Fluoropyrimidine particles for the treatment of cancer

Abstract

A new class of multi-functional lipid nanoparticles comprising a fluoropyrimidine-based therapeutic and a combination of helper lipids is disclosed. The disclosed lipid nanoparticles are formulated for efficient delivery of therapeutic agents to target cells and can be used in the treatment of cancers, including colorectal cancer. The disclosed lipid nanoparticles exhibit improved cellular uptake and increased toxicity to cancerous cells compared to free- fluoropyrimidine therapeutic and other conventional therapeutics. A method for treating cancer by administering the disclosed lipid nanoparticles is also disclosed herein.

Description

FLUOROPYRIMIDINE PARTICLES FOR THE TREATMENT OF CANCERSTATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant Number CA284083 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS REFERENCE OT RELATED APPLICATION

[0002] This application claims the benefit of priority under 35 U. S. C. §119(e) to U. S. Application No. 63 / 711,507, filed October 24, 2024, which is incorporated herein by reference in its entirety.FIELD

[0003] The invention relates generally to the fields of medicine, oncology, and molecular biology. In particular, the invention relates to cytotoxic fluoropyrimidine polymer nucleic acid molecules, compositions, and methods for treating cancer in an individual.BACKGROUND

[0004] Colorectal cancer (CRC) ranks as the third leading cause of cancer-related deaths in the U. S. and the world. CRC that metastasizes to the liver is rarely resectable, and fluoropyrimidine (FP)-based chemotherapy is often the preferred treatment option. 5 -Fluorouracil (5-FU) is the FP drug most widely used to treat metastatic CRC (mCRC) and is used in combination with leucovorin and oxaliplatin in the FOLFOX regimen and with leucovorin and irinotecan in the FOLFIRI regimen. While 5-FU is effective in the context of FOLFOX and FOLFIRI (5-FU, irinotecan, and leucovorin regimen) combination chemotherapy, it is inefficiently converted to the primary active metabolite, 5-fluoro-2'-deoxyuridine monophosphate (FdUMP), and it is readily converted to metabolites that contribute to systemic toxicities, including fluorouridine triphosphate (FUTP) and alpha-fluoro-beta-alanine (FBAL).

[0005] As a consequence, the antitumor activity of 5-FU-based therapy is sub-optimal, and many patients treated with 5-FU-based regimens experience high-grade toxicities.6 A subset of patients who inefficiently catabolize 5-FU are at high risk for serious toxicities or death if dose adjustments are not made prior to initiating treatment. Due to these limitations, there is a need to develop next-generation FP drugs and drug delivery vehicles that provide an improved therapeutic response and reduced toxicity profile.

[0006] While FP-based drugs or compounds remain among the most effective drugs used to treat mCRC, particularly, there are limitations of 5-FU that decrease its clinical efficacy. Specifically, 5-FU is rapidly degraded and excreted (~15 min half-life; 85% degraded or excreted intact), and it affects RNA function through misincorporation of the ribonucleotide form FUTP into RNA, which causes gastrointestinal (GI) toxicities that are often dose-limiting and may be life-threatening. Moreover, while FP-based drugs show promise for mCRC treatment with delivery as a “naked” DNA-based nanomaterial, in principle, packaging of these drugs in a nanocarrier could further improve their efficacy advantage relative to their base form by increasing plasma half-life and decreasing extracellular degradation, ultimately reducing the required dosage to achieve a comparable therapeutic effect.

[0007] Given the foregoing, the development of a drug carrier that overcomes these limitations, achieves efficient delivery of these drugs to the cells, and reduces the high mortality rate associated with advanced CRC is greatly needed. This document describes the composition and method that address some or all of the problems described above.SUMMARY

[0008] First aspect of the disclosure relates to a lipid nanoparticle comprising a nucleic acid molecule comprising FdUMP having a polyethylene glycol (PEG) spacer appended to a 5’-terminus and a moiety appended to a 3’-terminus, wherein the moiety is not recognized by 3’-exonucleases; an ionizable lipid; a phospholipid; a sterol-derived lipid; and a pegylated lipid.

[0009] In some embodiments, the moiety may bee, wherein R₁ is H or OH; R₂ is H, halogen or OH. In some embodiments, and is a PEG spacer, wherein the PEG spacer has a molecular weight ranging from about 5,000 to about 40,000 daltons.

[0010] In some embodiments, the nucleic acid molecule iswherein X is 1 to 10, Y is 1 to 5.

[0011] In some embodiments, the ionizable lipid is selected from the group consisting of l'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 2-[2,2-Di-[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N, N-dimethyl-2,3-bis[(9Z, 12Z)-octadeca-9, 12-dienoxy]propan-l -amine (HGT4003), (15Z, 18Z)-N, N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-15,18-dien-l-amine, (HGT5000), (15Z, 18Z)-N, N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-4, 15, 18- trien-1 -amine (HGT5001), l,2-dioleoyl-3-trimethyl ammonium-propane (DOT AP), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), and a combination thereof.

[0012] In some embodiments, the phospholipid is selected from the group consisting of Dilauroyl lecithin (DLPC), Dimyristoyl phosphatidylcholine (DMPC), Dioleoyl lecithin (DOPC), Dipalmitoyl phosphatidylcholine (DPPC), Distearoyl phosphatidylcholine (DSPC), Dioleoyl phosphatidylcholine (DUPC), Palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-Di-O-octadecyl-sn-glycero-3 -phosphocholine, l-Oleoyl-2-cholesteryldimethylsuccinoyl-sn-glycero-3-phosphocholine, 1 -Hexadecyl-sn-glycero-3 -phosphocholine, 1,2-Divinyl-sn-glycero-3 -phosphocholine, l,2-Diarylacyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3 phosphorylethanolamine (DOPE), 1, 2-Di-phytanoyl-sn-glycero-3 -phosphoethanolamine, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, l,2-Diethenol-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl-sn-glycero-3 -phosphoethanolamine, 1,2-Diaryl-sn-glycero-3 -phosphoethanolamine, l,2-Dithiohexaenoate-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate-(l -glycerol) sodium salt (DOPG), and a combination thereof.

