Multi-dimensional nanoparticles

Abstract

A quaternary multi-dimensional nanoparticle (qMDNP) includes (a) a nanoparticle, of a cyclodextrin and biocompatible polymer, having a biocompatible shell protecting an internal core wherein the cyclodextrin is selected from a group consisting of (a positively charged functional group)-substituted cyclodextrin, (a negatively charged functional group)-substituted cyclodextrin, and combinations thereof, (b) a polyion adapted to stabilize the internal core containing components with various charges and (c) a therapeutic agent entrapped in the cavity of the cyclodextrin protected in the internal core of the nanoparticle.

Description

MULTI-DIMENSIONAL NANOPARTICLESGOVERNMENT INTEREST

[0001] This invention was made with government support under grant numbers AG073122, TR001998, and CA177558 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0002] The presently-disclosed subject matter generally relates to new quaternary multidimensional nanoparticles (qMDNPs) for use in drug therapy including combination drug delivery.BACKGROUND

[0003] Nanoparticles, such as polymer micelles, liposomes, solid-lipid nanoparticles, dendrimers, and extracellular vesicles, have garnered great attention as drug delivery vehicles that can carry various therapeutic agents. However, there are various drawbacks that have been associated with existing systems. For example, known systems have difficulty in delivering a combination of various drugs, such as combinations of small molecule drugs, either hydrophobic or hydrophilic, metal ions, proteins, nucleotides, and plasmid DNAs. Furthermore, chemical modifications of drug molecules and nanoparticles are often needed to address issues such as sudden changes in physicochemical properties, particle integrity, or size of the drug-loaded nanoparticles, which affect in vivo performance whenever a new drug is introduced.

[0004] Thus, there remains a need in the medical arts for more versatile and efficient techniques to prepare drug-loaded nanoparticles for improving the solubility, stability, bioavailability and / or the pharmacokinetics of various therapeutic agents so that better in vivo therapeutic outcomes can be achieved. It would be particularly advantageous if a nanoparticle can be developed that will easily load either hydrophobic or hydrophilic therapeutic agents in the core of the nanoparticle without any need to alter the structures of the drug payload or the nanoparticle carrier. Toward this end, this document relates to new qMDNPs that incorporate (a) selfassemblies, (b) inclusion complexes, allowing for the loading of hydrophobic therapeutic agents, (c) polyion complexes, allowing for the loading of hydrophilic therapeutic agents, and (d) polypeptide complexes that tend to better stabilize the cores of the qMDNPs. The self-assembliesare formed through micellization among amphiphilic block copolymers. The inclusion complexes are formed between hydrophobic therapeutic agents / small molecule drugs and cyclodextrins (either positively or negatively charged). The polyion complexes are formed between the charged cyclodextrins and the block copolymers with counter charges. The polypeptide complexes are formed between added polyions and the block copolymers. As a result, the inclusion complexes with charged cyclodextrins can be entrapped in the polyion complexes. See FIG. 1.

[0005] Advantageously, the self-assemblies, inclusion complexes, polyion complexes and polypeptide complexes function together in a synergistic relationship to provide a number of benefits and advantages. The resulting qMDNPs allow for versatile and efficient combination drug delivery that can maximize therapeutic outcomes when hydrophobic, hydrophilic, chemically unmodifiable, or biologically unstable small molecule drugs are used in combination. At the same time, the qMDNPs minimize potential side effects caused by toxic drugs, protecting normal organs and cells from non-specific drug exposure. Still further, the qMDNPs also enhance combination drug efficacy, by allowing one to finely tune the drug mixing ratio within a narrow range.

[0006] As will be further described below, the new qMDNPs allow for (a) active cell targeting, (b) loading of both hydrophobic and hydrophilic drug types, (c) hydrophobic, ionic and charge competition molecular interaction and (d) entrapment of multiple drugs for combination therapy.

[0007] Thus, the new qMDNPs are able to address shortcomings in the prior art by providing a unique drug delivery system that allows for targeted combination delivery of various drugs with enhanced safety and efficacy to treat human diseases including, but not limited to, various cancers.SUMMARY

[0008] In accordance with the purposes and benefits set forth herein, new quaternary multidimensional nanoparticles (qMDNPs) are provided. The qMDNPs comprise, consist of or consist essentially of a nanoparticle, of a cyclodextrin and a biocompatible polymer, having a biocompatible shell protecting an internal core wherein the cyclodextrin forms polyion complexes with the biocompatible polymer and is selected from a group consisting of a positively-charged functional group-substituted cyclodextrin, a negatively-charged functional group-substituted cyclodextrin, and combinations thereof;a polyion adapted to stabilize the internal core; and a therapeutic agent entrapped in a cavity of the cyclodextrin and / or the internal core of the nanoparticle. The therapeutic agent may be one or more hydrophobic therapeutic agents or one or more hydrophilic therapeutic agents. In some embodiments, the qMDNPs include one or more hydrophobic therapeutic agents and one or more hydrophilic therapeutic agents.

[0009] Positively-charged functional groups include, but are not limited to, amines, while negatively-charged functional groups include, but are not limited to, carboxyl groups.

[0010] In at least one possible embodiment, the polyion is selected from a group consisting of block copolymers, such as polyethylene glycol)-poly(L-aspartic acid), poly(ethylene glycol)- poly(L-glutamic acid), poly(ethylene glycol)-poly(L-lysine), poly(ethylene glycol)-poly(L- histidine), and homopolymers such as, poly(L-lysine), linear polyethylene imine, poly(L-histidine) and combinations thereof. In other embodiments, other polyions are used.

[0011] In at least some embodiments, the therapeutic agent is selected from a group consisting of small molecule drugs (< 1 kDa) such as SN-38, PX-866, carfilzomib (CFZ), fluorouracil, oxaliplatin, capecitabine, temozolomide, dacarbazine, streptozotocin, doxorubicin, gemcitabine, etoposide, cisplatin, carboplatin, paclitaxel, docetaxel and combinations thereof. In other embodiments, other therapeutic agents are used.

[0012] In at least some embodiments, the qMDNPs include a targeting agent conjugated to the nanoparticle. More specifically, the targeting agent may be conjugated to a surface of the biocompatible shell. The targeting agent may be selected from a group consisting of cancerspecific ligands such as octreotide (Oct) targeting somatostatin receptor 2, folic acid targeting folate receptor alpha, molecules targeting hormone receptor and PD-L1, and combinations thereof. In other embodiments, other targeting agents or cancer targeting peptides (CTPs) may be used.

[0013] In one particularly useful embodiment, the cyclodextrin is an amine-substituted P- cyclodextrin.

[0014] The biocompatible polymer may be a block copolymer such as poly(ethylene glycol)- poly(L-aspartic acid), polyethylene glycol)-poly(L-glutamic acid) (PEG-PLE), poly(ethyleneglycol)-poly(L-lysine), poly( ethylene glycol)-poly(L-histidine) and mixtures thereof. In other embodiments, other block copolymers are used.

[0015] In at least some embodiments, the cyclodextrin is an amine-substituted P-cyclodextrin and the biocompatible polymer is polyethylene glycol)-poly(L-glutamic acid) (PEG-PLE).

[0016] In accordance with an additional aspect, the qMDNPs comprise, consist of or consist essentially of a therapeutic agent-cyclodextrin inclusion complex enveloped in a biocompatible polymer. The qMDNP also includes self-assemblies, polyion complexes and polypeptide complexes. The inclusion complexes are formed between the therapeutic agents / small molecule drugs and the cyclodextrins (either positively or negatively charged). The polyion complexes are formed between the charged cyclodextrins and the block copolymers with counter charges. The polypeptide complexes are formed between added polyions and the block copolymers. In some embodiments, a hydrophobic therapeutic agent is entrapped in the cyclodextrin inclusion complex while the hydrophilic therapeutic agent is entrapped in the polypeptide complex within the internal core of the qMDNPs.

[0017] The therapeutic agent may be selected from a group consisting of SN-38, PX-866, carfilzomib (CFZ), fluorouracil, oxaliplatin, capecitabine, temozolomide, dacarbazine, streptozotocin, doxorubicin, gemcitabine, etoposide, cisplatin, carboplatin, paclitaxel, docetaxel and combinatios thereof. The biocompatible polymer may be poly(ethylene glycol)-poly(L- aspartic acid), poly(ethylene glycol)-poly(L-glutamic acid) (PEG-PLE), polyethylene glycol)- poly(L-lysine), poly(ethylene glycol)-poly(L-histidine) or mixtures thereof. Still further, the cyclodextrin may be an amine-substituted P-cyclodextrin.

[0018] In accordance with yet another aspect, a quaternary multi-dimensional nanoparticle (qMDNP), comprises, consists of or consists essentially of: (a) self-assemblies of micellized block copolymers forming a biocompatible shell protecting an internal core, (b) inclusion complexes, of charged functional group-substituted cyclodextrin and a small molecule therapeutic agent of less than 1 kDa, held in the internal core and stabilized by polyion complexation between the charged functional group-substituted cyclodextrin and the block copolymers, and (c) polyions held in the internal core, said polyions forming polypeptide complexes with the block copolymers.

[0019] In accordance with yet another aspect, a method of making qMDNPs, comprises, consists of or consists essentially of: mixing a therapeutic agent with a cyclodextrin to create a therapeutic agent- cyclodextrin inclusion complex wherein the cyclodextrin is selected from a group consisting of (a positively-charged functional group)-substituted cyclodextrin, (a negatively-charged functional group)-substituted cyclodextrin, (a positively-charged functional group and a negatively-charged functional group)-substituted cyclodextrin and combinations thereof; adding biocompatible polymers with counter charges to the therapeutic agent- cyclodextrin inclusion complex and sonicating to produce polyion complexes and form the qMDNPs; and lyophilizing the qMDNPs to obtain a fine powder of nanoparticles wherein the therapeutic agent is held in a core of a biocompatible shell.

[0020] The method may further include conjugating a targeting agent to a surface of the biocompatible shell.

[0021] The mixing of the therapeutic agent with the cyclodextrin may include (a) adding the therapeutic agent in alcohol to the cyclodextrin dissolved in deionized water to create a reaction mixture, (b) adding a facilitating agent, such as citric acid, to facilitate complexation between the therapeutic agent and the cyclodextrin while preventing the inclusion complexes from precipitating, (c) sonicating the reaction mixture, and (d) removing the alcohol by evaporation.

[0022] In at least one of the many possible embodiments, the method may further include using mixing ratios of the polyion to neutralize charges in the core. In some embodiments, the method may include using a cryoprotectant during the lyophilizing.

[0023] In accordance with still another aspect, a method of delivering a therapeutic agent to a patient, comprises, consists of or consists essentially of administering to the patient a pharmaceutically effective amount of the qMDNPs described in this document.

[0024] In accordance with yet another aspect, a method of delivering a therapeutic agent to an organ or a tumor, comprises, consists of or consists essentially of administering the qMDNPs described in this document to the organ or cell, wherein the biocompatible polymers of thosenanoparticles dissociate or hydrolyze to release the therapeutic agent upon encountering a pH of less than about 7.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are used, and the accompanying drawings of which:

[0026] FIG. 1 illustrates a quaternary multi-dimensional nanoparticle including the number of parameters, the specific complexes involved, the design, the active cell targeting, the loadable drug type, the molecular interactions and the feasibility to entrap multiple drugs.

[0027] FIG. 1A is a schematic illustration of a qMDNP loaded with a hydrophobic therapeutic agent.

[0028] Fig. IB is a schematic illustration of a qMDNP loaded with a hydrophilic therapeutic agent.

[0029] FIG. 1C is a schematic illustration of a qMDNP loaded with a hydrophobic therapeutic agent and a hydrophilic therapeutic agent.

[0030] FIG. 2 CFZ-loaded qMDNPs stabilized by polyions with varying pKa values.

[0031] FIG. 3. Particle size distribution of empty and CFZ-loaded qMDNPs determined by dynamic light scattering measurement. The He-Ne laser ( = 633 nm) was used to measure the speed of nanoparticles undergoing Brownian motion.

