPs). In addition, a small amount of PEG was included to minimize nonspecific interactions with serum proteins. As shown in Figure 2E, increasing the amount of CS gradually neutralized the positive charge. At a NP / sulfate (S) ratio of 10 / 1 / 2.25, the size of the resulting NPs was 136.5 nm and slightly positively charged (4.2 mV). Under these conditions, incorporation of small amounts of CS-PEG (CS / CS-PEG ratios from 2.5 / 0.1 to 2.25 / 0.25) further neutralized / shielded the positive charges to near charge neutrality (Figure 2F). Further increasing the amount of CS-PEG led to the formation of negatively charged NPs. The NPs with CS-PEG (~120 nm) were smaller than those without CS-PEG (~140 nm) (Figure 2F).

[0092] Figure 2G shows that there was minimal change in the size of PMAOB-CP ​​NPs after 2 weeks in PBS at room temperature. No obvious change in size was observed after incubating PMAOB-CP ​​NPs in 50% mouse serum for 24 hours (Figure 2G). Figure 2H shows that free siRNA was completely degraded upon treatment with RNase III at 37°C for 1 hour. In contrast, siRNA loaded on PMAOB-CP ​​NPs was well protected from degradation by RNase III (Figure 2H). These data indicate that PMAOB-CP ​​NPs loaded with FuOXP / siRNA are likely stable in blood for a sufficient time to achieve effective tumor targeting after systemic administration.

[0093] An sc tumor model (CT26) was used for initial representative optimization studies. PMAOB-CP ​​NPs were prepared with a mixture of Cy5-labeled and unlabeled siRNA (1:1, w / w), and the in vivo distribution of labeled siRNA in tumors was examined by fluorescence microscopy 24 h after tail vein injection. As the liver is the major organ that nonselectively takes up NPs, we also investigated siRNA uptake in the liver. Figure 3C shows the tissue distribution of Cy5-siRNA after treatment with various PMAOB-CP ​​NPs prepared at a NP ratio of 10 / 1 but coated with various amounts of CS / CS-PEG, respectively. Increasing the NP / S (CS) ratio from 1 / 10 / 1 to 1 / 10 / 2.5 led to a gradual increase in the Cy5.5 signal in tumors and a concomitant decrease in the signal in the liver (Figure 3C). Further increasing the amount of CS led to a decrease in the signal in tumors and an increase in the signal in the liver (Figure 3C). Next, it was studied whether the tumor targeting efficiency of CS-coated NPs could be further improved by incorporating CS-PEG. In most studies, the amount of CS-PEG was gradually increased while keeping the NP / S(CS) ratio at 10 / 1 / 2.25. Increasing the CS / CS-PEG ratio from 2.25 / 0.2 to 2.25 / 0.5 further increased the Cy5.5 signal in the tumor but decreased the signal in the liver (Figure 3C). Further increasing the amount of CS-PEG decreased the signal in the tumor and increased the signal in the liver (Figure 3C). Subsequent studies were performed with PMAOB-CP ​​NPs prepared with a NP / S(CS) / S(CS-PEG) ratio of 10 / 1 / 2.25 / 0.5.

[0094] Figure 4A to 4C show NIR images at different times after iv injection of Cy5-siRNA-loaded PMAOB-CP ​​NPs. Whole-body imaging was performed first. Then, the tumor and major organs were harvested and ex vivo imaging was performed. The fluorescence intensity was also quantified. Whole-body imaging showed that the CY5 signal was concentrated in the tumor area (Figure 4A). The ex vivo imaging data (Figure 4B) were consistent with the results of whole-body imaging. The tumor showed the highest level of fluorescence signal. The liver also showed obvious NPs uptake, but the level of Cy5 signal in the liver was significantly lower compared with that in the tumor. Little signal was found in other organs, including the heart, kidney, spleen, and lung. Figure 4C shows that a significant amount of siRNA signal was detected in the tumor at 12 h after iv injection. The level peaked at 24 h and then slowly decreased (Figure 4C). The NPs remained in the blood significantly longer than free siRNA (Figure 4D).

[0095] Following the demonstration of effective tumor targeting of the PMAOB-CP ​​NPs herein in the scCT26 model, studies were conducted to determine whether the nanocarriers herein could also mediate selective delivery to other sc tumor models, including human colon cancer (WiDr), human breast cancer (BT-474), mouse pancreatic cancer (Panc02), and mouse breast cancer (4T1.2). Figure 4E shows that similar to what was seen in the scCT26 model, PMAOB-CP ​​NPs loaded with Cy5 siRNA were primarily concentrated in the tumor tissue in all other sc tumor models examined. Similar results were observed in an orthotopic mouse colon cancer model (Figure 4F). Figure 4G shows the distribution of Cy5 siRNA in tumor sections 24 hours after iv administration of siRNA NPs. A widespread distribution of Cy5 signal was observed in the tumor tissue, with the signal being predominantly localized in tumor cells and tumor ECs (CD31 +Both the endosomes and the nucleus of the cells appeared to have taken up the NPs. At higher magnification, the colocalization of DAPI and Cy5 was clearly visualized, indicating that the siRNA was effectively released from the endosomes and reached the nucleus after intracellular delivery. Together, these data indicate that PMAOB-CP ​​NPs can be effectively targeted to various types of cancer.