[0013] In some embodiments, the sterol-based lipid is selected from the group consisting of a cholesterol, coprosterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, bile acid, and a combination thereof.

[0014] In some embodiments, the pegylated lipid is selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000 (14:0 PEG2000 PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DOPE-PEG), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG),PEG-disteryl glycerol (DSG-PEG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (DAG-PEG), PEG-dipalmitoyl phosphatidylethanolamine (DPPE-PEG), or PEG-l,2-dimyristyloxlpropyl-3-amine (DMA-c-PEG), and a combination thereof.

[0015] In some embodiments, the ionizable lipid comprises l'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), the phospholipid comprises 1,2 Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the sterol-derived lipid may comprise cholesterol, and the pegylated lipid may comprise l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000.

[0016] In some embodiments, a mass ratio between total lipid component and the nucleic acid molecule is about 5 to about 30.

[0017] In some embodiments, the ionizable lipid is present in an amount ranging from about 5% to about 95%, by total weight of the lipid nanoparticle, the phospholipid is present in an amount ranging from about 1% to about 80% by total weight of the lipid nanoparticle, the sterol -derive lipid is present in an amount ranging from about 1% to about 80%, and the pegylated lipid is present in an amount ranging from about 1% to about 20%, by total weight of the lipid nanoparticle.

[0018] In some embodiments, the lipid nanoparticle comprises an additional therapeutic agent, wherein mass ratio between the lipid nanoparticle and the additional therapeutic agent is from about 5 / 1 to about 20 / 1. In some embodiments, the additional therapeutic agent forms a non-covalent complex with the lipid nanoparticle. In some embodiments, the additional therapeutic is selected from the group consisting of irinotecan, leucovorin, oxaliplatin, Ataxia-telangiectasia andRad3-related (ATR) inhibitors, Weel inhibitors, SLFN11 mRNA, GEM-DEC or siRNA targeting TS, and a combination thereof.

[0019] In some embodiments, the lipid nanoparticle is modified at surface of the nanoparticle with a surface modifier selected from the group consisting of cell surface receptors, antibodies, antibody fragments, peptide, aptamers, small molecules, cleavable linkers, cellpenetrating peptides, and a combination thereof. In some embodiments, the nanoparticle surface may be modified with folinic acid.

[0020] In another aspect, the present disclosure relates to a method of treating cancer comprising administering to a subject an effective amount of the lipid nanoparticle disclosed herein. In some embodiments, the cancer is a colorectal cancer.

[0021] In some embodiments, the method further comprises administering to the subject an additional therapy selected from the group consisting of immunotherapy, chemotherapy, radiotherapy, surgery, and a combination thereof. In some embodiments, the cancer is resistant to prior treatment of 5-fluorouracil (5-FU).

[0022] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 Illustrates a reaction scheme for functionalizing folinic acid to a pegylated lipid through click chemistry.

[0024] FIG. 2 illustrates a reaction scheme for attaching Cy5.5 to CF10 through click chemistry.

[0025] FIG. 3A illustrates cellular uptake of CF10 lipid nanoparticles and free-CFlO in LS174T cells.

[0026] FIG. 3B illustrates cellular uptake of CF10 lipid nanoparticles and free-CFlO in HCT116 cells.

[0027] FIG. 4A illustrates cytotoxic potency of CF10 lipid nanoparticles, free-CFlO, and 5-FU inLS174T cells.

[0028] FIG. 4B illustrates cytotoxic potency of CF10 lipid nanoparticles, free-CFlO, and 5-FU in HCT116 cells.

[0029] FIG. 5 illustrates thymidylate synthase inhibition of CF10 lipid nanoparticles, free-CF10, and 5-FU.

[0030] FIG. 6 illustrates in vivo tumor reduction of CF10 lipid nanoparticles, free-CFlO, and 5-FU in MC38 cells.DETAILED DESCRIPTION

[0031] The following discussion omits or only briefly describes conventional features of the disclosed technology that are apparent to those skilled in the art. Reference to various embodiments does not limit the scope of the claims attached hereto. Additionally, any examples set forth in this specification are intended to be non-limiting and merely set forth some of the many possible embodiments for the appended claims. Further, particular features described herein can be used in combination with other described features in each of the various possible combinations and permutations. A person of ordinary skill in the art would know how to make and use the disclosed technology, in combination with routine experiments, to achieve other outcomes not specifically disclosed in the examples or the embodiments.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methodsare described herein. In describing and claiming the present disclosure, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0033] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, recitation of “a cell”, for example, includes a plurality of cells of the same type. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0034] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20%, + / - 10%, + / - 5%, + / -1%, or + / - 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0035] As used herein, the terms “agent” or “therapeutic agent” are meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. Nonlimiting terms include small molecule compounds, antisense reagents, mRNA, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fab’, F(ab’)2 fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domainpeptides and monobodies), peptoids, aptamers; hormones, oligonucleotides, enzymes, peptides, organic or inorganic small molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the disclosure at any stage during clinical trials, during pre-trial testing, or following FDA approval.

[0036] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open-ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. include those specified elements— or, as appropriate, equivalents thereof— and that other elements can be included and still fall within the scope / defmition of the defined item, composition, apparatus, method, process, system, etc.

[0037] An “effective amount,” as used herein means an amount that provides a therapeutic or prophylactic benefit.

[0038] The term “fully encapsulated” as used herein indicates that the payload in the nanoparticles is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10%, and most preferably less than 5% of the particle nucleic acid is degraded. Fully encapsulated also indicates that the particles are serum stable, that is, that they do not rapidly decompose into their parts upon in vivo administration.