[0032] FIG. 4 Effects of pH on the release of CFZ from qMDNPs. Drug release experiments were performed at 37°C in buffers with pH values mimicking the conditions in blood stream (pH 7.4), acidic tumor microenvironment (pH 6.0), and intracellular lysosomes (pH 5.0), respectively.

[0033] FIG. 5. Cytotoxicity of CFZ-loaded qMDNPs in comparison to free drug. Viability of CFZ-resistant DLD1 cells was measured after treating the cells with serial dilutions of free CFZ in DMSO or CFZ / qMDNPs in aqueous media.

[0034] FIG. 6 Confirmation of proteasome activity inhibition by CFZ-loaded qMDNPs. Proteasome activity remaining was measured in CFZ-resistant DLD1 cells after treating the cells with free CFZ or CFZ-loaded qMDNPs for 6, 48, or 96 h. Data was analyzed to determine statistical significance in rapid (< 6 h) and prolonged (> 48 h) inhibition of proteasome activity for CFZ / qMDNPs with respect to free CFZ (*: p < 0.01, **: p < 0.05).

[0035] FIG. 7. Illustrates the formulation and synthesis of a second possible embodiment of qMDNPs including the therapeutic agent SN-38.

[0036] FIG. 8. Is a graph illustrating the size of the qMDNPs after drug loading.

[0037] FIG. 9. Is a graph illustrating sustained drug release after the initial burst with a drug release half-life of approximately 20 hours.

[0038] FIG. 10. Illustrates comparison of neuroendocrine cancer cells viability treated with qMDNPs- / SN-38 vs Oct-qMDNPs / SN-38 nanoparticles. IN A and B, BON and BON-SSTR2 cells were treated with qMDNPs / SN-38 and Oct-qMDNPs / SN-38 nanoparticles; viabuility was measured 72 hours after treatment with nanoparticles. In C and D, BON and BON-SSTR2 were treated with qMDNPs- / SN-38 vs Oct-qMDNPs / SN-38 nanoparticles; viability was measured 72 hours after treatment with nanoparticles.

[0039] FIG. 11. Illustrates flow cytometry analysis of qMDNP-Alexa647 or Oct-qMDNP- Alexa647 binding BON SSTR2-GFP cells.DETAILED DESCRIPTION

[0040] The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0041] The presently-disclosed subject matter includes quaternary multi-dimensional nanoparticles (qMDNPs) for combination drug delivery, and methods of making and using such qMDNPs.

[0042] In some embodiments, the presently-disclosed subject matter includes a composition as disclosed herein for use in combination drug delivery. In some embodiments, the presently- disclosed subject matter includes a multi-dimensional nanoparticle (MDNP) system as disclosed herein.

[0043] In some embodiments, the presently-disclosed subject matter includes a method of delivering a combination of drugs, comprising administering a composition or MDNP system as disclosed herein.

[0044] In some embodiments, the presently-disclosed subject matter includes a method of making a composition or MDNP as disclosed herein.

[0045] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0047] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0048] Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0049] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognizedabbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9): 1726-1732).

[0050] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0051] The present application can “comprise” (open ended) or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0052] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a therapeutic agent” includes a plurality of such therapeutic agents, and so forth.

[0053] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently- disclosed subject matter.

[0054] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, in some embodiments ±0.1%, in some embodiments ±0.01%, and in some embodiments ±0.001% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0055] As used herein, ranges can be expressed as from “about” one particular value, and / or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0056] A new quaternary multi-dimensional nanoparticle (qMDNP) for use in drug therapy including combination drug delivery includes: (a) a nanoparticle, of a cyclodextrin and a biocompatible polymer, having a biocompatible shell protecting an internal core wherein the cyclodextrin is selected from a group consisting of (a positively charged functional group)- substituted cyclodextrin, (a negatively charged functional group)-substituted cyclodextrin, and combinations thereof, (b) a polyion adapted to stabilize the internal core, and (c) a therapeutic agent entrapped in the cavity of the cyclodextrin and / or internal core of the nanoparticle.

[0057] FIG. 1A illustrates a qMDNP loaded with a hydrophobic therapeutic agent. It shows the nanoparticle 10, the charged cyclodextrin 12, the block copolymer 14, the shell 16, the core 18, the polyion 20 and the hydrophobic therapeutic agent 22. FIG. IB illustrates a qMDNP loaded with a hydrophilic therapeutic agent. It shows the nanoparticle 10, the charged cyclodextrin 12, the block copolymer 14, the shell 16, the core 18, the polyion 20 and the hydrophilic therapeutic agent 24. FIG. 1C illustrates a qMDNP loaded with both a hydrophobic therapeutic agent and a hydrophilic therapeutic agent. It shows the nanoparticle 10, the charged cyclodextrin 12, the block copolymer 14, the shell 16, the core 18, the polyion 20, the hydrophobic therapeutic agent 22 and the hydrophilic therapeutic agent 24. In these FIGS. 1A-1C, the polyion complexes are represented by the action arrows PC, the polypeptide complexes are represented by the action arrows PPC and the inclusion complexes are generally shown at IC.

[0058] As indicated, the cyclodextrins used in the qMDNPs include positive or negative charges by substitution with functional groups, such as amines for a positive charge and carboxyl groups for a negative charge. The functional group used should match the type of polymer used, which, in turn, will be selected according to the type of drug / therapeutic agent payload. Thus, amine substitution is needed when anionic polymers are used while carboxyl group substitution is needed when cationic polymers are used so as to cancel out charges in the nanoparticles as they are formed.

[0059] The biocompatible polymers used in the quaternary nanoparticles include both positively and negatively charged polymers. Block copolymers are particularly useful in the construction of the quaternary nanoparticles. “Block copolymer” refers to a polymer with repeating units of one type adjacent to each other in a linear manner to form a block, which is linked, for example, through a covalent bond to a second block made up of repeating units of a second type, which are adjacent to one another in a linear manner to form a second block of the block copolymer. Block copolymers referred to in this document may have a natural, synthetic or semisynthetic polymer backbone (e.g. polyester, polyether, polyamide, polypeptide, fatty acid, and combinations thereof). Each polymer chain may have a molecular weight of 1-50 kDa. The block copolymers include a hydrophilic segment and a hydrophobic segment, a hydrophilic segment and a charged segment, or one that is biologically inert and one that is chemically modifiable. Block copolymers useful in the present invention include, but are not limited to protected form block copolymers, anionic form block copolymers, cationic form block copolymers, cross-linked form block copolymers, drug-binding linker form block copolymers, drug-conjugated form block copolymer and mixtures thereof as is known in the art. Such block copolymers include, but are not necessarily limited to poly(ethylene glycol)-poly(L-aspartic acid), poly(ethylene glycol)-poly(L- glutamic acid) (PEG-PLE), polyethylene glycol)-poly(L-lysine), poly(ethylene glycol)-poly(L- histidine) or mixtures.

[0060] The block copolymers used in the qMDNPs include di- and tri-block copolymers to further optimize physicochemical properties of the molecular assemblies, while the hydrophilic block of poly(ethylene glycol) can be replaced with other biocompatible hydrophilic polymer alternatives such as polyoxazoline, polysarcosine, or polyvinylpyrrolidone.

[0061] The polyion useful in the quaternary nanoparticles includes those with a positive and those with a negative charge. Examples of polyions useful in the construction of the quaternary nanoparticles include but are not necessarily limited to a group including poly(ethylene glycol)- poly(L-lysine), poly(L-lysine), poly(L-histidine), linear polyethylene imine and combinations thereof. Here, it should be appreciated that this list is merely representative of the types of polyions that may be used and should not be considered as being restrictive in scope.

[0062] For the purposes of this document, the term "therapeutic agent" refers to biologically active agents, prodrugs, or drugs, including, for example, any organic or organometallic small molecule compound (e.g., a molecule with a molecular weight of less than about 1 kDa), polymeric species (including nucleic acids (DNA and RNA), proteins, peptides, hormones, carbohydrates, and derivatives thereof), lipids and mixtures thereof, wherein said drug or agent can be administered in vivo (in humans or animals) for the treatment of a disease, condition, or disorder.

[0063] The therapeutic agents useful in the quaternary nanoparticles include any known therapeutic agent or agents that may be loaded into a nanoparticle and used for the treatment of any particular disease or malady that may treated with the quaternary nanoparticles. This includes, for example, chemotherapeutic agents for the treatment of a wide range of cancers such as neuroendocrine neoplasms, a diverse group of neoplasms that can occur in various areas throughout the body. Such neoplasms can prove challenging to treat and, therefore, various chemotherapy combinations have been developed and used to treat neuroendocrine tumors (NETs), especially metastatic NETs.

[0064] More specifically, chemotherapeutic agents such as fluorouracil, oxaliplatin, SN-38 and combinations thereof (e.g., FOLFOX) may be used in the qMDNPs to treat neuroendocrine, colorectal and pancreatic cancer. Chemotherapeutic agents such as capecitabine and oxaliplatin and combinations thereof (e.g., CAPOX) may be used in the qMDNPs to treat neuroendocrine and colorectal cancer. Chemotherapeutic agents such as capecitabine and temozolomide and combinations thereof (e.g., CAPTEM) may be used in the qMDNPs to treat neuroendocrine tumors.

[0065] Chemotherapeutic agents such as temozolomide, dacarbazine, streptozotocin, doxorubicin, gemcitabine, PX866 and combinations thereof may be used in the qMDNPs to achieve alkylating therapy of neuroendocrine, lung, breast, ovary, leukemia, lymphoma, Hodgkin’s disease, multiple myeloma and sarcoma.

[0066] Chemotherapeutic agents such as etoposide, cisplatin, carboplatin and taxenes, such as paclitaxel and docetaxel, and combinations thereof may be used in the qMDNPs to achieve platinum chemotherapy of neuroendocrine, testicular, ovarian, and bladder cancer.

[0067] Chemotherapeutic agents such as carfilzomib (CFZ), daratumumab, dexamethasone, pomalidomide and combinations thereof may be used in the qMDNPs to treat relapsed or refractory multiple myeloma.

[0068] The above therapeutic agents are merely representative examples and others may be used in the qMDNPs to treat these and other cancers. At least some of the other therapeutic agents may be found, for example, listed in the BC Cancer Pharmacy Education Program Cancer Drug Pharmacology Table as updated on July 16, 2024, incorporated herein by reference.

[0069] For purposes of this document, the term “targeting agent” refers to any anti-tumor antibody, anti-tumor aptamer, anti-tumor binding peptide, hormone, cytokine, growth factor or substance preferentially taken up by a tumor cell. In at least some embodiments, the qMDNPs include a targeting agent conjugated to the nanoparticles. More specifically, the targeting agent may be conjugated to a surface of the biocompatible shell of the nanoparticles. The targeting agent used may be substantially any known in the art to be useful for the targeting of the cancer to be treated. Thus, for example, the targeting agent octreotide (Oct) may be conjugated to the surface of a quaternary nanoparticle loaded with a chemotherapeutic agent useful to treat neuroendocrine cancer cells.

[0070] In other examples, the nanoparticles can be modified with cancer-specific ligands such as octreotide (Oct) targeting somatostatin receptor 2, folic acid targeting folate receptor alpha, molecules targeting hormone receptor and PD-L1, cancer targeting peptides (CTPs) and combinations thereof.

[0071] In accordance with yet another aspect, a method of making quaternary multidimensional nanoparticles (qMDNPs) may be generally described as including the steps of: (a) mixing a therapeutic agent with a cyclodextrin to create a therapeutic agent-cyclodextrin inclusion complex wherein the cyclodextrin is selected from a group consisting of (a positively charged functional group)-substituted cyclodextrin, (a negatively-charged functional group)-substituted cyclodextrin, and combinations thereof, (b) adding a biocompatible polymer to the therapeutic agent-cyclodextrin inclusion complex and sonicating in presence of a polyion to produce the qMDNPs, and (c) lyophilizing the qMDNPs to obtain a fine powder of nanoparticles wherein the therapeutic agent is held in a core of a biocompatible shell.