[0096] Without being limited to any mechanism, the decreased tumor uptake associated with increased PEG shielding (>0.5 CS-PEG) may indicate that CS-mediated active targeting is likely to play a role in overall tumor targeting. CS is a natural ligand for CD44, which is known to be overexpressed on both tumor cells and tumor endothelial cells (ECs). To investigate whether CD44-mediated targeting of ECs is involved in effective tumor targeting by PMAOB-CP ​​NPs, we analyzed CD44 - / - NIR imaging was also performed in mice and the results were compared with those in CD44 wild-type (WT) mice. - / - Most studies used the MC38 colon cancer model because the mice have a C57BL / 6 genetic background and are not suitable for establishing CT26 tumors. As shown in Figure 5A, Cy5 siRNA-loaded PMAOB-CP ​​NPs accumulated very effectively in tumor tissues of WT mice. Its levels in tumors were significantly higher than those in liver. However, the Cy5 signal in tumor tissues was not significantly different from that in CD44 - / - The uptake of Cy5 siRNA-loaded PMAOB-CP ​​NPs was also significantly decreased in CD44 mice (Figures 5B and 5D). - / - In mice, the Cy5 siRNA signal was reduced in the liver. - / - The expression of PMAOB-CP ​​NPs loaded with Cy5 siRNA was increased in mice (Figures 5C and 5E). These results indicate that PMAOB-CP ​​NPs loaded with Cy5 siRNA are highly stable in the blood and that CD44-mediated tumor ECs contribute to the targeting of the entire tumor. Meanwhile, CD44 in liver sinusoidal ECs (LSECs) may also contribute to the uptake of PMAOB-CP ​​NPs loaded with Cy5 siRNA in the liver.

[0097] The tumor targeting efficiency of Cy5 siRNA-loaded PMAOB-CP ​​NPs was also evaluated in zombie mice, whose systemic vasculature, including tumor vasculature, was perfused and fixed with 4% paraformaldehyde before tail vein injection of NPs. In this model, passive targeting mechanisms such as ERP are effective. As shown in Figure 5F to Figure 5G, the accumulation of Cy5 siRNA-loaded PMAOB-CP ​​NPs in tumors was significantly reduced in the zombie mouse model, indicating that both active and passive targeting mechanisms contribute to tumor-wide targeting by PMAOB-CP ​​NPs.

[0098] To further explore the respective roles of CD44 in tumor ECs and LSECs interacting with the representative PMAOB-CP ​​NPs, we examined the uptake of Cy5 siRNA-loaded PMAOB-CP ​​NPs by isolated mouse LSECs and human umbilical vein endothelial cells (HUVECs). HUVECs cultured in the absence of growth factors (basic fibroblast growth factor: bFGF) are quiescent and express low levels of CD44. On the other hand, HUVECs cultured in the presence of bFGF are activated and express high levels of CD44. As shown in Figure 5H, NPs coated with CS only (without CS-PEG) were effectively taken up by both activated HUVECs and LSLECs, with more NPs taken up by activated HUVECs (69.3% vs. 61.5%). It is also evident that quiescent HUVECs take up significantly less NPs compared to activated HUVECs (Figure 5H). Similar results were obtained for quiescent HUVECs with significantly lower CD44 expression compared to WT LSECs. - / - It was also shown for LSECs, indicating that CD44-mediated endocytosis likely plays a role in the intracellular uptake of CS-coated NPs by both tumor ECs and LSECs.

[0099] The uptake of CS-PEG decreased the cellular uptake of NPs in both activated HUVECs and LSECs in a PEG dose-dependent manner. However, CS-PEG clearly showed more effect on uptake by LSECs compared to activated HUVECs. At a CS / CS-PEG ratio of 2.5 / 0.25, the level of uptake by LSECs decreased to 39.8%, whereas it remained at 58.7% in activated HUVECs (Figure 5H). At a CS / CS-PEG ratio of 2.5 / 1.0, the uptake level of PMAOB-CP ​​NPs in both types of cells decreased to comparable levels (24.4% vs. 26.8%).

[0100] CD44 has been shown to have the ability to mediate transcytosis. To explore the potential role of transcytosis in tumor targeting by the representative PMAOB-CP ​​NPs, we performed co-culture experiments with HUVECs and CT26 cells using transwell plates. Cy5.5 after 12 h + As revealed by flow analysis of CT26 cells, application of Cy5.5-siRNA NPs to HUVECs grown in the upper chamber effectively transfected CT26 cells grown in the lower chamber (Figure 5I). Transfection was significantly inhibited by dynasore, an endocytosis inhibitor, indicating the effectiveness of our NPs in mediating transcytosis through vascular ECs.