[0039] As used herein, the term '7 / 7 vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0040] As used herein, the term “w vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0041] The term “lipid nanoparticle” refers to a nanoparticle that includes lipids and that is stable and dispersible in aqueous media. As used herein, the term “nanoparticle” refers to a particle having one or a plurality of components, the particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties as compared to a bulk sample of the same material or component materials. Routinely, nanoparticles have one structural feature on a scale of less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, or less than about 100 nm. In exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10 nm to about 500 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10 nm to about 1000 nm. A spherical nanoparticle would have a diameter, for example, of between 10 nm to about 100 nm or 10 nm to about 1000 nm.

[0042] A nanoparticle most often behaves as a unit in terms of its physical or biophysical properties, e.g., transport. It is noted that novel properties that differentiate nanoparticles from the corresponding bulk material typically develop at a size scale of under 1000 nm, or at a size of under 500 nm, but nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. The size at which materials display different properties as compared to the bulk material is material-dependent and can be seen for many materials much larger in size than 100 nm and even for some materials larger in size than 1000 nm. Nanoparticles can be employed in a variety of drug delivery technologies (e.g., siRNA delivery technologies) and can be employed for various purposes, including, but not limited to, controlled drug delivery, protection of the drugs from degradation, and protection of the body from the toxic effects of the drugs.

[0043] As used in this specification and the appended claims, the term “or” is generally employed in its sense, including “and / or” unless the content clearly dictates otherwise.

[0044] As used herein, the term “comprising”, which is synonymous with “including”, “containing”, and “characterized by”, is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. “Comprising” is a term of art that means that the name elements and / or steps are present, but that other elements and / or steps can be added and still fall within the scope of the relevant subject matter.

[0045] As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0046] As used herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0047] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous, intravenous, intramuscular, intrathecally, or intrastemal injection, or infusion techniques.

[0048] The terms “patient” and / or “individual” and / or “subject” are used interchangeably herein, and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the disclosure find use in experimental animals, in veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents, including mice, rats, hamsters, and primates.

[0049] As used herein, a “pharmaceutically acceptable” component / carrier, etc., is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0050] Treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those for whom the disorder is to be prevented. As defined herein, a “therapeutically effective” amount of a compound or agent (i.e., an effective dosage) means an amount sufficient to produce a therapeutically (e.g., clinically) desirable result. The compositions can be administered from one or more times per day to one or more times per week, including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, the treatment of a subject with a therapeutically effective amount of the compounds of the disclosure can include a single treatment or a series of treatments.

[0051] Ranges: Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, or 6. This applies regardless of the breadth of the range.

[0052] Efficient delivery of potent FP-based drugs in patients diagnosed with colorectal cancer remains a significant challenge. In this regard, the present document provides novel lipid nanoparticles (LNPs) as a preferred nanocarrier for nucleic acid therapeutics because of their biocompatibility and efficient delivery of diverse nucleic acid cargo, including mRNA and siRNA. The packaging of nucleic acid cargo in LNPs is based on ionic interactions between an ionizable cationic lipid, such as l,l'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C 12-200), and the phosphodiester backbone of the nucleic acid. The use of LNPs for the delivery of low molecular weight, small molecule drugs such as 5-FU or other FP-based drugs, however, remains unproven, and it is uncertain whether LNPs would form efficiently and if the resulting LNPs would display sufficient stability for uptake into cells and in vivo delivery. In the case of FP-based compounds, the packaging into LNPs also raises concerns over whether the drug would undergo efficient release from the LNP following cell uptake with conversion to the active component nucleotides.

[0053] In contrast to liposomes and cationic liposomes, lipid nanoparticles have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in their interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound, thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size, ranging from about 10 nm to 1000 nm.

[0054] In the present document, a novel FP-based lipid nanoparticle system that provides efficient delivery of potent therapeutic agents for the treatment of mCRC is disclosed. The disclosed system is formulated by adapting LNP technology with a mixture of an ionizable lipid, a phospholipid lipid, a sterol-derived lipid, and a pegylated lipid in an optimized ratio. The disclosed system forms in the desired target range for drug delivery to take advantage of the enhanced permeability and retention effect (80-200 nm) and displays sufficient stability for cell uptake and in vivo delivery. Further, the disclosed LNP system presents a more effective, efficient, and selective therapeutic option relative to a free FP-based drug, as the FP-based LNPs are more potent than the naked drug delivering equivalent FP content, and FP-based LNPs are preferentially internalized by cancer cells relative to non-malignant cells due to improved uptake via endocytosis.

[0055] In the context of the present disclosure, a lipid nanoparticle delivery vehicle typically serves to transport a desired therapeutic agent to a target cell or tissue. In certain embodiments, the lipid formulation encapsulates the therapeutic agent.Nucleic Acid Molecule

[0056] In one aspect, the lipid nanoparticle formulation comprises a nucleic acid molecule comprising a cytotoxic fluoropyrimidine (CF) polymer. In some embodiments, a typical cytotoxic fluoropyrimidine polymer comprises a nucleic acid molecule including 5-fluoro-2’deoxyuridine monophosphate (FdUMP) having a polyethylene glycol (PEG) spacer appended to the 5’ terminus and a nucleotide appended to the 3 ’terminus. In some embodiments, a nucleic acid molecule for treating cancer includes FdUMP

[0010] (ten FdUMP nucleotides serially connected) having a PEG spacer (or other modification that promotes cell uptake including promoting exosome-mediated uptake) appended to the 5’-terminus and a AraC nucleotide (or any moiety that is not recognized by 3 ’-exonucleases) appended to the 3 ’-terminus. In some embodiments, cleavage of the terminalmoiety appended to the 3 ’-terminus occurs substantially in cancer cells. Synthesis of cytotoxic fluoropyrimidine polymers may be carried out using synthesis method described in, for example, U. S. Patent No. 12,319,915B2, titled “Cytotoxic Fluoropyrimidine Polymers and Methods of Use Thereof,” granted on June 3, 2025, the entire disclosure of which is incorporated herein by reference in its entirety

[0057] In some embodiments, the moiety may have Formula (I):Formula (I)wherein R₁ is H or OH; R₂ is H, halogen or OH. In some embodiments, andmay be a PEG spacer.