[0072] The mixing of the therapeutic agent with the cyclodextrin may include (a) adding the therapeutic agent in alcohol to the cyclodextrin dissolved in deionized water to create a reaction mixture, (b) adding a facilitating agent, such as citric acid, to facilitate complexation between the therapeutic agent and the cyclodextrin while preventing the inclusion complexes from precipitating, (c) sonicating the reaction mixture, and (d) removing the alcohol by evaporation.

[0073] The method may further include using mixing ratios of the polyion to neutralize charges in the core. In some embodiments, the method may include using a cryoprotectant during the lyophilizing. The method may also further include conjugating a targeting agent, of the type described above, to a surface of the biocompatible shell.

[0074] In accordance with still another aspect, a method of administering a therapeutic agent to a patient includes administering to the patient a pharmaceutically effective amount of the qMDNPs. Alternatively, a method of delivering a therapeutic agent to an organ or a tumor, includes administering the qMDNPs to the organ or cell, wherein the biocompatible polymer hydrolyzes to release the therapeutic agent upon encountering a pH of less than about 7.

[0075] For the purposes of this document, the term "pharmaceutically effective amount" is intended to qualify the amount of a therapeutic agent required to relieve to some extent one or more of the symptoms of a disease or disorder, including, but not limited to: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition of (i.e., slowing to some extent, preferably stopping) cancer cell infiltration into peripheral organs; 3) inhibition of (i.e., slowing to some extent, preferably stopping) tumor metastasis; 4) inhibition, to some extent, of tumor growth; 5) relieving or reducing to some extent one or more of the symptoms associated with the disorder; and / or 6) relieving or reducing the side effects associated with the administration of anticancer agents. The terms "treat" and "treatment" refer to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly. The term "inhibition," in the context of neoplasia, tumor growth or tumor cell growth, may be assessed by delayed appearance of primary or secondary tumors, slowed development of primary or secondary tumors, decreased occurrence of primary or secondary tumors, slowed or decreased severity of secondary effects of disease,arrested tumor growth and regression of tumors, among others. In the extreme, complete inhibition can be referred to as prevention or chemoprevention.

[0076] More specifically, qMDNPs can be suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in certain embodiments, a pharmaceutical composition is provided that includes qMDNPs as described herein, in admixture with a suitable diluent or carrier. Suitable diluents or carriers include saline or aqueous dextrose, for example, a 5% aqueous dextrose solution. Such formulations can be prepared so that they are isotonic with human fluids, such as blood, or various tissue environments. In certain embodiments, it may also be desirable to prepare hypertonic or hypotonic preparations. In other embodiments, the composition can be prepared and used for in vitro experimentation, for example, in various screens and diagnostic procedures. The compositions containing qMDNPs can be prepared by known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the therapeutic agent within the qMDNPs is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003, 20thEd.), in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999, and in the Handbook of Pharmaceutical Additives (compiled by Michael and Irene Ash, Gower Publishing Limited, Aidershot, England (1995)). On this basis, the compositions include, albeit not exclusively, solutions of the qMDNPs in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. In this regard, reference can be made to U.S. Patent No. 5,843,456 (Paoletti et al.). In one embodiment, the pharmaceutical compositions can be used to enhance biodistribution and drug delivery of therapeutic agents, such as a drug linked to a polymer of the qMDNPs.

[0077] The qMDNPs described herein can be administered to a subject in a variety of forms depending on the route of administration selected, as is readily understood by those of skill in the art. The qMDNPs can be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, intrasternal, transepithelial, nasal, intrapulmonary, intrathecal, rectaland infusion modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0078] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can 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 can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents can also be useful.

[0079] A qMDNP may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the qMDNP may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. A qMDNP may also be administered parenterally.

[0080] Solutions of a qMDNP can be prepared in water suitably mixed with suitable excipients. Under ordinary conditions of storage and use, these preparations may contain a preservative, for example, to prevent the growth of microorganisms.

[0081] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The formulation should be sterile and should be fluid to the extent that the solution or dispersion can be administered via syringe.

[0082] Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension ofthe active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.

[0083] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

[0084] The compositions described herein can be administered to an animal alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice. In an embodiment, the pharmaceutical compositions are administered in a convenient manner such as by direct application to the infected site, e.g. by injection (subcutaneous, intravenous, parenteral, etc ). In case of respiratory infections, it may be desirable to administer the qMDNPs and compositions comprising same, through known techniques in the art, for example by inhalation. Depending on the route of administration (e. g. injection, oral, or inhalation, etc.), the pharmaceutical compositions or qMDNPs or biologically active agents in the qMDNPs may be coated in a material to protect the qMDNPs or agents from the action of enzymes, acids, and other natural conditions that may inactivate certain properties of the composition or its encapsulated agent.

[0085] In addition to pharmaceutical compositions, compositions for non- pharmaceutical purposes are also included within the scope of the invention. Such non-pharmaceutical purposes may include the preparation of cosmetic formulations, or for the preparation of diagnostic or research tools. In one embodiment, the therapeutic agents or qMDNPs comprising such agents canbe labeled with labels known in the art, such as florescent or radio-labels, or the like. In some embodiments, one or more of the drugs of the polymer can be replaced with a diagnostic agent.

[0086] The invention also provides a delivery system that can be used to deliver biologically active agents or formulations or pharmaceutical compositions. In one possible embodiment, the invention includes the delivery of a combination of cancer therapeutic agents. In another embodiment, the invention includes delivery of therapeutic agents by linking the agents to polymers that self-assemble into qMDNPs comprising an amphiphilic or hydrophobic core and a hydrophilic outer surface, thus improving their delivery in aqueous mediums, such as blood, body fluids, tissues, and organs.

[0087] In other aspects, the invention includes the delivery of biologically active agents while reducing their toxicity profile. This is often effectuated by the synergy derived from the administration of a combination of therapeutic agents, thereby reducing the dose required for an equivalent therapeutic effect. The invention also includes a method for reducing aggregation or precipitation of drugs in delivery vehicles, a common problem associated with currently used vehicles for drug solubilization and delivery. As such, the invention provides improved biodistribution of therapeutic agents, resulting in decreased toxicity and / or improved therapeutic efficacy at lower doses. For example, the combined dose used in the combination therapy of the invention can be used to deliver a larger amount of drugs than could be provided as a single dose of one drug, without concomitant toxicity issues that would be encountered if that larger dose was provided by the single drug.

[0088] Another aspect of the invention includes a method of delivering biologically active agents to treat a disease, condition, or disorder in a subject in need thereof comprising administering an effect amount of an agent-loaded qMDNPs to a subject. In one embodiment, the disease, condition or disorder is cancer or drug resistant cancers, infectious disease or an autoimmune disease.

[0089] The dosage of the qMDNPs can vary depending on many factors such as the pharmacodynamic properties of the qMDNPs, the biologically active agent, the rate of release of the agent from the qMDNPs, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type ofconcurrent treatment, if any, and the clearance rate of the agent and / or qMDNP in the subject to be treated.

[0090] For example, in some embodiments, a dose of a qMDNP formulation equivalent to about 1 mg mL1to about 100 mg mL1can be administered to a patient. In certain other embodiments, the qMDNP formulation includes about 2- 20, about 5-15, or about 10 mg mL"1. The specific doses of the compounds administered according to this invention to obtain therapeutic and / or prophylactic effects will, of course, be determined by the particular circumstances surrounding the case, including, for example, the compounds administered, the route of administration, the condition being treated and the individual being treated. A typical daily dose (administered in single or in divided doses) can contain a dosage level of from about 0.01 mg / kg to about 150 mg / kg of body weight of an active therapeutic agent described herein. In some embodiments, about 5-10, about 10-20, about 20-40, about 25-50, about 50-75, about 75-100, or about 100-150 150 mg / kg of body weight of a therapeutic agent are provided in a dose. In other embodiments, about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 140, or 150 mg / kg of body weight of a therapeutic agent are delivered in a dose. Often times, daily doses generally will be from about 0.05 mg / kg to about 20 mg / kg and ideally from about 0.1 mg / kg to about 10 mg / kg.

[0091] One of skilled in the art can determine the appropriate dosage based on the above factors. The qMDNPs may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. For ex vivo treatment of cells over a short period, for example for 30 minutes to 1 hour or longer, higher doses of qMDNPs may be used than for long term in vivo therapy. The qMDNPs can be used alone or in combination with other agents that treat the same and / or another condition, disease or disorder. In another embodiment, where either or both the qMDNP or biologically active agent is labeled, one can conduct in vivo or in vitro studies for determining optimal dose ranges, drug loading concentrations and size of qMDNPs and targeted drug delivery for a variety of diseases.

[0092] The polymers, qMDNPs, and qMDNP formulations of the invention also provide advantageous methods for the delivery of a combination of poorly water-soluble therapeutic agents, which are frequently incompatible in commonly encountered delivery vehicles. Becausethe drugs linked to the polymers are only released in a specific condition (e.g., acidic environment), the delivery of the therapeutic agent is very controlled. The qMDNPs can accommodate high levels of drug loading while maintaining low toxicity because the drugs are not released at an appreciable rate when not in the vicinity of a tumor. In addition to their tumor specific accumulation, the qMDNPs also offer long circulation in the blood, and the regeneration of active drugs from prodrugs at the targeted site. Furthermore, the use of qMDNPs capable of controlling drug release can reduce non-specific drug distribution, thereby enhancing both the safety of the anticancer drugs and the efficiency of the tumor-targeted delivery, all while delivering two or more drugs simultaneously.

[0093] The delivery of two or more therapeutic agents is commonly known as combination therapy. The phrase "combination therapy" (or "co-therapy") embraces the administration of two different therapeutic agents as part of a specific treatment regimen intended to provide a beneficial effect from the co- action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).

[0094] Combination drug therapy typically has inherent difficulties with suitable administration because most drugs are highly water insoluble. Accordingly, oral and intravenous administration can be problematic and ineffective. A significant advantage of the combination therapy that can be administered using the qMDNPs described in this document is that two or more otherwise difficult-to-administer agents, such as low solubility agents, can be administered in a simultaneous manner. Simultaneous administration can be accomplished, for example, by administering to the subject a single qMDNP formulation having a fixed ratio of each therapeutic agent. Simultaneous administration of the combination of therapeutic agents can be achieved by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. Separate co-therapies can be administered by the same route or by different routes.

[0095] Since the new qMDNPs provide hydrophobic, ionic and charge competition molecular interaction and thereby allow for loading of both hydrophobic and hydrophilic drug types and entrapment of multiple drugs in a single nanoparticle, the qMDNPs are particularly effective and useful for combination therapy. Advantageously, the qMDNPs also allow for active cell targeting.

[0096] "Combination therapy" also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients (such as, but not limited to, a third and different therapeutic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

[0097] The phrases "low dose" or "low dose amount", in characterizing a therapeutically effective amount of the therapeutic agents in the combination therapy, defines a quantity of such agent, or a range of quantity of such agent, that is capable of improving the disorder or disease severity while reducing or avoiding one or more therapeutic-agent-induced side effects, such as myelosuppression, cardiac toxicity, alopecia, nausea or vomiting. Many synergistic drug combinations can be administered using the qMDNP compositions of the invention. In one synergistic combination of significant importance the qMDNP includes one drug that is effective to sensitizes tumor cells while apoptosis is induced by the second drug. These synergistic effects can be especially valuable for treating breast cancer.

[0098] It is particularly advantageous to deliver combinations of therapeutic agents in a ratio that is non-antagonistic, and especially that is non-antagonistic over a wide range of concentrations. As described in PCT publication PCT / CA02 / 01500, algorithms are available such that, based on the results of in vitro tests, non-antagonistic ratios may be determined. Examples of suitable synergistic drug combinations and further discussion of determining non- antagonistic ratios over a wide range of drug concentrations can be found in WO 2006 / 014626 (Mayer et al.), which is incorporated herein by reference.