[0101] Nanocarriers have the potential to enhance the delivery of various types of anticancer drugs, especially the co-delivery of different types of therapeutic agents. The nanocarriers are suitable for the co-delivery of, for example, siRNA and chemotherapeutic agents. In addition to enhancing the delivery of both types of therapeutic agents to the tumor, this strategy has the advantage of selectively delivering siRNA to tumor cells exposed to the chemical drug. An embodiment of this co-delivery approach is effective, for example, to antagonize the mRNA induced in situ by the co-delivered chemotherapeutic agent. Several amphiphilic polymers (POEG-st-Pmor and PMet-P(cdmPEG)) have been shown to be effective for co-forming nucleic acids (plasmids and siRNA) with hydrophobic anticancer drugs (PTX and DOX). 2K ) have been previously studied. Although these carriers are highly effective for delivery to the lung, including lung metastases, their cationic surface has limited effectiveness for selective delivery to distant solid tumors, such as sc tumors. To facilitate the co-delivery of siRNA and small molecule drugs to distant solid tumors, a new type of polymer with several novel features (PMAOB being a representative example) was developed in this study (Figure 2A). In that regard, in many embodiments, lipid motifs were introduced to promote interaction with cell membranes and improve transfection. It also serves to improve loading of a representative chemotherapeutic agent, FuOXP, into the hydrophobic / lipophilic core. In many embodiments, in vivo cationic biguanidine was introduced into the polymer to enhance the interaction with siRNA. Furthermore, in many embodiments, CS / CS-PEG 2Kwas used to coat FuOXP / siRNA co-loaded PMAOB micelles, generating PMAOB-CP ​​NPs with neutral or slightly anionic surfaces, minimizing "non-specific" uptake by RES. The data from this study demonstrated that the amount and ratio of CS / CS-PEG can be adjusted in a readily determinable manner for effective tumor targeting. NPs without CS-PEG showed limited efficacy in tumor targeting as a result of substantial "non-specific" uptake by the liver. In a representative study, increasing the CS / CS-PEG ratio from 2.5 / 0.2 to 2.5 / 0.5 reduced liver uptake and increased tumor accumulation. However, further increasing the amount of CS-PEG reduced tumor uptake. These data support the finding that CD44 - / - Together with the mouse data, this strongly indicates that CD44-mediated tumor EC targeting / metastasis plays a key role in global tumor targeting.

[0102] CD44 is known to be overexpressed on both tumor ECs and tumor cells and is highly effective in mediating transcytosis. Hyaluronic acid (HA) and chondroitin sulfate (CS) are endogenous polysaccharide ligands present in the extracellular matrix. A barrier limiting the effectiveness of HA- or CS-mediated targeting of tumor ECs is the expression of CD44 on LSECs, which are abundant and therefore remove most of the NPs in circulation. Indeed, many reported HA- and CS-coated NPs showed extensive liver uptake at levels significantly higher than that in tumors. Based on the fact that tumor ECs express higher levels of CD44 than LSECs, it was hypothesized that if NPs could reach tumor cells via EPR, their interaction with LSECs could be minimized, for example, by incorporating an appropriate amount of PEG, without significantly compromising binding to tumor ECs or tumor cells. Using an excess amount of PEG would inhibit the interaction of CS-coated NPs with tumor ECs, resulting in NPs being taken up by Kupffer cells and possibly LSECs via a CD44-independent mechanism. Based on hypothesis-driven research, a representative PMAOB-CP-based nanocarrier was developed and demonstrated high tumor targeting efficacy in multiple tumor models, including an orthotopic colon cancer model. The tumor accumulation level was significantly higher than that in the liver in all tumor models examined. More importantly, this nanocarrier was capable of tumor targeting through both EPR and transcytosis, providing potential for clinical application.

[0103] Delivery of siRNA via PMAOB-CP ​​and other polymer-based NPs leads to effective knockdown of target genes in vitro and in vivo. For example, co-delivery of siRNA can significantly reduce chemotherapy-induced upregulation of specific mRNAs. Furthermore, co-delivery of siRNA with chemotherapeutic compounds / drugs can result in significantly improved antitumor activity in many cancer models over using NPs containing only chemotherapeutic drugs. Various flow studies have demonstrated that PMAOB-CP ​​significantly reduces the expression of CD45 cells, IFNγ, and IFNγ receptors in tumor cells. +CD8 + T cells and GzmB + CD8 + Increased numbers of T cells, increased M1 / M2 ratio, and decreased Treg cells were observed, indicating a likely role for an improved tumor immune microenvironment in enhancing overall antitumor activity.

[0104] Our hypothesis-driven study led to the development of a nanocarrier that is highly effective in tumor targeting by effectively targeting tumor ECs while minimizing LSEC-mediated liver uptake. In addition to enhancing tumor delivery of nucleic acid and chemotherapeutic drugs, this strategy has the advantage of selectively delivering nucleic acid therapeutics, such as siRNA, to tumor cells exposed to chemical drugs. Thus, the co-delivery approach here may be particularly effective in antagonizing mRNA induced in situ by the co-delivered chemotherapeutic drug, for example. A representative study of co-delivery of siRNA and FuOXP led to significant improvement of the tumor microenvironment and enhanced antitumor activity. Targeting various immunological targets in combination with chemotherapy may provide novel and effective immunochemotherapeutics for the treatment of various types of cancer, including colon and pancreatic cancer, for example.