[0058] In some embodiments, moiety according to Formula (I) may be selected from the group consisting of following:

[0059] In some embodiments, the moiety is cytosine arabinoside (Ara-C).

[0060] One example of such a nucleic acid molecule is cytarabine-fluorodeoxyuridine monophosphate “X” (i.e., CF“X”) having Formula (II):or an analogue or a derivative thereof, wherein X ranges from 1 to 500, Y ranges from 1 to 5, and a Cytosine arabinoside (AraC) nucleotide appended to the 3 ’-terminus. Additionally, “X” in CF“X” refers to value of X in Formula (II). In some embodiments, X may be up to 500, up to 100, up to 50, or up to 10. The longer CF“X” polymers would package more efficiently in LNPs and be internalized into cancer cells, whereas without LNP delivery this would not happen.

[0061] In some embodiments, X may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, Y may be 1, 2, 3, 4, or 5. For instance, when a CF“X” derivative or CF“X” analogue may include a 3’ terminus other than AraC, for example, any nucleotide analog that is not recognized by d’exonucleases (e g., dideoxynucleotides). A CF“X” derivative or CF“X” analogue may include a 5’ terminus other than PEG, for example, any modification that promotes cell uptake, including promoting exosome-mediated uptake. A CF“X” derivative or CF“X” analogue may include backbone modifications, such as phosphorothioate, being active. Further discussion regarding fluoropyrimidine polymer compounds and is described in, for example, U. S. Patent Application No. 19 / 226,041, titled “Cytotoxic Fluoropyrimidine Polymers and Methods of Use Thereof,” filed on June 2, 2025, the entire disclosure of which is incorporated herein by reference in its entirety

[0062] In some embodiments, the moiety appended to the 3’-terminusis 3 ’-deoxy-adenine, 3 ’-deoxy-cytosine, 3’-deoxy-guanine, 3 ’ -deoxy-thymine, or any analog or derivative thereof. In some embodiments, the moiety is an anti-nucleoside analog with anti-viral activity. Non-limiting examples include carbovir, acyclovir, 3TC (Lamivudine), AZT (Zidovudine), (-)-FTC, ddl (Didanosine), ddC (zalcitabine), abacavir (ABCTm), tenofovir (PMPATm), DD4FCTM (Reverset), (Stavudine), Racivir, L-FddCTM, L-FD4C, NVP (Nevirapine), DLVTM (Delavirdine), EFVTM (Efavirenz), SQVMTm (Saquinavir mesylate), RTVTm (Rifonavir), IDVTM (Indinavir), SQVTM (Saquinavir), NFVTM (Nelfinavir), APVTM (Amprenavir), and LPVTM (Lopinavir). In some embodiments, the moiety is an L-nucleoside. Non-limiting examples include 2'-deoxy-L-nucleosides, beta-L-2’-deoxythymidine, L-2'-deoxyuri dines. L-FMAU (2'-fIuoro-5-methyl-P-L-arabinofuranosyluridine), L-FIAU (2'-fluoro-5-iodo-0-L-arabinofuranosyluridine), L-FC (2'-fluoro-P-L-arabinofuranosylcytosine), L-FIAC (2'-fluoro-5-iodo-P-L-arabinofuranosylcytosine), L-2-Cl-2'-F-2'-deoxyadenine, L-FEAU (2'-fluoro-5-ethyl-P-L-iarabinofuranosyluridine), L-arathymidine, L-fludarabine, L-araguanosine, and L-ara-inosine.

[0063] Such a nucleic acid molecule (e.g., Formula (I) or Formula (II)) is an anti-cancer agent, and can be used to treat any of a plurality of cancers. The nucleic acid molecules described herein are capable of being internalized by cancer cells, of inhibiting Thymidylate Synthase (TS) activity, and of inducing Topl-mediated DNA damage in the cancer cells. In one embodiment, the PEG spacer has 2-2,000, 5-1,000, 10-1,000, 10-1,000, or 100-1,000 repeating units of CH2CH2O. Non-limiting examples of the number of repeating units of CH2CH2O include 2, 5, 10, 15, 20, 30, 40, 50, 80, 100, 200, 400, 500, 1,000, 1500, 2,000, and any range between any two of the aforementioned numbers. In one embodiment, the PEG spacer has 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeating units of CH2CH2O. The PEG spacer may be linear or branched. In one embodiment, alinear PEG or an individual arm of a branched PEG, has a MW ranging from about 200 Da to about 1,000 Da, from about 500 Da to about 1,000 Da, from about 500 Da to about 5,000 Da, from about 500 Da to about 10,000 Da, from about 500 Da to about 20,000 Da, from about 500 Da to about 30,000 Da, from about 500 Da to about 50,000 Da, from about 500 Da to about 100,000 Da, from about 1,000 Da to about 20,000 Da, from about 5,000 Da to about 20,000 Da, from about 5,000 Da to about 10,000 Da, from about 10,000 Da to about 20,000 Da, or from about 5,000 Da to about 15,000 Da. Non-limiting examples of the MW of the PEG include about 100 Da, about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 1,000 Da, about 2,000 Da, about 3,000 Da, about 4,000 Da, about 5,000 Da, about 6,000 Da, about 8,000 Da, about 10,000 Da, about 15,000 Da, about 20,000 Da, about 30,000 Da, about 40,000 Da, about 45,000 Da, about 50,000 Da, about 80,000 Da, about 100,000 Da, or any range between any two of the aforementioned values.