[0099] The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.EXAMPLESExample 1Introduction

[0100] The utility and benefits of the multi-dimensional nanoparticle (MDNP) system will be further described with reference to an embodiment that makes use of carfilzomib.

[0101] Carfilzomib (CFZ) is an epoxomicin analog that has demonstrated great efficacy in treating patients with relapsed or refractory multiple myeloma who had at least two prior therapies [1, 2], and it is the second-in-class proteasome inhibitor approved by the FDA in 2012 [3, 4], Unlike first-generation proteasome inhibitors such as Bortezomib, CFZ binds selectively and irreversibly to the 20S proteasome to inhibit its chymotrypsin-like activity [5-7], Through this mechanism, CFZ prevents the proteasome from tagging proteins with ubiquitin for cellular degradation, which intracellularly builds up defective proteins that induces cell stress, cell cycle arrest, apoptosis and ultimately inhibits the growth of cancer cells [6], Furthermore, CFZ has been effective in reducing dose limiting toxicity associated with Bortezomib among patients that developed resistance to the first-generation proteasome inhibitor [8],

[0102] The great efficacy and potential benefits of CFZ have garnered attention to treat other cancers such as advanced solid and metastatic cancers using the drug, but there have been limited success in the clinic due to poor biostability of the tetrapeptide (Phe-Leu-Phe-Leu) with an epoxyketone ring that undergoes protonation and subsequently ring opening catalyzed under acidic microenvironment to make the pharmacophore inactive in tumors with acidosis [9-11], The amide bonds linking the peptides were found also susceptible to enzymatic degradation in vivo

[0012] , In addition to poor biostability, CFZ has other pharmaceutical limitations such as low water solubility and efflux by P-glycoprotein (P-gp) [13-16], which justify the development of injection formulations for CFZ. Currently available injection formulations for CFZ, such as Kyprolis, require a large amount of solubilizers such as cyclodextrin and more than 50 times morecyclodextrin molecules are needed to dissolve one drug molecule in water, which is a ratedetermining factor for the development of injection formulations for CFZ and other small molecule drugs.

[0103] To mitigate the pharmaceutical limitations and maximize therapeutic potential of CFZ, ternary polypeptide nanoparticles (tPNPs) were previously developed that consist of drug- cyclodextrin inclusion complexes enveloped by biocompatible polymers, which are further stabilized through polyion complexation with small molecule organic acids such as citric acid and lactic acid [17-25], Preclinical studies revealed that CFZ-loaded tPNPs (CFZ / tPNPs) significantly improved biostability of the tetrapeptide in vitro, yet tPNPs would require further improvement in particle stability to suppress initial burst drug release and thus achieve prolonged inhibition of proteasome activity with CFZ against tumor cells in vivo.

[0104] The aforementioned tPNPs are one type of MDNPs stabilized with organic acids while qMDNPs are stabilized with polyions. As disclosed herein, new quaternary multi-dimensional nanoparticles qMDNPs were developed, which include amine-substituted P-cyclodextrins (CD) and biocompatible poly(ethylene glycol)-poly(L-glutamic acid) (PEG-PLE) that self-assemble into nanoparticles in solution while polyions with varying pKa values were used to stabilize the assemblies through polyion complexation (FIG. 2). It was conceived that cationic polymers would stabilize the core of the new qMDNPs by neutralizing charges between positive CD with amines and negative PEG-PLE with carboxylic acids to ensure a well-defined structure of a nanoparticle with uniform size, high drug loading and stability in aqueous solutions. The cationic polymers were also expected to modulate the release of CFZ from CFZ / qMDNPs in a pH-dependent manner due to their pKa values determining the dissociation of qMDNPs through disruption of charges in the nanoparticle core. Such pH-sensitive nanoparticles hold great promise to develop smart drug delivery systems that remain stable in the physiological pH 7.4 yet trigger drug release when exposed to acidic environment such as tumors with acidosis (pH 6.8 - 7.2) or endosomes and lysosomes in the cell (pH 5 - 6.8) [26-28], Thus, the new qMDNPs provide a unique option to deliver CFZ to tumor cells in the body while increasing therapeutic efficacy and reducing toxicity of the proteasome inhibitor.

[0105] Based on the background and rationale, three polyions were tested with varying pKa values as stabilizing agents for CFZ / qMDNPs: poly(ethylene glycol)-poly(L-lysine) (PLL, pKa ~ 10), poly(L-histidine) (PLH, pKa « 6), and linear polyethylene imine (PEI, pKa ~ 7, where pKal = 4.5 and pKa2 = 10). PLL, PLH, and PEI are polyions that are widely used as building components for various drug and gene delivery systems due to their biocompatibility and safety [29-40], Thus, with ensured safety of the selected polyions, the exemplary CFZ study focuses on investigating if CFZ / qMDNPs stabilized by polyions would show uniform physiochemical properties such as particle size, surface charge and drug loading while improving drug release profdes to enhance proteasome inhibition for CFZ.

[0106] Carfilzomib (CFZ) is a second-generation proteasome inhibitor showing great efficacy in multiple myeloma treatment, yet its clinical applications for other diseases such as solid cancers are limited due to low aqueous solubility and poor biostability. Quaternary multi-dimensional nanoparticles (qMNDPs) are drug carriers that overcome these pharmaceutical limitations by entrapping CFZ in the core of the nanoparticles and protecting the drugs from degradation in biological media. Further, the CFZ-loaded qMNNPs are stabilized by polyions which have varying pKa values and thus differently modulate nanoparticle stability in response to solution pH. Through polyion complexation, the polyions appear to stabilize the core of qMDNPs entrapping CFZ-cyclodextrin inclusion complexes while allowing for uniform particle size before and after freeze drying. Interestingly, CFZ-loaded qMDNPs (CFZ / qMDNPs) showed pH-dependent drug release kinetics, which accelerated CFZ release as solution acidity increased (pH < 6) without compromising particle stability at the physiological condition (pH 7.4). In vitro cytotoxicity and proteasome activity assays confirmed that qMDNPs stabilized with cationic polymers improved bioactivity of CFZ against CFZ-resistant cancer cells, which would be greatly beneficial in combination with pH-dependent drug release for treatment of solid cancers with drug resistance and tumor microenvironment acidosis by using CFZ and other proteasome inhibitors.Materials

[0107] Potassium biphthalate sodium hydroxide buffer and HPLC grade water were obtained from Fisher Scientific (Fair-lawn, NJ). Slide-A-Lyzer Mini Dialysis Units (20,000 MWCO) were obtained from Thermo Scientific (Rockford, IL). Heptakis (6-amino-6-deoxy)-P- CD heptahydrochloride (CD) was purchased from Cyclolab R&D Laboratory Ltd.(Hungary). Citric acid was from EM Science (Gibbstown, NJ). CFZ was purchased from LC laboratories (Woburn, MA). Methoxy-poly(ethylene glycol)-poly(L-glutamic acid) (PEG-PLE), methoxy-poly(ethylene glycol)-poly(L-lysine) (PLL) was purchased from Alamanda Polymers (Huntsville, AL). a-Q-bis-(amino)-terminated poly(L-histidine) (PLH) was purchased from Polymer Source Inc (Canada). Linear polyethyleneimine (PEI) was purchased from Polysciences Inc (Warrington, PA).Cell culture

[0108] Human colorectal adenocarcinoma cell (DLD1) was from ATCC and cultured in RPML1640 medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO2. The cells were maintained in an exponential growth phase by periodic sub-cultivation. For CFZ-resistant DLD1 cells, parent DLD1 cells were treated with increasing concentration of CFZ for about 4 months until cells became > 95% survival at a concentration of 1 pM CFZ.Preparation of qMDNPs

[0109] qMDNPs were prepared by using a solvent evaporation method as reported previously with slight modification. Briefly, CFZ in ethanol was mixed with CD dissolved in deionized water at a 1 :4 mass ratio in a 50 mL round bottom flask. Citric acid was added to the mixture to facilitate complexation between CFZ and CD while preventing the inclusion complexes from precipitating. After sonicating the solution for 2 minutes, ethanol was removed by rotary evaporation at 60°C with reduced pressure. Following the removal of ethanol, PEG-PLE was added to the aqueous solution of CFZ / CD inclusion complexes and the solution was sonicated for 5 minutes to prepare CFZ / qMDNPs in the presence of PLL, PLH or PEI at mixing ratios to neutralize charges in the nanoparticle core. CFZ / qMDNPs were then frozen in dry ice and lyophilized overnight (Labconco Freezone®) using 2% (w / v) sucrose as a cryoprotectant to obtain a fine powder of nanoparticles. The primary and secondary drying was performed at -50°C and 25°C for 24 h, followed by storage at -20°C prior to use.Particle size and zeta potential determination

[0110] The particle size and surface charge of qMDNPs with and without CFZ were determined by dynamic light scattering (DLS) and zeta-potential measurements (Zetasizer NanoZS, Malvern, UK). Freeze-dried CFZ / qMDNPs were resuspended and sonicated in normal saline at 2 mg / mL. The resultant suspension was placed in a folded capillary cuvette and DLS was measured with 173° backscatter at 25°C. Data including the polydispersity index (PDI) of particle size were also calculated with the accompanying software.Drug loading and encapsulation efficiency[00111J Drug loading of CFZ / qMDNPs was determined by high performance liquid chromatography (HPLC, Agilent XDB-C 18). The mobile phase consisted of water and acetonitrile (ACN) with 0.1% formic acid. Using an isocratic method of 55:45 of ACNiFFO at a flow rate of 0.5 mL / min, CFZ was eluted at a retention time of 3.6 minutes. Drug concentrations were measured by integrating peaks at a wavelength of 210 nm and the peak area was compared to a calibration curve established with a serial dilution of CFZ. Drug loading efficiency was defined as the percentage of CFZ encapsulated to the weight of nanoparticles, and encapsulation efficiency was defined as the percentage of drug encapsulated to drug added. Data are represented as mean ± standard deviation (SD).Drug release confirmation

[0112] CFZ release kinetics was determined as previously reported. Briefly, freeze-dried CFZ / PLL-qMDNP, CFZ / PLH-qMDNP, and CFZ / PEI-qMDNP were dissolved in PBS (pH 7.4, 10 mM) at 10 mg / mL and placed in dialysis cups (MWCO 20 kDa). The dialysis cups were then placed in 4 L buffers at pH 7.4, 6 or 5 at 37°C to simulate conditions in blood stream, acidic tumor microenvironment, and intracellular lysosomes. Nanoparticle solutions were retrieved at 0, 0.5, 1, 3, 6, 24, 48 and 72 h, and drug remaining was measured using the HPLC method described above. Percent drug remaining at each time point was calculated as a normalized value of drug remaining compared to drug concentration at t = 0 h. The drug release profiles were fitted to an exponential decay with GraphPad Prism 9 software.Cell viability determination

[0113] CFZ-resistant DLD1 cells were cultured in drug-free media for 9 days prior to the cell viability assay. DLD1 cells were seeded in a 96-well plate at 5,000 cells per well and incubated overnight for attachment. The cells were treated with serial dilutions of free CFZ in DMSO and CFZ / qMDNPs in aqueous solutions. After treating the cells for 96 h, 20 pL of MTS dyes wasadded to each well and the plates were placed in an incubator at 37°C and 5% CO2 for 2 h to determine cell viability. Absorbance was measured at 490 nm using a plate reader (Spectra Max M5). The MTS assay (Promega) was used to determine half-maximal inhibitory concentration (IC50) values for CFZ / PLL-qMDNP, CFZ / PLH-qMDNP, and CFZ / PEI-qMDNP, which were calculated by a non-linear regression method using GraphPad Prism 9 software.Proteasome activity assay