[0105] Experimental Method

[0106] material Dulbecco's modified Eagle's medium (DMEM) and trypsin-EDTA solution were purchased from Sigma-Aldrich (MO, USA). Fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Invitrogen (NY, USA). Antibodies used for flow cytometry were purchased from reputable vendors such as BioLegend and BD Biosciences.

[0107] Cells and animals All cell lines used in this study were obtained from ATCC (Manassas, VA). The CT26 mouse CRC, HT29 human CRC Panc02 mouse PCa, PANC-1 human PCa, WiDr human CRC, BT-474 human BCa, and 4T1.2 mouse BCa SC models, as well as the MC38 mouse CRC orthotopic CRC model, were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

[0108] Four- to six-week-old female C57BL / 6 mice, BALB / c mice, and B6.129(Cg)-CD44tm1Hbg / J (CD44 - / - ) mice were purchased from The Jackson Laboratory. All animals were housed under pathogen-free conditions in accordance with AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) guidelines. Experiments involving mice were performed in full compliance with institutional guidelines and approved by the University of Pittsburgh Animal Use and Care Management Advisory Committee.

[0109] RNA-seq analysis BALB / c mice bearing CT26 tumors (n=3, approximately 200 mm 3 ) were treated with FuOXP NPs via tail vein injection once every 5 days for three doses with PBS as a control. Tumors were harvested 24 hours after the last dose. Samples were sent to the Health Sciences Sequencing Core at the University of Pittsburgh for RNA extraction, library construction, and sequencing. RNA-seq data were aligned to the mouse reference genome GRCm38 using STAR. Gene expression levels were quantified, and a count expression matrix was created from the aligned reads using RSEM. Counts per million were used for further analysis.

[0110] cDNA was generated from purified RNA extracted from cultured cells, isolated tumor and stromal cells, or tumor tissues using the QuantiTect Reverse Transcription Kit (Qiagen, MD, USA) according to the manufacturer's instructions. Quantitative real-time PCR was performed using SYBE Green mix on a 7900HT Fast Realtime PCR System. Relative target mRNA levels were analyzed using the delta-delta Ct method and normalized to GAPDH.

[0111] Synthesis scheme of PMAOB PMAO (compound 1) is sold with an average Mn of 30,000 to 50,000, resulting in a polymer with about 100 to about 140 repeat units per polymer molecule. Mn is the molecular weight of the repeat unit. To synthesize derivatives of compound 1, calculations of the molar ratio of reactants were based on repeat monomer unit A with a molecular weight of 350 g / mol. 7 grams of compound 1 (20 mmol of repeat units) were added to a 250 mL glass bottle equipped with a magnetic bar and placed under nitrogen atmosphere. The polymer was dissolved in 150 mL of dry degassed DMSO. 6.67 mL of ethylenediamine (100 mmol) in 50 mL of dry degassed DMSO solution was then added to the solution. The solution was stirred at 160 °C under nitrogen for 48 hours. After the reaction, the solution was cooled to room temperature. 1 L of hydrochloric acid solution (2 mol / L) was added to the DMSO solution, and the precipitate was filtered, washed three times with water, and dried under vacuum at 50 °C (compound 2). The yield was quantitative. 392 mg of compound 2 (1 mmol of repeat unit) and 200 mg of PEG were 2K-NHS (0.1 mmol) was added to a 50 mL bottle equipped with a magnetic bar, and the solid was dissolved in 10 mL of dry DMSO and 1 mL of TEA (triethylamine). The solution was stirred at room temperature for 48 hours. After the reaction, the solution was transferred to a dialysis bag (MWCO 12,000-14,000) and dialyzed in water for 24 hours. After the reaction, the solution was transferred to a dialysis bag (MWCO 12,000-14,000) and dialyzed in water for 24 hours. After dialysis, the solution was filtered with P5 filter paper, and the filtrate was subjected to freeze drying (PEG derivative). The yield is about 10-20%. 100 mg of PEG derivative and 840 mg of dicyandiamide (10 mmol) were added to a 50 mL bottle equipped with a magnetic bar. The solid was dissolved in 10 mL of tert-BuOH. The solution was then reflected and stirred for 12 hours. After the reaction, the solution was transferred to a dialysis bag (MWCO 12,000-14,000) and dialyzed in water for 24 hours. After dialysis, the solution was subjected to freeze-drying (Compound 3: PMAOB). The yield was quantitative.