[0064] In some embodiments, the PEG spacer has multiple arms (e.g. 2, 3, 4, 5, 6, 7, or 8 arms). For instance, the PEG spacer can have a central core and multiple PEG chains (arms) extending from it. Each arm can vary in molecular weight, independently ranging, for example, from 100 Da to 20,000 Da or more. The MW range of each PEG arm is as described in the immediate above paragraph. One or more arms can be linked to an active moiety. In one embodiment, each arm is linked to an active moiety. The PEG spacer can also include one or more PEG components that are connected via one or more linkers. A core PEG component is thus linked to one or more arm PEG components and contains for example 1 to 100 (CH2CH2O) units. The arm PEG component can have, for example 1 to 300 (CH2CH2O) units. Non-limiting examples for the molecular weight for the core PEG component and each arm PEG component is as illustrated above for the single arm PEG spacer.

[0065] It was discovered that CF“X” of Formula (II) displays therapeutic advantages supporting its use for the clinical management of colorectal cancer, including being well-tolerated in vivo, more rapid internalization into malignant cells, and an improved cytotoxicity to cancer cells relative to previous chemotherapeutic drugs, including 5-FU.

[0066] As disclosed throughout this document, all stereoisomers of the compounds are encompassed by the aforementioned formula. When no particular stereoisomer or stereochemistry is indicated, it is understood to mean all possible stereoisomers that could be produced from a reaction are present. A person of ordinary skill in the art will recognize that the reactions can be optimized to give one isomer preferentially, or new schemes may be devised to produce a single isomer.Lipid Nanoparticles (LNPs)

[0067] In one aspect, the present disclosure also features a lipid nanoparticle composition comprising a CF“X” compound according to Formula (II) as described herein.

[0068] As used herein, a “lipid component” is that component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may comprise a compound according to Formula (I) or Formula (II) and a helper lipid component including but not limited to one or more of an ionizable lipid, PEGylated-lipid, structural-lipid, or phospholipid.

[0069] In some embodiments, the lipid nanoparticle formulation may comprise a CF“X” compound encompassed or encapsulated in at least one helper lipid. In some embodiments, the helper lipid may comprise an ionizable lipid, phospholipid, a sterol-derived lipid, a pegylated (i.e., PEG-modified)-lipid, or a combination thereof. In some embodiments, the LNP may comprise comprises mass / weight ratio of total lipid mass to therapeutic agent (e.g., CF“X”) ranges from about 5 to about 50. In some embodiments, the LNP comprises a mass ratio of total lipid mass totherapeutic agent ranges from about 5 to about 30, about 10 to about 25, about 10 to about 20, or between any two aforementioned values.

[0070] In some embodiments, the mass ratio between the CF“X” and the total helper lipid component (i.e., ionizable lipid + phospholipid + sterol- derived lipid + pegylated lipid) is from about 1 / 10 to about 1 / 5, about 1 / 9, about 1 / 8, about 1 / 7, about 1 / 6, about 1 / 5 or between any two aforementioned values of CF“X / helper lipid component.

[0071] As used herein, the term “ionizable lipid” refers to a lipid that, at a given pH, is in an electrostatically neutral form and that may either accept or donate protons, thereby becoming electrostatically charged, allowing for complexation with nucleic acids and eventual endosomal escape into the cytoplasm. In some embodiments, the ionizable lipid is positively charged at low pH to allow complexation with the negatively charged mRNA and may also help with cellular uptake and endosomal escape.

[0072] In some embodiments, the ionizable lipid may comprise 1, 1 '-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C 12-200), 2-[2, 2-Di-[(9Z,12Z)-octadeca-9, 12-dienyl]- 1,3-dioxolan-4-yl]-A', N'-dimethylethanamine (DLin-KC2-DMA), l,2-dioleoyl-3 -dimethylammonium -propane (DODAP), N, N-dimethyl-2,3-bis[(9Z, 12Z)-octadeca-9, 12-dienoxy]propan-l -amine (HGT4003), (15Z, 18Z)-N, N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-15,18-dien-l-amine, (HGT5000), (15Z, 18Z)-N, N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-4, 15, 18- trien-1 -amine (HGT5001), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), 9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 3-(didodecylamino)-N 1, N 1,4-tridodecyl-l -piperazineethanamine (KL 10), N 1 -[2-(didodecylamino)ethyl]-Ni, N4, N4-tridodecyl- 1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), l,2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-ylox y]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl }oxy)-N, N-dimethyl-3 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl oxy] propan- 1 -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9,l 2-dien- l-yloxy]propan-l -amine (Octyl-CLinDMA (2S)) or a combination thereof.

[0073] In some embodiments, the ionizable lipid may comprise a compound having Formula (A):Formula (A)or salts or isomers thereof, wherein Ri is — R'M'R' or C5-20 alkenyl; R2and R?>are each independently selected from C1-14 alkyl and C2-14 alkenyl; R4is — (CH2)nQ, wherein Q is OH and n is selected from 3, 4, and 5; M and M' are each independently — C(O)O — or — OC(O) —; Rs, Re, and R7 are each H; R' is a linear C1-12 alkyl, or C1-12 alkyl substituted with Ce-9 alkyl; R" is C3-14 alkyl; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. Further discussion regarding ionizable lipid based on Formula (A) is described in, for example, U. S. Patent No. 12,151,995, titled “Compounds and Compositions for Intracellular Delivery of Therapeutic Agents,” issued on November 26, 2024, the entire disclosure of which is incorporated herein by reference in its entirety.

[0074] As used herein, the term “alkyl” or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three,, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation “C1-14 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms. Unless otherwi se specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.

[0075] As used herein, the term “alkenyl” or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. ’The notation “C?.-14 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example. Cis alkenyl may include one or more double bonds. A Cis alkenyl group including two double bonds may be a linoleyl group Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

[0076] As used herein, the term “alkynyl” or “alkynyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted The notation “C2-14 alkynyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds For example, Cis alkynyl may include one or more carbon-carbon triple bonds Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

[0077] As used herein, the term “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, e.g., unsaturated fatty acid chains. The phospholipid may comprise one or more multiple (e.g., double or triple bond) bonds (e.g., one or more unsaturated bonds). The phospholipids promote fusion with the membrane and fortify the bilayer structure of the LNP, and promote endosomal escape. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). The fusion of the phospholipid to the membrane may allow one or more elements of the lipid-containing composition to pass through the membrane, thereby allowing, for example, the delivery of one or more elements to the cell.