[0114] Proteasome activity was determined by measuring the chymotrypsin-like activity in CFZ-resistant DLD1 cells treated with free CFZ, CFZ / PLL-qMDNP, CFZ / PLH-qMDNP or CFZ / PEI-qMDNP. The cells were cultured for 9 days without CFZ and then seeded in a 6-well plate at 150,000 cells / well for additional 24 h. The cells were incubated with free CFZ or CFZ / qMDNPs at CFZ equivalent concentrations (0.5 pM) at 37°C with 5% CO2 for 6, 48 and 96 h. At each time point, cell pellets were washed with DPBS and harvested for storage at -20°C until they were lysed with lx Passive Lysis Buffer. The total protein concentrations were determined by the Bradford assay. The proteasome activity was measured by treating 5 pg of protein collected from each sample with 100 pM of Suc-LLVY-AMC substrate. Fluorescence was quantified with a plate reader (Spectra Max M5) at a rate of 1 reading per minute with excitation and emission wavelengths of 360 and 460 nm, respectively. The percent of proteasome activity remaining was determined as the relative fluorescence between treated and untreated cells. Multiple unpaired t- tests were performed for statistical analysis to compare the means of groups for a time point. Statistical significance was determined with a p value < 0.01 or < 0.05 with respect to cells treated with free CFZ using GraphPad Prism 9 software.ResultsPhysiochemical properties of qMDNPs

[0115] PLL, PLH and PEI were used in this study because they induced no precipitation of qMDNPs after CFZ loading in preliminary experiments. All freeze-dried CFZ / qMDNPs readily dissolved in aqueous solutions with simple vortexing and showed a unimodal particle size distribution (FIG. 3). The particle size of empty and CFZ / qMDNPs was less than 100 nm and zeta potentials were ± 5 mV (Table 1).Table 1. Physiochemical properties of qMDNPsCF7 / PT FT 91.9 ± 13.5 0.63 ± 0.05 -1.59 ± 0.29 9.80 ± 0.98 91.0 ± 3.2 qMDNPEmqMSDxNTPn1-34.0 ± 3.3 0.20 ± 0.03 -1.07 ± 0.37CF7 / PFI-56.52 ± 9.8 0.48 ± 0.04 2.86 ± 0.49 8.95 ± 0.92 80.3 ± 4.5 qMDNPDrug loading and entrapment efficiency

[0116] The amounts of CFZ loaded in qMDNPs were calculated using the formulas described in the methods section. CFZ / PLL-qMDNP, CFZ / PLH-qMDNP and CFZ / PEI-qMDNP had drug loading of 11.44%, 9.80% and 8.95% respectively. The encapsulation efficiency for CFZ / PLL- qMDNP, CFZ / PLH-qMDNP, CFZ / PEI-qMDNP was 95.3%, 91.0% and 80.3% respectively. These results confirmed that polyions showed no adverse effect on loading and entrapment of CFZ in qMDNPs.Drug release of CFZ from qMDNPs

[0117] Drug release experiments were performed at 37° C in buffers with three pH values mimicking the conditions in blood stream (pH 7.4), acidic tumor microenvironment (pH 6.0), and intracellular lysosomes (pH 5.0). At pH 7.4, none of qMDNPs showed burst drug release, which suggests the particle stabilizing effect of polyions. CFZ / qMDNPs, however, accelerated drug release as pH decreased below 6.0 while complete disposal of drug was observed at pH 5.0 for CFZ / PEI-qMDNP after 48 h incubation and CFZ / PLH-qMDNP after 3 h incubation (FIG. 4). Drug release rates were further analyzed by determining drug release half-life with biphasic curve fitting (Table 2). For all CFZ / qMDNPs, drug release underwent short transitions from initial fastdrug release (ti / 2 fast < 1 h) to the second phase of modest drug release (ti 2 slow > 30 h) at pH 7.4, while ti / 2 slow values significantly dropped as drug release was accelerated at pH 6.0 and 5.0. Interestingly, at pH 7.4, CFZ / PLL-qMDNP showed no ti / 2 slow yet maintained a sustained release over 72 h while CFZ / PEI-qMDNP and CFZ / PLH-qMDNP demonstrated biphasic drug release profiles. These results showed that all CFZ / qMDNPs stabilized with polyions were stable at pH 7.4 with drug release half-life greater than 30 h while accelerating drug release in acidic conditions (pH 6.0) in the order of PELqMDNP (fastest), PLH-qMDNP and PLL-qMDNP (slowest). Only PLH induced complete dissociation of qMDNPs at pH 5.0 in the time frame of 72 h.Table 2. Drug release kinetics of qMDNPsPIC pH ti / 2 fast (h) ti / 2 slow (h)7.4 N.D. 44.04 ± 5.25CFZ / PLL-qMDNP 6.0 0.54 ± 0.12 25.27 ± 2.705.0 0.63 ± 0.08 19.98 ± 11.57.4 0.30 ± 0.01 48.73 ± 1.01CFZ / PLH-qMDNP 6.0 0.32 ± 0.12 17.64 ± 4.545.0 0.34 ± 0.11 0.34 ± 0.067.4 0.39 ± 0.23 30.72 ± 5.63CFZ / PEI-qMDNP 6.0 0.49 ± 0.05 16.09 ± 1.315.0 0.45 ± 0.17 7.54 ± 1.63Cell viability

[0118] FIG. 5 shows the results of cytotoxicity assays in CFZ-resistant human colorectal cancer cells. Drug resistance of DLD1 cells was confirmed by > 10% of cell viability at 100 pM of free CFZ in DMSO. The cell viability curve shows that CFZ / PLL-qMDNP effectively inhibited the growth of CFZ-resistant cells in comparison to free CFZ. CFZ / PLH-qMDNP was as equally effective as free CFZ yet CFZ / PEI-qMDNP failed to suppress more than 50% of cell viability even at the maximum aqueous solubility achieved for CFZ using qMDNPs. Among qMDNPs, CFZ / PLL-qMDNP had the lowest IC50 value (3.9 ± 0.2 pM), which was approximately 1.8 and >3.6 times more effective than CFZ / PLH-qMDNP (7.0 ± 0.5 pM) and CFZ / PEI-qMDNP (> 14 pM), respectively.Proteasome activity

[0119] Free CFZ used as a positive control induced a rapid reduction in proteasome activity in CFZ-resistant DLD1 cells following 6 h of treatment, but the proteasome activity recovered after 48 post incubation (FIG. 6). CFZ / PLL-qMDNP inhibited the proteasome activity by 50% as effectively as free CFZ while CFZ / PLL-qMDNP and CFZ / PLH-qMDNP showed mediocre proteasome activity inhibition (< 30%) at 6 h post incubation. Interestingly, all qMDNPs showed proteasome activity inhibition greater than free CFZ at 48 h, although proteasome activity recovered in the next 48 h. CFZ / PLL-qMDNP was the only formulation that remained to suppress proteasome activity more than 50% at 96 h post incubation. These results revealed that CFZ / PLL- qMDNP induced the best combination of effective inhibition and prolonged suppression of proteasome activity.Discussion

[0120] CFZ has demonstrated great therapeutic efficacy in treating multiple myeloma yet achieved limited success in treating solid cancers due to pharmaceutical limitations such as low water solubility and poor biostability. To address these problems and maximize therapeutic potential of CFZ, injection formulations that control the delivery and release of the proteasome inhibitor to solid tumors were developed by using qMDNPs. qMDNPs are biocompatible nanoparticles with an average diameter < 50 nm, which is suitable for tumor-specific drug delivery following intravenous injection. Previous studies suggest that qMDNPs have the advantage of low immunogenicity, better stability, versatile structures, and higher drug loading compared to other nano formulations like liposomes and micelles. When designed with optimized particle size and surface characteristics, qMDNPs can increase in vivo blood circulation time, stealth, and drug accumulation in tumors. However, CFZ-loaded qMDNPs still need improvement in suppressing initial burst drug release, which appeared critical to maximize therapeutic outcomes for CFZ against solid cancers.

[0121] In this study, a self-assembled pH sensitive qMDNPs with a high CFZ loading capacity and encapsulation was developed by using polyions as stabilizers (FIG. 2). qMDNPs entrap CFZ- CD inclusion complexes in the core through polyion complexation, which is further stabilized with polyions that neutralize the charge in the nanoparticle core. The inclusion complex ensures highloading of CFZ while polyion complexation stabilized with polyions modulates the electrostatic interactions between the positively charged CFZ-CD inclusion complexes with the negatively charged PEG-PLE to finely tune drug release while improving particle stability without compromising the uniformity of qMDNPs (FIG. 3). All qMDNPs exhibited particle size < 100 nm and neutral surface charge even in the high loading capacity of CFZ (Table 1). The sub-100 nm particles are expected to facilitate the delivery of CFZ to tumors by taking advantage of the leaky vasculature and poor lymphatic drainage in tumor tissue to improve nanoparticle accumulation in tumors, which is also known as the enhanced permeation and retention effect. It must be noted that the surface charge of CFZ / qMDNPs was neutral (± 5 mV) regardless of the polyion used, which is favorable for prolonged retention of the nanoparticles in biological systems by avoiding nonspecific binding to plasma proteins or cellular uptake by the mononuclear phagocytic system. In addition, qMDNPs showed a high encapsulation efficiency of > 80%, which can deliver a large quantify of CFZ to tumors for better efficacy. This is a clear difference from many other nanoparticle formulations with low drug encapsulation that often show insufficient accumulation of cytotoxic agents in disease sites and thus poor therapeutic outcomes.

[0122] Drug release experiments revealed that qMDNPs released CFZ at a slow rate without showing burst drug release at the physiological pH 7.4, which may prolong drug exposure to tumor cells for better efficacy (FIG. 4). The slow drug release was probably due to the core of qMDNPs stabilized by polyions that neutralized the charges between the CFZ-CD inclusion complexes and PEG-PLE polymers and thus made it difficult for the hydrophobic drugs to escape qMDNPs. Interestingly, polyions appeared to trigger dissociation of qMDNPs to accelerate drug release depending on the degree of ionization as a function of pKa that induces charge repulsion in the core of qMDNPs. PLL (pKa ~ 10) indeed maintained particle stability and sustained drug release in a broad pH range (7.4 - 5.0), whereas PLH (pKa ~ 6) and PEI (pKa ~ 7) triggered fast drug release as pH decreased below 6.0. In comparison to PLH that abruptly dissociated at pH 5.0 within 3 h post incubation, PEI showed better stability in acidic conditions up to 24 h post incubation, which was probably due to the buffering effect of the polymer with two pKa values (pKal = 4.5 and pKa2 = 10). Thus, the unique two-phase decay of CFZ / qMDNPs in acidic conditions might be attributed to secondary assembly formation between anionic PLE and cationic CFZ-CD complexes as polyions move around during particle dissociation, although further investigation on the inter- and intra-molecular interactions among polymers within the qMDNP particle is needed.

[0123] The acid-sensitive drug release profiles are potentially beneficial for qMDNPs to deliver CFZ to solid tumors because most tumor sites have acidic microenvironment compared to normal tissue. To this end, qMDNPs may stably travel through the bloodstream under normal physiological conditions (pH 7.4) while accelerating CFZ release in solid tumors with acidosis (pH < 6.8) or endosomes / lysosomes in cancer cells (pH 5-6.5). Such a drug release scenario is partially supported by cytotoxicity assays shown in FIG. 5. Although three qMDNP formulations showed similar particle sizes, drug entrapment yields, and drug release half-life at pH 7.4, they showed distinctive cytotoxic effects against CFZ-resistantDLDl cells. CFZ / PLL-qMDNP showed the highest cytotoxicity as the formulation showed the greatest difference in drug release at varying pH conditions. CFZ / PLL-qMDNP with the greatest drug remaining at 72 h post incubation appeared to effectively kill DLD1 cells that have a doubling time of approximately 20 h, which is consistent with previous findings that drug release rates synchronized with cell growth would be most effective to suppress the growth of tumor cells in vitro and in vivo. In addition, qMDNPs showed a clear pH dependent drug release profile. Even CFZ / PLL-qMDNPs that showed no burst release at physiological pH exhibited a sustained release with half-life of about 44 h (Table 2). When the pH was reduced to 6 and 5, CFZ / PLL-qMDNPs showed a biphasic release profile with the initial drug release half-life less than one hour for both pH values followed by a slow-release half-life of about 25 h at 6.0 and 20 h at 5.0. These results suggest that MDNPs would release CFZ fast in endosomes or lysosomes and increase intracellular drug accumulation for improved inhibition of proteasome activity.