[0112] Preparation of FuOXP / siRNA-colloidized PMAOB NPs FuOXP-loaded PMAOB NPs were prepared using a film hydration method. Briefly, PMAOB polymer and FuOXP were mixed in a ratio of 10:1 (w / w) in dichloromethane. After evaporating the solvent, nanowater was added to hydrate the film, resulting in FuOXP / PMAOB NPs. siRNA diluted in nanowater was then mixed with FuOXP-loaded micelles to form FuOXP / siRNA / PMAOB complexes. After incubation with various ratios of CS / CS-PEG, CS / CS-PEG-decorated FuOXP / siRNA co-loaded PMAOB-CP ​​NPs were formed. The complexation of siRNA with PMAOB polymer was confirmed by gel retardation assay. The loading capacity (DLC) and drug loading efficiency (DLE) of FuOXP were measured by high performance liquid chromatography (HPLC). The particle size and zeta potential were measured by dynamic light scattering (DLS). In vitro drug release studies were performed by dialysis according to published protocols. FuOXP released into the dialysate was measured by HPLC. The colloidal stability of FuOXP / siRNA co-loaded NPs was investigated in PBS (with or without 50% mouse serum) by following the changes in size and surface charge. The integrity of siRNA after exposure to RNase was examined by electrophoresis.

[0113] In vitro drug release The release of FuOXP from FuOXP-loaded PMAOB-CP ​​with or without siRNA complex was examined using dialysis. Briefly, 2 mL of FuOXP / siRNA / PMAOB-CP ​​and FuOXP / PMAOB-CP ​​micelles containing 1 mg FuOXP and 10 mg PMAOB-CP ​​were placed in a dialysis bag (MWCO 3.5 kDa) and immersed in 40 mL of 0.1 M PBS solution containing 0.5% (w / v) Tween 80. The experiment was performed at 37°C and 100 rpm in an incubation shaker. At regular time intervals, 10 μL of the solution in the dialysis bag and 1 mL of the medium outside the dialysis bag were withdrawn and replaced with the same amount of fresh dialysis solution. The concentration of FuOXP was examined by HPLC. Free FuOXP was added as a control.

[0114] Whole-body near-infrared (NIR) fluorescence imaging and in vitro imaging Groups of five female BALB / c mice were each administered 5 × 10 5 CT26 cells were inoculated into the right flank. The tumor was approximately 300 mm 3 At the time of growth, mice were intravenously administered PMAOB-CP ​​NPs loaded with Cy5.5-siRNA at a concentration of 1 mg / kg. At 12, 24, 36, and 48 hours, mice were imaged with an IVIS200 system (Perkin Elmer, USA) at an excitation wavelength of 678 nm and an emission wavelength of 694 nm with an exposure time of 60 seconds. Tumors and various organs were excised for ex vivo imaging according to a previously published protocol. Blood was collected at 5 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 48 h, and 72 h, and serum samples were prepared and imaged with the IVIS200 system.

[0115] Microscopic study of tumor distribution of NPs. For in vivo tumor biodistribution studies, MC38 tumor-bearing mice (approximately 300 mm 3 Cy5.5-siRNA-loaded NPs were intravenously (i.v.) injected into the tumor. Mice were sacrificed 24 hours after injection. Tumor frozen sections were prepared, stained with Hoechst, and observed under a fluorescent microscope (BZ-X710, Japan). Blood vessels were stained with FITC-anti-CD31 antibody.

[0116] Fixation of zombies and circulation of nanoparticles. The zombie mouse assay was performed according to a previously published protocol. Mice were fixed using transcardial perfusion with TEM solution (4% formaldehyde and 0.5% glutaraldehyde in 1x PBS) for 20 min. Cy5.5-siRNA-loaded PMAOB-CP ​​NPs were then added at an siRNA concentration of 1 mg / mL and allowed to circulate. The concentration of nanoparticles was the same as that expected for 1.8 mL of blood from a control animal. Each of these nanoparticle solutions was pumped at a physiologically relevant flow rate (6 ml min ) using a peristaltic pump that varied the pressure during circulation. -1) was allowed to circulate in the fixed mice for 4 hours, after which the mice were imaged using the IVIS200 system to detect Cy5.5.

[0117] Cellular uptake Murine hepatic sinusoidal endothelial cells (LSECs) were cultured from wild-type C57BL / 6 mice and B6.129(Cg)-CD44tm1Hbg / J mice (CD44 - / - Perfused mouse livers were isolated according to previously published protocols from both WT and CD44 mice. Perfused mouse livers were excised and mashed to release cells. The cell suspension was centrifuged several times at different speeds and the suspended pellet was loaded onto a Percoll gradient. Nonparenchymal cells (NPCs) were collected from the interface of two density cushions of 25% and 50% Percoll, and Kupffer cells were removed by selective adhesion. LSECs were harvested by seeding on collagen-coated cell culture plastic dishes. WT and CD44 mice were isolated from CD44 mice. - / - Mouse LSECs and subconfluent HUVECs treated with or without growth factor (bFGF) were incubated with Cy5.5-siRNA-loaded PMAOB-CP ​​NPs at various PEG-CS ratios, and cellular uptake was examined by flow cytometry after 4 h.

[0118] Investigation of the therapeutic effect of FuOXP / siRNA-colloidal NPs In mice bearing subcutaneous CT26 tumors, BALB / c mice (n=5. Jax) were administered 5×10 5 CT26 cells were injected subcutaneously. The tumor volume was approximately 50 mm 3 At the time of reaching 100% tumor size, the mice were intravenously administered DPBS (CT), siCT NPs, siRNA NPs, FuOXP NPs, FuOXP / siCT NPs, or FuOXP / siRNA NPs three times at 5-day intervals. Tumor volume and body weight were monitored on specific days.