[0078] In some embodiments, the phospholipid may comprise Dilauroyl lecithin (DLPC), Dimyristoyl phosphatidylcholine (DMPC), Dioleoyl lecithin (DOPC), Dipalmitoyl phosphatidylcholine (DPPC), Distearoyl phosphatidylcholine (DSPC), Dioleoyl phosphatidylcholine (DUPC), Palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-Di-O-octadecyl-sn-glycero-3 -phosphocholine, l-Oleoyl-2-cholesteryldimethylsuccinoyl-sn-glycero-3-phosphocholine, 1 -Hexadecyl-sn-glycero-3-phosphocholine, 1,2-Divinyl-sn-glycero-3-phosphocholine, l,2-Diarylacyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero- 3phosphorylethanolamine (DOPE), l,2-Di-phytanoyl-sn-glycero-3 -phosphoethanolamine, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, l,2-Diethenol-sn-glycero-3-phosphoethanolamine, l,2-Divinyl-sn-glycero-3 -phosphoethanolamine, l,2-Diaryl-sn-glycero-3-phosphoethanolamine, l,2-Dithiohexaenoate-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate-(l -glycerol) sodium salt (DOPG), or a combination thereof.

[0079] As used herein, the term “sterol-derived lipid” refers to a lipid component that enhances LNP stability and promotes membrane fusion. In some embodiments, the sterol-based lipid may comprise cholesterol, coprosterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, bile acid, or a combination thereof.

[0080] As used herein, the term “PEG lipid” or “pegylated lipid” refers to a lipid comprising a polyethylene glycol component to reduce aggregation and nonspecific endocytosis. In some embodiments, the pegylated-lipid may comprise l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DOPE -PEG), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-disteryl glycerol (DSG-PEG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (DAGPEG), PEG-dipalmitoyl phosphatidylethanolamine (DPPE-PEG), or PEG- 1,2-dimyristyloxlpropyl-3-amine (DMA-c-PEG), and a combination thereof. In some embodiments, the lipid moiety of the PEG lipids includes those having lengths from about C14 to about C22. In some embodiments, a PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000, or 20,000 daltons, i.e., PEG1000, PEG2000, PEG5000, PEG10000, PEG15000, orPEG20000, respectively.

[0081] In some embodiments, the pegylated lipid may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, the PEG lipid is a PEG-azide lipid. As generally defined herein, a “PEG-azide lipid” is a PEGylated lipid having one or more azide groups on the lipid. In some embodiments, the surface of the lipid nanoparticles may be covalently modified, i.e., functionalized, with a surface modifier / targeting moiety, such as cell surface receptors, antibodies, antibody fragments, peptides, aptamers, small molecules, cleavable linkers, cell-penetrating peptides, or a combination thereof. In some embodiments, mass ratio between the lipid nanoparticle and the surface modifier ranges from about 1 / 9 to about 1 / 1, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 6, about 1 / 7, about 1 / 8, about 1 / 9, about 1 / 10, or between any two aforementioned values of lipid nanoparti cl e / surface modifier.

[0082] In some embodiments, the lipid nanoparticle may be functionalized at the surface with folinic acid. The inhibition of TS occurs through the formation of a ternary complex between folinic acid (Leucovorin; LV), a reduced folate co-factor that binds to TS, and Fluoro- 2'-deoxyuridine-5'-O-monophosphate (FdUMP) that irreversibly inhibits TS enzymatic activity. In some embodiments, folinic acid is modified with an alkyne group through a reaction betweenfolinic acid and an amino alkyne (e g., propargyl amine) as seen in the reaction scheme 1 below:Scheme 1

[0083] Such modification provides reaction site at the alkyne moiety for functionalization with a PEG-azide surface lipid such as DOPE-PEG-azide through click chemistry as seen in the FIG. 1. In some embodiments, mass ratio between the lipid nanoparticle and the folinic acid modifier ranges from about 1 / 1 to about 5 / 1, about 2 / 1, about 3 / 1, about 4 / 1, about 5 / 1, or between any two aforementioned values of lipid nanoparti cl e / surface modifier.

[0084] In some embodiments, the mass ratio between the CF“X” and ionizable lipid is from about 1 / 4 to about 1 / 2, about 1 / 2, about 1 / 3, about 1 / 4, or between any two aforementioned values of CF“X” / ionizable lipid.

[0085] In some embodiments, the mass ratio between the CF“X” and phospholipid is from about 1 / 1 to about 3 / 1, about 1 / 1, about 2 / 1, about 3 / 1, or between any two aforementioned values of CF“X” / phospholipid.

[0086] In some embodiments, the mass ratio between the CF“X” and sterol-derived lipid is from about 1 / 2 to about 1 / 1 of CF“X” / sterol-derived lipid.

[0087] In some embodiments, the mass ratio between the CF“X” and pegylated lipid is from about 1 / 1 to about 5 / 1, about 1 / 1. about 2 / 1, about 3 / 1, about 4 / 1, about 5 / 1, or between any two aforementioned values of CF“X” / pegylated lipid.

[0088] In some embodiments, the mass ratio between the CF“X” and each component of the helper lipid i.e., ionizable lipid, phospholipid, sterol-derived lipid, and pegylated lipid is from about 5 / 20 / 3 / 5 / 1 of CF“X” / ionizable lipid / phospholipid / sterol-derived lipid / pegylated lipid.

[0089] In some embodiments, the mass ratio between each component of the helper lipid i.e., ionizable lipid, phospholipid, sterol-derived lipid, and pegylated lipid (ionizable lipid / phospholipid / sterol-derived lipid / pegylated lipid) is from about 10 / 1.5 / 2.5 / 0.5 of ionizable lipid / phospholipid / sterol-derived lipid / pegylated lipid.