[0124] Related to the observed drug release and cytotoxicity patterns, qMDNPs demonstrated intriguing effects in proteasome activity inhibition as shown in FIG. 6. At early time points of 6 h post incubation, free CFZ and CFZ / PLL-qMDNP were equally effective in suppressing proteasome activity in DLD1 cells. However, CFZ / PLH-qMDNP and CFZ / PEI-qMDNP had mediocre effects on proteasome activity inhibition, which might be attributed to their drug release patterns discussed above. CFZ / PLL-qMDNPs showed no biphasic drug release at pH 7.4, which could allow for qMDNPs to internalize inside the cell without being recognized by P-gp as was previously observed. For CFZ / PLH-qMDNPs and CFZ / PEI-qMDNP, the initial release might have facilitated P-gp activation to reduce intracellular drug concentrations. Over longer period (>48 h), all three qMDNPs suppressed proteasome activity, which is a clear improvement over free CFZ. Targeting the ubiquitin-proteasome pathway has drawn more attention recently as apromising pathway for cancer treatment. Suppression of proteasome activity has been effective for cancer treatment by inducing cell cycle arrest and subsequently apoptosis. It was postulated that CFZ / PLL-qMDNPs killed CFZ-resistant DLD1 cells and suppressed proteasome activity for a prolonged period of time better than free CFZ as they modulated drug release in a pH-dependent manner and avoid drug efflux mediated by P-gp. From this perspective, qMDNPs have great potential to improve the efficacy and toxicity of CFZ in future preclinical applications.Conclusions

[0125] CFZ-loaded qMDNPs stabilized by cationic polymers were developed, which improve biostability and modulate the release of the proteasome inhibitor from the nanoparticles in response to pH conditions, which would be beneficial to increase drug concentrations in solid tumors of acidosis or inside tumor cells overexpressing P-gp drug efflux pumps to greatly improve therapeutic outcomes of CFZ or other proteasome inhibitors.Example 2Abstract

[0126] Neuroendocrine tumors are a class of malignant tumors that arise from cells throughout the diffuse endocrine tumors. One of the most common forms of neuroendocrine tumors, pancreatic neuroendocrine tumors (pNET) frequently present with advanced disease and is often linked to aggressive metastasis to the liver as well as other distant sites. Surgical resection is the main therapy for pNET, but there are limited treatment options for metastatic disease. Nanotherapy using targeted polypeptide nanoparticles have emerged as a viable strategy to deliver potent drugs to metastatic disease. Here, we designed quaternary multi-dimensional nanoparticles (qMDNPs) entrapping a potent chemotherapeutic (SN-38) to treat pNET. To enhance specific delivery, octreotide (Oct), a somatostatin analog targeting somatostatin receptor 2 (SSTR2) is conjugated onto the surface of qMDNPs. These nanoparticles have the advantage of easy formulation, increased drug solubility and targetability. qMDNPs were prepared by a modified solvent evaporation method and characterized for particle size, zeta potential and drug release kinetics. Our results revealed Oct conjugated SN-38 loaded qMDNPs (Oct-qMDNPs / SN-38) having sub- 100 nm size with neutral surface charge. Additionally, Oct-qMDNPs / SN-38 released drug in vitro and produced a reduction in the viability of pNET cells. Octreotide ligand-SSTR2 receptorinteraction on pNET cell surface was confirmed with confocal microscopy and flow cytometry. In vivo treatment of BON lung metastasis with Oct-qMDNP / SN-38 or Oct-qMDNP / PX866 significantly reduced cancer cells viability and metastatic burden. Therefore, taken together, Oct- qMDNPs show ability to efficiently encapsulate potent drugs and preferentially target SSTR2 which holds promise for the treatment of pNET.Introduction

[0127] Neuroendocrine tumors (NET) are a broad family of heterogenous neoplasms that is found in many parts of the body. They are thought to arise from cells of the endocrine system that can diffuse to other organs. The prevalence of NET patients in the United States is over 100,000 with an estimated 16,000 new cases every year. Due to its evasive nature, it is estimated that there are many more undiagnosed cases of NET. Given the wide distribution of NE cells, NET have been found in the respiratory tract, the central nervous system, the gastrointestinal (GI) tract, breast, thyroid, and many other systems. Generally, NET can be categorized into indolent lymphomas and aggressive carcinomas. Patients presenting with indolent carcinoids show a lower risk of metastases and better long-term prognosis, while patients with carcinomas show rapid disease progression, distant metastases, and poor long-term prognosis. Typically, surgical resection is the treatment of choice for low grade tumors, but unresectable carcinomas are treated with chemotherapy such as somatostatin analogues (SSA). FDA approved peptide receptor radionucleotide therapy (PRRT) Lutathera® for gastropancreatic NET have proven to extend patients’ survival, however, they have relatively low efficacy on rapidly dividing tumors.

[0128] In the carcinogenesis of pancreatic NETs, many hormones, growth factors and receptors have been extensively studied as potential culprits. One of the hormones implicated in pNET tumorigenesis is somatostatin (SST). Structurally, SST is a neuropeptide that shows inhibition against many endocrine systems. It is responsible for regulating activities like cell division, secretion, apoptosis, and proliferation. In the blood, naturally occurring SST has a very short half-life of about 1-3 minutes. This has led to efforts to develop synthetic SST that have longer blood half-lives. Several somatostatin octapeptide analogs such as vapreotide, lanreotide and octreotide have been developed and found to be clinically useful. These specifically bind to somatostatin receptors and inhibit cell growth. The expression of a subtype of somatostatin receptor called somatostatin 2 receptor (SSTR2) on the cell membrane of NET is over 20-foldmore than that of normal cells. This makes SSTR2 a useful therapeutic target for treatment of NET. Octreotide is a somatostatin analog that is a substrate of SSTR2. In one study, radiolabeled octreotide 123I-[Tyr3]-octreotide was used as a diagnostic tool to detect the presence of small NET. In another study, radiolabeled octreotide was used to detect tumors that would respond to therapeutic doses of octreotide. Additionally, studies show that somatostatin analogs binding to SSTR2 induce no immunogenicity, therefore octreotide can serve as a useful targeting moiety for targeted delivery. This strategy can be combined with cytotoxic drugs that can induce tumor suppression.

[0129] SN-38 is the active metabolite of Irinotecan which is a topoisomerase I inhibitor. This inhibition leads to loss of DNA replication and transcription. When delivered into the body, Irinotecan is metabolized by carboxylesterases to form SN-38, however less than 10% of the administered Irinotecan is converted to SN-38 therefore high doses of the drug will need to be delivered to achieve minimum efficacious concentration. Furthermore, enzymatic conversion of Irinotecan to SN-38 is dependent of genetic variability of carboxylesterase activity. Therefore it is more attractive to directly administer SN-38 which has been shown to be over 1000 times more active than Irinotecan. However, SN-38 have very poor aqueous solubility which has limited its clinical use since development. Additionally, it has been reported that at biologically relevant pH, the lactone ring in SN-38 is hydrolyzed to form an inactive carboxylate metabolite thus reducing the efficacy of SN-38. Hence, it is important to develop new therapies to improve patient outcomes.

[0130] Nanotherapy has emerged as a promising tool to deliver chemotherapeutics. The need for new formulations that can address clinical limitations like solubility and stability associated with many potent drugs is in demand. Nanoparticle based formulation can mitigate these problems as well as reduce toxicity associated with free drug delivery. Additionally, due to the need for nutrient supply to rapidly forming tumors, the vasculature supplying tumors is poorly developed. They therefore have leaky pores and poor lymphatic drainage. These allow nanoparticles to extravasate into these tumors and enhance accumulation and blood circulation time. This is called the enhanced permeation and retention (EPR) effect. The surface of nanoparticles can also be modified to enhance their targetability to solid tumors. The objective of this study was to develop and assess a novel targeted therapy to deliver cytotoxic agents and treat pNETs. A supram olecul ar assembly comprising of cyclodextrin, and polypeptides was used to develop quaternary multi-dimensional nanoparticles (qMDNPs). Octreotide, a substrate of SSTR2 was used as a targeting ligand to deliver SN-38 or PI3K inhibitor PX-866 loaded qMDNPs to NET cells and the efficacy of this formulation was evaluated in vitro and in vivo.Materials and methodsMaterials

[0131] Octreotide acetate, SN-38 and PX866 were purchased from Med Chem Express (Monmouth Junction, NJ). Heptakis (6-amino-6-deoxy)-pCD heptahydrochloride (HapCD) was purchased from Cyclolab R&D Laboratory Ltd. (Budapest, Hungary). Azido-poly (ethylene glycol)-block-poly (L-glutamic acid sodium salt) (N3-PEG-PLE), methoxy-poly (ethylene glycol)- block-poly (L-lysine hydrochloride salt) (MeO-PEG-PLL) and methoxy-poly (ethylene glycol)- block-poly (L-glutamic acid sodium salt) (MeO-PEG-PLE) were purchased from Alamanda Polymers (Huntsville, AL). Potassium biphthalate sodium hydroxide buffer, formic acid, and HPLC grade water were obtained from Fisher Scientific (Fair-lawn, NJ). Dialysis cups (20,000 MWCO). Alexa-fluor 647-Dibenzocyclooctyne (AFDye 647 DBCO) and Dibenzocyclooctyne-PEG-NHS ester (DBCO-PEG-NHS) were purchased from Click Chemistry Tools (Scottsdale, AZ). Somatostatin Receptor 2 (SSTR2) (NM 001050) Human Tagged ORF Clone Lentiviral Particle was purchased from Origene (Rockville, MD; #RC205146L4V). Octreotide acetate was purchased from MedChemExpress (Monmouth Junction, NJ; #HY-17365).Cell culture

[0132] BON-SSTR2 and QGP-SSTR2 (ATCC) cell lines were cultured in DMEM / F-12 medium supplemented with 10% FBS and 1% Gibco antibiotic-antimycotic in a humidified incubator at 37°C and 5% CO2. The cells were maintained in an exponential growth phase by periodic sub-cultivation.Preparation of Octreotide-qMDNPs

[0133] For preparation of qMDNPs / SN-38, HapCD was incubated with excess of SN-38 at room temperature on a laboratory shaker at 700 rpm for 3 days. After incubation, the complex was sterile filtered through a 0.2pm Acrodisc® syringe filter. MeO-PEG-PLL was added to improve complex stability followed by addition of MeO-PEG-PLE to form qMDNPs. ForqMDNPs / PX866, HapCD was mixed with PX866 dissolved in ethanol at a 2: 1 molar ratio. Ethanol was removed by rotary evaporation under reduced pressure at 60°C. MeO-PEG-PLL was added to the drug-cyclodextrin complex followed by addition of MeO-PEG-PLE to form qMDNPs. For nanoparticle functionalization, octreotide was conjugated to qMDNPs via [3+2] cycloaddition chemistry under sterile conditions. Briefly, 2.05 mg of DBCO-PEG-NHS ester dissolved in DMSO was allowed to react withlO mg of N3-PEG-PLE dissolved in MES buffer pH 6.0 for 3 hours. Afterwards, the reaction mixture was dialyzed against deionized water for 2 h twice and then overnight. Following dialysis, 1.5 mg of octreotide acetate was added to react with the activated ester and reacted for 3 h at room temperature. The resulting reaction mixture was freeze-dried and collected as a dry powder. Reaction was monitored and confirmed by SDS-PAGE.Particle size and Zeta potential

[0134] Size distribution, polydispersity index (PDI) and zeta potential of qMDNPs / SN38 and Oct-qMDNPs / SN-38 were determined by dynamic light scattering (DLS) using the Zetasizer Nano ZS (Malvern Instruments, UK). Freeze-dried samples were dissolved in deionized water and sonicated to a concentration of 2 mg / mL. Measurements were obtained in triplicates using a capillary folded cuvette at 25°C and incidence angle of 173° backscatter.Drug release