[0119] In subcutaneous Panc02 tumor-bearing mice, C57BL / 6 mice (n=5, Jax) were inoculated with 5×10 5 Panc02 cells were injected subcutaneously into the right flank. Tumor volumes were approximately 70 mm3 At the time of reaching 100% tumor size, the mice were intravenously administered DPBS (CT), siCT NPs, siRNA NPs, FuOXP NPs, FuOXP / siCT NPs, or FuOXP / siRNA NPs three times at 5-day intervals. Tumor volume and body weight were monitored on specific days.

[0120] Tumor-infiltrating immune cells BALB / c mice bearing CT26 tumors were administered DPBDS as a control via tail vein injection three times, once every 5 days. Tumors and spleens were harvested 24 hours after the last treatment. Tumor-infiltrating immune cells were isolated and single-cell suspensions were prepared and analyzed by flow cytometry using annexin V, CD45, CD8 (IFN-γ) and IFN-γ antibodies. + / - and granzyme B + / - ), CD4(IFN-γ + / - and granzyme B + / - ), FoxP3, and macrophages (F4 / 80 and CD206).

[0121] toxicity The body weight of mice after treatment was tracked as an index of systemic toxicity. After the end of the in vivo treatment study, blood samples were collected and ALT and AST were measured by ALT / SGPT or AST / SGPT liquid UV assay kits according to the manufacturer's protocol. Serum cytokine levels (TNF-α and IL-6) were measured using a mouse cytokine assay kit. Tumors and major organs, including heart, liver, spleen, lungs, and kidneys, were excised and fixed in PBS containing 10% formaldehyde, and then embedded in paraffin. Paraffin-embedded samples were sliced ​​at 4 μm using a HM325 rotary microtome. Tissue slices were then subjected to hematoxylin-eosin staining for histopathological examination under a Zeiss Axiostar plus Microscope (PA, USA).

[0122] statistical analysis All values ​​were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using two-tailed Student's t-test for comparisons between two groups and one-way analysis of variance (ANOVA) for comparisons between multiple groups. Results were considered statistically significant when p < 0.05.

[0123] The foregoing description and accompanying drawings set forth a number of representative embodiments presently. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope of the present specification, which is set forth by the following claims rather than the foregoing description. All changes and variations that come within the meaning and range of equivalency of the claims are embraced within their scope.

Claims

1. a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers containing cationic groups; an application agent added to the nanostructures, The application agent comprises a negatively charged CD44 ligand and a hydrophilic polymer compound. formulation.

2. The formulation according to claim 1 , wherein the hydrophilic polymer compound has a negative charge.

3. The formulation according to claim 2 , wherein the hydrophilic polymer compound comprises a conjugate of a negatively charged molecule and a hydrophilic polymer.

4. The formulation of claim 3 , wherein the negatively charged molecule attached to the hydrophilic polymer is a CD44 ligand.

5. 10. The formulation of claim 1, wherein each of the nanostructures comprises an interior hydrophobic domain and an exterior hydrophilic domain.

6. The formulation of claim 1 , wherein the CD44 ligand is osteopontin, collagen, matrix metalloproteinase, chondroitin sulfate, hyaluronic acid, or a derivative thereof.

7. The formulation of claim 6 , wherein the CD44 ligand is chondroitin sulfate.

8. 7. The formulation of claim 6, further comprising a therapeutic compound associated with the nanostructure.

9. The formulation of claim 8 , wherein the therapeutic compound comprises a nucleic acid added to the nanostructure before the negatively charged CD44 ligand and the hydrophilic polymer compound are applied.

10. 9. The formulation of claim 8, wherein the therapeutic compound comprises a hydrophobic or lipophilic therapeutic compound.

11. The formulation of claim 10 , wherein the therapeutic compound is a small molecule therapeutic compound.

12. 12. The formulation of claim 11, wherein the therapeutic compound has a molecular weight of less than 1 kDa.

13. 13. The formulation of claim 12, wherein the therapeutic compound is a chemotherapeutic compound.

14. 11. The formulation of claim 10, further comprising a second therapeutic compound different from the therapeutic compound, wherein the second therapeutic compound comprises a nucleic acid.

15. The formulation of claim 14 , wherein the therapeutic compound is a small molecule therapeutic compound.

16. 16. The formulation of claim 15, wherein the therapeutic compound has a molecular weight of less than 1 kDa.

17. 17. The formulation of claim 16, wherein the therapeutic compound is a chemotherapeutic compound.

18. 10. The formulation of claim 9, wherein the nucleic acid comprises RNA or DNA.

19. The formulation of claim 18, wherein the nucleic acid is a gene or siRNA.

20. 20. The formulation of claim 19, wherein the nucleic acid is an siRNA.

21. 2. The formulation of claim 1, wherein the cationic group comprises an intrinsically cationic group or a group that forms a cation in vivo.

22. 22. The formulation of claim 21, wherein the group that forms a cation in vivo is an amine group, and the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group.