[0090] In some embodiments, an LNP may comprise ionizable lipid ranging from about 5% to about 95%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% by total weight of LNP, or between any two aforementioned values.

[0091] In some embodiments, an LNP may comprise phospholipid ranging from about 1% to about 80%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% by total weight of LNP, or between any two aforementioned values.

[0092] In some embodiments, an LNP may comprise sterol -derived lipid ranging from about 1% to about 80%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% by total weight of LNP, or between any two aforementioned values.

[0093] In some embodiments, an LNP may comprise PEGylated lipid ranging from about 1% to about 20% by weight, about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20% by total weight of LNP, or between any two aforementioned values.

[0094] In certain embodiments, the lipid nanoparticle (LNP) comprises a CF10, an ionizable lipid, phospholipid, sterol -derived lipid, and pegylated lipid as disclosed above. In someembodiments, CF10, the ionizable lipid may be C12-200, phospholipid may be DOPE, sterolderived lipid may be a cholesterol, and pegylated lipid may be l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000 (14:0 PEG2000 PE) in a mass ratio of about 0.5 / 2 / 0.3 / 0.5 / 0.1 of CF10 / C12-200 / DOPE / cholesterol / l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000.

[0095] In some embodiments, the disclosed LNP may further comprise the incorporation of an additional therapeutic for the co-delivery of multiple agents. Non-limiting examples of additional anti-cancer therapy may include, but are not limited to, irinotecan, leucovorin, oxaliplatin, inhibitors of the DNA damage response such as Ataxia-telangiectasia and Rad3-related (ATR) inhibitors, Weel inhibitors, or other nucleic acid cargo such as SLFN11 mRNA, GEM-DEC or siRNA targeting TS.

[0096] In some embodiments, one or more additional therapeutics may be an anti-cancer agents that modulate the DNA damage response. Non-limiting examples an anti -cancer agents may include, but are not limited to poly(ADP ribose) polymerase (PARP) inhibitors and Bromodomaincontaining protein 4 (BRD4) inhibitors. PARP inhibitors are known in the art, and include, for example, Olaparib, Niraparib, Talazoparib, Veliparib, Iniparib, and Rucaparib. BRD4 inhibitors are also known in the art and include, as examples, BMS-986158, TG 1601, 1-BET-761, and JQ1.

[0097] Another example of an anti-cancer agent that may be included in the compositions is an agent that blocks one or more immune checkpoints (e.g., a “checkpoint inhibitor”) may be included in the compositions. Checkpoint inhibitors are known in the art, and include, for example, Pembrolizumab, nivolumab, durvalumab, atezolixumab, avelumab, etc.

[0098] In some embodiments, the additional therapeutic agent may be loaded / encapsulated into the LNP in a non-covalent complex. As used herein, the term “non-covalent complex” refersto encapsulation or loading of the aforementioned therapeutic without forming a chemical bond. The additional therapeutics are held in the LNP and stabilized by an electrostatic interaction between the negatively charged moiety of a therapeutic (e.g., siRNA) and positively charged ionizable lipids, hydrophobic interactions, Van der Waals forces, or hydrogen bonding. Non-covalent interactions allow additional therapeutics to be encapsulated within the LNP structure without altering its chemical structure, maintaining the biological activity of the therapeutic and LNP. In certain embodiments, the disclosed lipid nanoparticle may comprise a total mass ratio between the lipid nanoparticle and the additional therapeutic agent from about 5 / 1 to about 20 / 1, about 5 / 1, about 6 / 1, about 7 / 1, about 8 / 1, about 9 / 1, about 10 / 1, about 11 / 1, about 12 / 1, about 13 / 1, about 14 / 1, about 15 / 1, about 16 / 1, about 17 / 1, about 18 / 1, about 19 / 1, about 20 / 1, or between any two aforementioned values.

[0099] All or a portion of the therapeutic agent nucleic acid may be encapsulated in the lipid nanoparticles. In some embodiments, the LNP may exhibit encapsulation efficiency of at least about 90% (e.g., at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%). The term “encapsulation efficiency” (EE) as used herein refers to the percentage of nucleic acid / therapeutic agent in the lipid nanoparticles that is not degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids. Encapsulation efficiency measures how effectively the LNPs trap the payload inside the particle. A high EE means less waste of sensitive payloads and better protection from degradation of the payload.

[0100] In some embodiments, encapsulation efficiency is measured as a ratio of the amount of the therapeutic agent encapsulated in the lipid nanoparticle to the total amount of therapeuticagent used. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

[0101] In some embodiments, the LNP may exhibit a loading efficiency (LE) of the therapeutic agent of at least 90% (e.g., at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%). The term “loading efficiency” as used herein refers to the percentage of nucleic acid / therapeutic agent to the total mass of lipid nanoparticles. Loading efficiency measures how much payload is packed into each lipid nanoparticle. A high LE means more potent particles, potentially reducing the required dose and improving delivery efficiency. In some embodiments, loading efficiency is measured as a ratio of the amount of the therapeutic agent encapsulated in the lipid nanoparticle to the total mass of the lipid nanoparticle.

[0102] In some embodiments, the LNP may have a mean hydrodynamic diameter ranging from about 10 nm to about 1000 nm, about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 to about 800 nm, about 400 nm to about 600 nm, or between any two aforementioned values. As used herein, the term “hydrodynamic diameter” refers to a measurement of a particle's size that takes into account the particle's movement through a liquid. “Hydrodynamic diameter” represents the size of a sphere that diffuses at the same rate as the particle under observation, taking into account any solvation layer or dynamic interaction with the solvent.

[0103] In some embodiments, the LNP may have a mean poly dispersity index (PDI) from about 0.01 to about 0.2, about 0.02 to about 0.2, about 0.04 to about 0.18, about 0.06 to about 0.16, about 0.08 to about 0.14, about 0.10 to about 0.12, or between any two aforementioned values. As used herein, the term “poly dispersity index” refers to a measurement of heterogeneity of particle sizes. If particles in the population are uniform, the resulting size distribution will be narrow andthe PDI small, indicating that the sample is monodisperse. For heterogeneous particle populations, the size distribution will be broader, and the PDI increases, indicating that the sample is polydisperse.