[0135] The release of SN-38 from Oct-qMDNPs was investigated using dynamic dialysis in PBS solution (pH 7.4, 10 mM) 37°C. Oct-qMDNPs / SN-38 were transferred into dialysis cups (20,000 MWCO), which were placed in the PBS solution. Aliquots of each sample were collected from the cups at 0, 1, 3, 6, 24, 48 and 72 hours. SN-38 concentrations were analyzed by HPLC (Shimadzu) equipped with Agilent Eclipse XDB-C18 column. The mobile phase was a mixture of acetonitrile (ACN) and HPLC-grade water each containing 0.1% formic acid. An isocratic method of 55:45 of ACN:H2O at a flow rate of 0.5 mL / min was used and SN-38 was eluted at a retention time of 3.4 minutes through a. A drug release profile was fitted to a first order exponential decay using GraphPad Prism 9 software.Cell viability

[0136] BON, BON-SSTR2, QGP and QGP-SSTR2 cells were harvested and seeded in a 96 well plate at a seeding density of 5,000 cells per well. Following attachment overnight, the cellswere treated with serial dilutions of free SN-38 and Oct-qMDNPs / SN-38. The resazurin assay was used to determine cell viability and calculate IC50 values. Briefly, after incubating cells with drug and formulation for 72 hours, 20 pL of resazurin dye solution was added to each well and the plates were placed in an incubator at 37°C and 5% CO2 for 2 h. Fluorescence measurement (Ex: 560 nm, Em: 590 nm) were taken using a plate reader (Spectra Max M5). IC50 values were calculated by a non-linear regression method using GraphPad Prism 9 software.Flow cytometry

[0137] BON-SSTR2 cells were seeded overnight in a 6 well plate at a seeding density of 1 million cells per well. Next day, the media was removed and washed twice with cold PBS. Cells were then digested with accutase and filtered through a cell strainer to remove clumps. Digested cells were diluted in FACS buffer and spun down. After the supernatant was removed, the cell pellets were resuspended in 10% FBS in PBS and placed on ice for 20 minutes to block Fc receptors. Next cell suspension was centrifuged, and after the removal of supernatant, cells resuspended in FACS buffer were treated with Alexa647 labelled Oct-qMDNPs or Alexa647 labelled qMDNPs for 30 minutes on ice. Fluorescence intensity was determined with a BD FACSymphony A3 flow cytometer (BD Biosciences) by counting 30,000 events per sample.

[0138] Confocal imaging. BON, QGP, BON-SSTR2 and QGP-SSTR2 cells were plated onto gelatin coated (0.1% gelatin in deionized H2O) glass coverslips and 24h later treated with qMDNPs or qMDNPs labeled with Alexa647. Cell were fixed with 4% paraformaldehyde (Pierce, #28908, ThermoFisher Scientific) and mounted with antifade mounting medium with DAPI (Vector Laboratories; #H-1800).

[0139] pNET cancer lung metastasis establishment and treatment with qMDNPs. NOD.Cg-RagltmlMom I12rgtmlWjl / SzJ (Strain # 007799) were injected with BON GFP-Luc LungM3 cells iv (IxlO6, 100 pl in PBS). Treatment with qMDNPs started 5 days after cancer cells injection in 200 pl of PBS, iv. Mice were treated twice a week for a total of 6 injections and imaged for bioluminescent signal with Lago SII (Spectral Instruments Imaging, Tucson, AZ).Results

[0140] Development of quaternary multi-dimensional nanoparticles. In this study we prepared a qMDNPs formulation for SN-38 delivery in vitro and in vivo. (Figure 7). qMDNPs entrap drugs through three molecular interactions (hydrophobic, ionic, and supra-molecular) in three steps: 1) SN-38 was entrapped in the cavity of heptakis(6-amino-6-deoxy)-P-cyclodextrin(- hepta-hydrochloride) (Ha0CD) through hydrophobic interaction, 2) the drug-cyclodextrin inclusion complexes were mixed with azido-poly( ethylene glycol)-block-poly(L-glutamic acid sodium salt) (N3-PEG-PLE) to form polyionic complex nano-particles, and 3) the nanoparticles were modified with fluorescent dyes (Alexa 647) for imaging and / or octreotide peptide for SSTR2 cell receptor targeting. The hydrophobic interaction between cyclodextrin and drug molecules provides a fourth molecular interaction that further ensures the entrapment of SN-38. The ionic interaction between SN-38-loaded cyclodextrins with positive amines and anionic carboxylates on N3-PEG-PLE polymers significantly reduces the amount of cyclodextrin used to solubilize and protect the drug in an aqueous solution. The supramolecular interactions among drug, cyclodextrin, and polymers are beneficial to control the shape, size, and other physicochemical properties of qMDNPs by minimizing the variations in nanoparticle batches and drug payloads. The octreotide peptide was used for receptor targeting of nanoparticles to the somatostatin receptors SSTR2 and SSTR5. SSTR2 is the most frequently expressed subtypes in both gastrointestinal NETs.Physiochemical properties of qMDNPs and drug release characterization

[0141] Dynamic light scattering was used to assess the particle size and zeta potential of qMDNPs. Particle histograms show that qMDNPs / SN-38 and Oct-qMDNPs / SN-38 had unimodal size distribution with sub-100 nm particle size (Figure 8). qMDNPs / SN-38 were approximately 44 nm while Oct-qMDNPs / SN-38 were 59 nm with both particles exhibiting neutral surface charge (Table 1)Table 1. Physiochemical properties of qMDNPsFormulation Particle size (nm) Pdl Zeta Potential(mV) qMDNPs / SN-38 43.88 ± 2.2 0.094 1.34 ± 0.02Oct-qMDNP / SN- 58.67 ± 3.4 0.290 1.79 ± 0.0538

[0142] qMDNPs achieved sustained release of SN-38 compared to natural PCD inclusion complexes used as a control. Both qMDNPs and control showed biphasic drug release profiles with initial burst drug release as shown in Figure 9. However, qMDNPs exhibited sustained drug release after the initial burst with a drug release slow half-life of ~ 20 h. Kinetic parameters for drug release profiles are summarized in Table 2.Table 2. Kinetic parameters of SN-38 release.Formulation ti / 2fast (hr) ti / 2siow (hr) Kfast (hr ^) Ksiow(hr ') pCD / SN-38 0.197 7.3690.018 qMDNPs / SN-38 0.513 18.73 0.291 0.007Increased cytotoxicity of SSTR2-targeted qMDNPs

[0143] To compare the effects of qMDNPs and Oct-qMDNP, BON and QGP cells were treated for 2h with qMDNPs, then cell culture media with nanoparticles was removed and replaced with fresh media. SSTR2 -targeted nanoparticles bind to cancer cell surface receptor and enhance cytotoxicity through increased retention and drug delivery into cancer cells compared to qMDNPs without octreotide. Our results confirmed that the Oct-qMDNPs / SN-38 treatment resulted in lower IC50 values in comparison to qMDNPs / SN-38 (Figure 10, Graph A). IC50 value in BON cells treated with qMDNPs / SN-38 was 80.47 nM and IC50 value in Oct-qMDNPs / SN-38 was significantly reduced to 37.32 nM (2.15x decrease). For QGP cell lines, qMDNPs / SN-38 IC50 value was 104.3 nm while cells treated with Oct-qMDNPs / SN-38 had an IC50 of 28.76 nM (3.62x). Finally, we examined IC50 in BON and QGP cells with SSTR2 overexpression; IC50 value in BON-SSTR2 cells was 2.4x lower in Oct-qMDNPs / SN-38 group compared to qMDNPs / SN-38 group. IC50 value in QGP-SSTR2 cells was 4.67x lower in Oct-qMDNPs / SN-38 group compared to qMDNPs / SN-38 group (Figure 10, Graph B). Table 3 shows a summary of IC50 values.Table 3. The half maximal inhibitory concentration (ICso) values of qMDNPs / SN-38, and Oct-qMDNP / SN-38 against BON, BON SSTR2, QGP and QGP SSTR2 cell lines after 72 h treatmentIC50 (nM, mean ± SD)FormulationBON BON SSTR2 QGP QGP SSTR2 qMDNPs / SN-38 80.47 ± 5.64 12.00 ± 1.02 104.3 ± 7.90 122.9 ± 8.80Oct-qMDNPs / SN-38 37.32 ± 1.99 5.01 ± 1.03 28.76 ± 3.51 26.29 ± 2.51Confirmation of qMDNP binding to SSTR2 receptor

[0144] Cytotoxicity analysis demonstrated increased cytotoxicity with SSTR2 -targeted drug delivery. Next, we used two more methods to demonstrate specificity of Oct-qMDNPs binding to SSTR2 receptor. First approach utilizes unique ability of SSTR2 receptor to internalize uponbinding to a ligand. BON and QGP cells were established with overexpression of GFP -tagged SSTR2 receptor. To confirm SSTR2-GFP tagged receptor function and response to octreotide, BON-SSTR2 and QGP-SSTR2 cells were treated with octreotide at 1000 nM concentration for 30 min, Ih, 2h, 3h and 24h. SSTR2 receptor translocation into cytoplasm was observed at all timepoints. SSTR2-GFP receptor translocation after Oct-qMDNP treatment had same pattern of GFP -tagged SSTR2 accumulation in cytoplasmic fraction of cancer cells observed after octreotide treatment. Analysis of fluorescently labeled qMDNPs binding to BON cells by confocal microscopy and flow cytometry also demonstrated specificity of Oct-qMDNP-Alexa647 binding to SSTR2 receptor compared to qMDNP-Alexa647. The qMDNP-AF647 and Oct-qMDNP- AF647 accumulated on the BON-SSTR2 cell surface within 30 minutes of incubation, displayed as blue and orange groups; Oct-qMDNP -AF647 showed receptor specific binding as indicated by a 2-fold increase in binding compared to qMDNP-AF647 (Figure 11).BON lung metastasis therapy with qMDNPs in vivo.

[0145] BON lung metastasis was established with iv injection of BON cell trained to metastasize to lungs (BON GFP-Luc LungM3). First, we examined distribution of fluorescently labeled qMDNPs in vivo. Mice received single injection of PBS, qMDNP-Alexa647 or Oct- qMDNP-Alexa647 (300 pl; iv); organs were collected 30 min after iv injection and analyzed for presence of metastatic tumors (GFP signal) and fluorescent nanoparticles accumulation (Alexa647). Oct-qMDNP-Alexa647 nanoparticles accumulated in a lung, organ most affected by metastatic disease, compared to qMDNP-Alexa647.

[0146] Next, we established BON lung metastasis and started treatment with qMDNPs 5 days after cancer cells injection. Mice were randomized into four groups and received Oct-qMDNP (200 pl; 7.5 mg / kg), Oct-qMDNP-PX866 (200 pl; 7.5 mg / kg), Oct-qMDNP-SN-38 (200 pl, 0.5 mg / kg). Combination therapy was administered by mixing of Oct-qMDNP-SN-38 (100 pl; 3.75 mg / kg) and Oct-qMDNP -PX-866 (100 pl; 0.25 mg / kg). Treatment was performed twice a week for a total of 6 treatments; combination therapy was administered for a total of 3 treatments. Metastatic burden was assessed on day 32 after cancer cells injection and demonstrated both reduction in whole body metastasis and lung metastasis burden. Whole body metastatic burden was reduced in Oct-qMDNP-SN-38, Oct-qMDNP -PX-866 and combination group by over 60% compared to a control group. Lung metastasis burden was decreased by approximately 70% compared to a controlgroup. Therefore, these results demonstrated successful in vivo application of qMDNPs to treat pNETs by SSTR2 -targeted delivery of chemotherapy agents.Discussion

[0147] There is an urgent need to develop precise delivery of drugs to metastatic tumor sites. Somatostatin receptor 2 is an ideal target for the precise treatment of cancers that overexpress SSTR2 receptor. We developed a targeting nano-drug delivery system comprising anti-SSTR2 octreotide surface-modified nanoparticles encapsulating drugs. qMDNPs were quickly internalized by SSTR2-ov erexpressing pNET cells, triggering the release of loaded drugs, cytotoxicity and reduced metastatic burden. qMDNPs were confirmed to have an average of 50 nm particle size, which remained unchanged after SN-38 entrapment or lyophilization. These properties are unique in comparison to other nanoparticle formulations that often alter particle size and stability during storage and use. The uniform size and improved stability of qMDNPs appeared beneficial to entrap SN-38 with a reduced amount of cyclodextrin as opposed to a commercially available CFZ formulation Captisol®. Our results show that qMDNPs can enhance drug encapsulation at a far lower ratio of drug: cyclodextrin (l : lor 1 :2 maximum) than Captisol® that requires >50-fold cyclodextrin molecules to dissolve the same amount of drug molecules. Captisol® is administered intravenously into patients for several hours of infusion, minimum 4 days per week. The long drug infusion time is attributed to potential side effects of excipients added in a large amount while maintaining plasma drug concentrations for effective treatment. Thus, the higher drug loading content of qMDNPs with a lower amount of excipients such as cyclodextrin would allow for shorter injection time for a larger amount of drug for rapid therapeutic outcomes. Nanoparticles stored in aqueous solutions often show reduced stability limiting their clinical use.