23. 23. The formulation of claim 22, wherein the amine group is selected from the group consisting of metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

24. 4. The formulation of claim 3, wherein the hydrophilic polymer is selected from the group consisting of polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides.

25. 4. The formulation of claim 3, wherein the hydrophilic polymer is polyethylene glycol.

26. 26. A formulation described in any one of claims 1 to 25, wherein the ratio of the negatively charged CD44 ligand to the hydrophilic polymer is such that uptake of the nanostructure in the patient's liver is maintained at a sufficiently low level to allow interaction of CD44 with the negatively charged CD44 ligand in tumors distant from the liver.

27. 26. The formulation of any one of claims 1 to 25, wherein each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one cationic group, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer.

28. 28. The formulation of claim 27, wherein the hydrophobic polymer backbone further comprises pendant lipid groups.

29. 28. The formulation of claim 27, wherein the hydrophobic polymer backbone is formed via free radical polymerization.

30. 30. The formulation of claim 29, wherein the hydrophobic polymer backbone is formed via reversible deactivation radical polymerization.

31. forming a nanostructure through self-assembly of a plurality of amphiphilic polymers comprising cationic groups in an aqueous medium; adding a negatively charged CD44 ligand and a hydrophilic polymer compound to the nanostructure; Methods for formulating compositions.

32. The method according to claim 31 , wherein the hydrophilic polymeric compound has a negative charge.

33. The method of claim 32, wherein the hydrophilic polymeric compound comprises a conjugate of a negatively charged molecule and a hydrophilic polymer.

34. 34. The method of claim 33, wherein the negatively charged molecule attached to the hydrophilic polymer is a CD44 ligand.

35. 32. The method of claim 31 , wherein each of the nanostructures comprises an interior hydrophobic domain and an exterior hydrophilic domain.

36. 32. The method of claim 31, wherein the CD44 ligand is osteopontin, collagen, matrix metalloproteinase, chondroitin sulfate, hyaluronic acid, or a derivative thereof.

37. 37. The method of claim 36, wherein the CD44 ligand is chondroitin sulfate.

38. 37. The method of claim 36, further comprising combining a therapeutic compound with the nanostructure.

39. The method of claim 38, wherein the therapeutic compound comprises a nucleic acid added to the nanostructure before the negatively charged CD44 ligand and the hydrophilic polymer compound are added.

40. 39. The method of claim 38, wherein the therapeutic compound is a hydrophobic or lipophilic compound mixed with the plurality of amphiphilic polymers.

41. 41. The method of claim 40, wherein the therapeutic compound is a small molecule therapeutic compound.

42. 42. The method of claim 41, wherein the therapeutic compound has a molecular weight of less than 1 kDa.

43. 43. The method of claim 42, wherein the therapeutic compound is a chemotherapeutic compound.

44. The method of claim 40, further comprising adding a second therapeutic compound comprising a nucleic acid to the nanostructure before adding the negatively charged CD44 ligand and the hydrophilic polymer compound.

45. 45. The method of claim 44, wherein the therapeutic compound is a small molecule therapeutic compound.

46. 46. ​​The method of claim 45, wherein the therapeutic compound has a molecular weight of less than 1 kDa.

47. 47. The method of claim 46, wherein the therapeutic compound is a chemotherapeutic compound.

48. 40. The method of claim 39, wherein the nucleic acid comprises RNA or DNA.

49. 49. The method of claim 48, wherein the nucleic acid is a gene or siRNA.

50. 49. The method of claim 48, wherein the nucleic acid is an siRNA.

51. 32. The method of claim 31, wherein the cationic group comprises an intrinsically cationic group or a group that forms a cation in vivo.

52. 52. The method of claim 51, wherein the group that forms a cation in vivo is an amine group, and the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group.

53. 53. The method of claim 52, wherein the amine group is selected from the group consisting of metformin, morpholine, piperazine, pyridine, pyrrolidine, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

54. 34. The method of claim 33, wherein the hydrophilic polymer is selected from the group consisting of polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides.

55. 34. The method of claim 33, wherein the hydrophilic polymer is polyethylene glycol.

56. A method described in any one of claims 31 to 55, wherein the ratio of the negatively charged CD44 ligand to the hydrophilic polymer is such that uptake of the nanostructure in the patient's liver is maintained at a sufficiently low level to allow interaction of CD44 with the negatively charged CD44 ligand in tumors away from the liver.

57. 56. The method of any one of claims 31 to 55, wherein each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one of the cationic groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer.

58. 58. The method of claim 57, wherein the hydrophobic polymer backbone further comprises pendant lipid groups.

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

60. 60. The method of claim 59, wherein the hydrophobic polymer backbone is formed via reversible deactivation radical polymerization.

61. a nanostructure formed from the self-assembly of a plurality of amphiphilic polymers containing cationic groups; an application agent added to the nanostructures, The application agent includes a negatively charged targeting agent that targets a target area within a patient's body. formulation.

62. 62. The formulation of claim 61, wherein the negatively charged targeting agent is selected from the group consisting of a ligand for a cell receptor, a peptide, an aptamer, a polysaccharide, and an antibody.