[0148] One of the most significant findings from this study is that qMDNPs improve cytotoxicity of SN-38 with SSTR2 -mediated cell targeting both in vitro and in vivo, due to enhanced intracellular uptake of the particles through SSTR2 targeting. The small particle size of qMDNPs maintained after octreotide conjugation was also unique. Surface modification of nanoparticles often results in variations in size and particle stability, which negatively affects in vivo drug delivery performance and therapeutic outcomes of nanoparticle formulations. Whereas, qMDNPs may prolong blood circulation with minimum disparity among formulation batches andensure reduced glomerular filtration from the kidney to potentially increase their residence time in tumors (57). High drug entrapment in qMDNPs may be beneficial to reduce the amount of material injected into the body for the same therapeutic outcomes. qMDNPs were designed to entrap SN- 38 by forming a strong inclusion complex in the lipophilic core of HapCD that are further enveloped in the core of a poly ion complex made from cationic cyclodextrin and anionic block copolymers. Ionic interactions between the amine groups of HapCD and carboxylic groups on N3- PEG-PLE self-assemble into nanoparticles producing the ‘ternary’ system. qMDNPs appeared more effective than pCDs to maintain particle integrity for a prolonged time in the presence of serum proteins. The improved particle integrity will be beneficial for in vivo applications of qMDNPs. qMDNPs showed a bimodal drug release profile with a rapid release followed by a sustained release. The ternary structure of qMDNPs seems to play a major role to finely tune drug release profiles, and thus further investigation is warranted to achieve more sophisticated drug release control.Conclusion

[0149] In this study, we have developed qMDNPs as a delivery formulation for SN-38 to address pharmaceutical limitations of the inhibitor such as solubility, stability and targeted delivery. qMDNPs significantly improved entrapment yields, biostability, storage, and in vitro efficacy of SN-38 as opposed to a cyclodextrin-based formulation commercially available. Furthermore, qMDNPs maintained nanoparticle integrity and stability regardless of drug entrapment and octreotide conjugation. These promising results provide a strong basis for future investigation of qMDNPs on their in vivo performance to treat cancers overexpressing SSTR2 receptor, including pNETs.Example 3

[0150] qMDNPs entrapping hydrophilic drugs are prepared by using a solvent evaporation method as reported previously with slight modification. Briefly, CD and PEG-PLE are mixed in deionized water in the presence of citric acid to prepare loosely aggregated complexes. Hydrophilic drugs (e.g., cisplatin, oxaliplatin, carboplatin, or gemcitabine) dissolved in deionized water are added to the solution. After sonicating the solution for 5 minutes, the internal core of qMDNPs is stabilized with polyions (e.g., poly(L-lysine), Poly(L-histidine) ,or polyethyleneimine)to cancel out any remaining charges and prepare tightly assembled polypeptide complexes. qMDNPs entrapping hydrophilic drugs are lyophilized overnight (Labconco Freezone®) using 2% (w / v) sucrose as a cryoprotectant to obtain a fine powder of nanoparticles. The primary and secondary drying is performed at -50°C and 25°C for 24 h, followed by storage at -20°C prior to use.

[0151] If desired a targeting agent may then be conjugated to the surface of the biocompatible shell of the qMDNPs in a manner known in the art.Example 4

[0152] qMDNPs co-entrapping hydrophobic and hydrophilic drugs are prepared by using a solvent evaporation method as reported previously with slight modification. Briefly, hydrophobic drugs (e.g., CFZ) in ethanol are mixed with CD dissolved in deionized water at a 1 :4 mass ratio in a 50 mL round bottom flask. Citric acid is added to the mixture to facilitate complexation between CFZ and CD while preventing the inclusion complexes from precipitating. After sonicating the solution for 2 minutes, ethanol is removed by rotary evaporation at 60°C with reduced pressure. Following the removal of ethanol, PEG-PLE is added to the aqueous solution of CFZ / CD inclusion complexes and hydrophilic drugs (e.g., cisplatin, oxaliplatin, carboplatin, or gemcitabine) are added. The solution was sonicated for 5 minutes to stabilize the internal core of qMDNPs with polyions (e.g., poly(L-lysine), Poly(L-histidine) ,or polyethyleneimine) to cancel out any remaining charges. qMDNPs co-entrapping hydrophobic and hydrophilic drugs are lyophilized overnight (Labconco Freezone®) using 2% (w / v) sucrose as a cryoprotectant to obtain a fine powder of nanoparticles. The primary and secondary drying is performed at -50°C and 25°C for 24 h, followed by storage at -20°C prior to use.

[0153] If desired a targeting agent may then be conjugated to the surface of the biocompatible shell of the qMDNPs in a manner known in the art.

[0154] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

What is claimed:

1. A quaternary multi-dimensional nanoparticle (qMDNP), comprising: a nanoparticle, of cyclodextrin and a biocompatible polymer, having a biocompatible shell protecting an internal core wherein the cyclodextrin is selected from a group consisting of (a positively charged functional group)-substituted cyclodextrin, (a negatively charged functional group)-substituted cyclodextrin, and both (a positively charged functional group)-substituted cyclodextrin and (a negatively charged functional group )-substituted cyclodextrin; a polyion adapted to stabilize the internal core; and a therapeutic agent entrapped in a cyclodextrin inclusion complex in the internal core.

2. The qMDNP of claim 1, wherein the therapeutic agent is a hydrophobic therapeutic agent.

3. The qMDNP of claim 1, wherein the therapeutic agent is a hydrophilic therapeutic agent.

4. The qMDNP of claim 1, wherein the polyion is selected from a group of block copolymers or homopolymers .

5. The qMDNP of claim 1, wherein the therapeutic agent is selected from a group consisting of a small molecule drug of less than 1 kDa.

6. The qMDNP of any of claims 1-5, further including a targeting agent conjugated to the nanoparticle.

7. The PNP of any of claims 1-5, further including a targeting agent conjugated to a surface of the biocompatible shell.

8. The qMDNP of claim 7, wherein the targeting agent is selected from a group consisting of octreotide (Oct) targeting somatostatin receptor 2, folic acid targeting folate receptor alpha, molecules targeting hormone receptor and PD-L1, cancer targeting peptides (CTPs) and combinations thereof.

9. The qMDNP of claim 8, wherein the cyclodextrin is amine-substituted.

10. The qMDNP of claim 9, wherein the biocompatible polymer is poly(ethylene glycol )-poly(L- glutamic acid) (PEG-PLE).

11. The qMDNP of claim 8, wherein the biocompatible polymer is poly(ethylene glycol)-poly(L- glutamic acid) (PEG-PLE).

12. The qMDNP of claim 8, wherein the cyclodextrin is carboxyl group-substituted.

13. The qMDNP of claim 8, wherein the cyclodextrin includes both amine-substituted cyclodextrins and carboxyl group- substituted cyclodextrins.

14. The qMDNP of claim 1, wherein the cyclodextrin is an amine-substituted P-cyclodextrin and the biocompatible polymer is poly(ethylene glycol)-poly(L-glutamic acid) (PEG-PLE).

15. The qMDNP of claim 1, further including a hydrophilic therapeutic agent entrapped in a polypeptide complex in the internal core.

16. A quaternary multi-dimensional nanoparticle (qMDNP), comprising a therapeutic agent- cyclodextrin inclusion complex enveloped in a biocompatible polymer and including selfassemblies, polyion complexes, and polypeptide complexes.

17. The qMDNP of claim 16, wherein the therapeutic agent is selected from a group consisting of SN-38, PX-866, carfilzomib (CFZ), fluorouracil, oxaliplatin, capecitabine, temozolomide, dacarbazine, streptozotocin, doxorubicin, gemcitabine, etoposide, cisplatin, carboplatin, paclitaxel, docetaxil and combinations thereof.

18. The qMDNP of claim 16, wherein the biocompatible polymer is poly(ethylene glycol)- poly(L-glutamic acid) (PEG-PLE).

19. The qMDNP of claim 16, wherein the cyclodextrin is an amine-substituted P-cyclodextrin.

20. A quaternary multi-dimensional nanoparticle (qMDNP), comprising: self-assemblies of micellized block copolymers forming a biocompatible shell protecting an internal core; inclusion complexes, of charged functional group-substituted cyclodextrin and a small molecule therapeutic agent of less than 1 kDa, held in the internal core and stabilized by polyion complexation between the charged functional group-substituted cyclodextrin and the block copolymers; andpolyions held in the internal core, said polyions forming polypeptide complexes with the block copolymers.

21. A method of making quaternary multi-dimensional nanoparticles (qMDNPs), comprising: mixing a therapeutic agent with a cyclodextrin to create a therapeutic agent-cyclodextrin inclusion complex wherein the cyclodextrin is selected from a group consisting of (a positively charged functional group)-substituted cyclodextrin, (a negatively charged functional group)- substituted cyclodextrin, (a positively charged functional group and a negatively charged functional group)-substituted cyclodextrin and combinations thereof; adding a biocompatible polymer with counter charges to the therapeutic agent- cyclodextrin inclusion complex and sonicating in the presence of a polyion to produce polyion complexes and form polypeptide nanoparticles (qMDNPs);and lyophilizing the qMDNPs to obtain a fine powder of nanoparticles wherein the therapeutic agent is held in a core of a biocompatible shell.

22. The method of claim 21, including conjugating a targeting agent to a surface of the biocompatible shell.

23. The method of claim 21, wherein the mixing of the therapeutic agent with the cyclodextrin includes (a) adding the therapeutic agent in alcohol to the cyclodextrin dissolved in deionized water to create a reaction mixture, (b) adding citric acid, or hydrophilic drugs with counter charges, to facilitate complexation between the therapeutic agent and the cyclodextrin while preventing the inclusion complexes from precipitating, (c) sonicating the reaction mixture, and (d) removing the alcohol by evaporation.

24. The method of any of claims 21-23, further including using mixing ratios of the polyion to neutralize charges in the core.

25. The method of claim 24, including using a cryoprotectant during the lyophilizing.

26. The method of claim 20 wherein the therapeutic agent is a hydrophobic therapeutic agent entrapped in a cyclodextrin inclusion complex in the internal core.

27. The method of claim 26, further including adding hydrophilic drugs, with counter charges to the biocompatible polymer, entrapped in a polypeptide complex in the internal core.

28. A method of delivering a therapeutic agent to a patient, comprising administering to the patient a pharmaceutically effective amount of the qMDNPs set forth in claim 1.

29. A method of delivering a therapeutic agent to an organ or a tumor, comprising administering the qMDNPs of claim 1 to the organ or cell, wherein the biocompatible polymer dissociates from the nanoparticles or hydrolyze upon encountering a pH of less than about 7 to release the therapeutic agent.

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