63. 62. The formulation of claim 61, wherein the negatively charged targeting agent is a ligand for a cell receptor.

64. 62. The formulation of claim 61, wherein the application agent further comprises a hydrophilic polymer compound.

65. The formulation of claim 64, wherein the hydrophilic polymer compound has a negative charge.

66. The formulation of claim 65, wherein the hydrophilic polymer compound comprises a conjugate of a negatively charged molecule and a hydrophilic polymer.

67. 67. The formulation of claim 66, wherein the negatively charged molecule attached to the hydrophilic polymer is the same compound as the targeting agent.

68. 62. The formulation of claim 61, wherein the nanostructure comprises an inner hydrophobic domain and an outer hydrophilic domain.

69. 62. The formulation of claim 61, further comprising a therapeutic compound associated with the nanostructure.

70. 70. The formulation of claim 69, wherein the therapeutic compound comprises a nucleic acid.

71. 70. The formulation of claim 69, wherein the therapeutic compound comprises a hydrophobic or lipophilic therapeutic compound.

72. 72. The formulation of claim 71, wherein the therapeutic compound is a small molecule therapeutic compound.

73. 73. The formulation of claim 72, wherein the therapeutic compound has a molecular weight of less than 1 kDa.

74. 74. The formulation of claim 73, wherein the therapeutic compound is a chemotherapeutic compound.

75. 72. The formulation of claim 71, further comprising a second therapeutic compound different from said therapeutic compound, said second therapeutic compound comprising a nucleic acid.

76. 76. The formulation of claim 75, wherein the therapeutic compound is a small molecule therapeutic compound.

77. 77. The formulation of claim 76, wherein the therapeutic compound has a molecular weight of less than 1 kDa.

78. 78. The formulation of claim 77, wherein the therapeutic compound is a chemotherapeutic compound.

79. 71. The formulation of claim 70, wherein the nucleic acid comprises RNA or DNA.

80. 80. The formulation of claim 79, wherein the nucleic acid is a gene or siRNA.

81. 81. The formulation of claim 80, wherein the nucleic acid is an siRNA.

82. 62. The formulation of claim 61, wherein the cationic group comprises an intrinsically cationic group or a group that forms a cation in vivo.

83. 83. The formulation of claim 82, wherein the group that forms a cation in vivo is an amine group, and the amine group is an acyclic amine group, a cyclic amine group, or a heterocyclic amine group.

84. 84. The formulation of claim 83, wherein the amine group is selected from the group consisting of a metformin group, a morpholine group, a piperazine group, a pyridine group, a pyrrolidine group, piperidine, thiomorpholine, thiomorpholine oxide, thiomorpholine dioxide, imidazole, guanidine, biguanidine, or creatine.

85. 67. The formulation of claim 66, wherein the hydrophilic polymer is selected from the group consisting of polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides.

86. 67. The formulation of claim 66, wherein the hydrophilic polymer is polyethylene glycol.

87. 68. The formulation of any one of claims 64 to 67, wherein the ratio of the negatively charged targeting agent to the hydrophilic polymer compound added to the nanostructure is such that uptake of the nanostructure in one or more regions other than the target region is maintained at a sufficiently low level to allow interaction of the negatively charged targeting agent in the target region.

88. 87. The formulation of any one of claims 61 to 86, wherein each of the plurality of amphiphilic polymers comprises a hydrophobic polymer backbone, a first plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one of the cationic groups, and a second plurality of pendant groups attached to the hydrophobic polymer backbone and comprising at least one hydrophilic polymer.

89. 89. The formulation of claim 88, wherein the hydrophobic polymer backbone further comprises pendant lipid groups.

90. 89. The formulation of claim 88, wherein the hydrophobic polymer backbone is formed via free radical polymerization.

91. 91. The formulation of claim 90, wherein the hydrophobic polymer backbone is formed via reversible deactivation radical polymerization.

92. 87. The formulation of claims 61-86, wherein the targeting agent is a CD44 ligand.

93. 89. The formulation of claim 88, wherein the hydrophilic polymer of the second plurality of pendant groups is selected from the group consisting of polyalkylene oxides, polyvinyl alcohols, polyacrylic acids, polyacrylamides, polyoxazolines, polysaccharides, and polypeptides.

94. 89. The formulation of claim 88, wherein the hydrophilic polymer of the second plurality of pendant groups is polyethylene glycol.

95. 89. The formulation of claim 88, wherein the ratio of the negatively charged targeting agent to the hydrophilic polymer added to the nanostructure is such that uptake of the nanostructure in one or more regions other than the target region is maintained at a sufficiently low level to allow interaction of the negatively charged targeting agent in the target region.

96. 96. The formulation of claim 95, wherein the targeting agent is a CD44 ligand.

97. forming nanostructures through self-assembly of a plurality of amphiphilic polymers comprising cationic groups in an aqueous medium; adding a negatively charged targeting agent to the nanostructures, thereby adding an application agent to the nanostructures; Methods of formulating compositions.

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