[0104] In some embodiments, a pharmaceutical composition comprising the present LNP is disclosed herein. A therapeutically effective amount of at least one therapeutic agent and / or one or more pharmaceutically acceptable excipient, carrier, or diluent. As used herein, the term “pharmaceutically acceptable excipient, carrier, or diluent” includes compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments, the pharmaceutical compositions may be in a form suitable for parenteral administration. Depending upon the therapeutic application, for in vivo administration, the disclosed LNP herein may be administered to a subject in need thereof intravenously, intradermally, intramuscularly, intrathecally, or subcutaneously.

[0105] In some embodiments, the pharmaceutical compositions may be in the form of a sterile injectable aqueous suspension, which may be formulated according to known procedures. A sterile injectable preparation may also be a sterile injectable suspension in a non-toxic parenterally acceptable buffer. In other embodiments, the pharmaceutical composition may be lyophilized, resulting in the form of a dry powder, wherein the dry powder can be later reconstituted for administration as needed. Dry powder compositions may further comprise bulking agents, for example, sucrose or trehalose.

[0106] Further provided herein is a pharmaceutical kit comprising a pharmaceutical composition comprising the disclosed LNP having a therapeutically effective amount of at leastone therapeutic agent. Such kits may further comprise various conventional pharmaceutical kit components, such as containers comprising pharmaceutically acceptable adjuvants, diluents, or carriers, and additional containers readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, can also be included in the kit.

[0107] In another aspect, the disclosed LNP, pharmaceutical composition, or kit may be used for intracellular delivery of a therapeutic agent. In some embodiments, the delivery of the LNP may comprise contacting a cell or administering to a subject a lipid nanoparticle comprising a therapeutically effective amount of a therapeutic agent. As used herein, the term “subject” encompasses any animal, but preferably a mammal, e.g., a human, non-human primate, a dog, a cat, or a mouse. More preferably, the subject is a human. As used herein, the term “delivery” refers to providing an entity to a target site. For example, delivering a therapeutic and / or prophylactic agent to a subject may include administering a nanoparticle composition containing a therapeutic and / or prophylactic agent to a subject (e g., by intravenous, intramuscular, intradermal, intrathecally, or subcutaneous routes). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the lipid nanoparticle composition.

[0108] In another aspect, methods of treating cancer in an individual in need thereof include administering to the individual a pharmaceutically effective amount of the disclosed LNP, pharmaceutical composition, or kit for treating (e.g., alleviating, ameliorating, curing) cancer in the individual and a pharmaceutically acceptable carrier. In the methods, the disclosed LNP, pharmaceutical composition, or kit is capable of being internalized by cancer cells in the individual. In a typical embodiment, administration of an LNP, a pharmaceutical composition, or a kit as described herein inhibits TS activity and induces Top1-mediated DNA damage in thecancer cells without affecting normal cell viability in the individual (i.e., viability of the individual’s normal “non-cancerous” cells), and reduces tumor growth rate in the individual. In some embodiments, administration of the LNP, pharmaceutical composition, or kit to the individual reduces metastatic spread of the cancer cells in the individual. In an embodiment, the individual in need of treatment has CRC (e.g., mCRC, stage I CRC, stage II CRC, stage III CRC, stage IV CRC). In such an embodiment, the individual may be resistant to prior treatment with 5-fluorouracil (5-FU).

[0109] The disclosed LNP, pharmaceutical composition, or kit for treating cancer (e.g., CF10 or derivative or analog thereof) may be administered as a monotherapy or as part of a combination therapy with any other anti-cancer agent in a method of treating cancer in an individual in need thereof. Some of the compositions described above are compositions that contain both a nucleic acid molecule for treating cancer as described herein and a second anticancer agent. In other embodiments of a combination therapy, a first composition may include an LNP, pharmaceutical composition, or kit for treating cancer as described herein, and a second composition may include the second anti -cancer agent. In such embodiments, the first composition may be administered at the same time point or approximately the same time point as the second composition. Alternatively, the first and second compositions may be administered at different time points.

[0110] In some embodiments, an LNP, pharmaceutical composition, or kit for treating cancer as described herein can be used in a combination therapy that includes one or more of immunotherapy, chemotherapy, radiotherapy, and surgery. In an embodiment in which the individual being treated is also receiving immunotherapy, the immunotherapy can include, for example, a checkpoint inhibitor. In some embodiments, the individual being treated is alsoreceiving an agent that modulates the DNA damage response (e.g., a PARP inhibitor, a BRD4 inhibitor, etc.). In some embodiments, the individual being treated with an LNP, pharmaceutical composition, or kit as described herein is also receiving radiation therapy (optionally with surgery). Methods of administering radiation therapy that use high-energy radiation (e.g., one or both of X-rays and gamma rays can be used) to shrink tumors and kill cancer cells are well known. The radiation may be delivered by a machine outside the body (external-beam radiation therapy), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy), or alternatively, they can be used in combination. The methods described herein also include using systemic radiation therapy, which uses radioactive substances, such as radioactive iodine, that travel in the blood to kill cancer cells, (e.g., combining radioactive iododeoxyuridine with CF10: LNP to promote tumor-specific radiation damage).

[0111] Any suitable method of administering an LNP, pharmaceutical composition, or kit as described herein to an individual may be used. In these methods, the LNP, pharmaceutical composition, or kit can be administered to an individual by any suitable route, e.g., oral, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces), rectal, vaginal, and transdermal administration. In an embodiment, LNP, pharmaceutical composition, or kit may be administered systemically by intravenous injection.

[0112] In another embodiment, an LNP, pharmaceutical composition, or kit may be administered directly to a target site, by, for example, surgical delivery to an internal or external target site, or by catheter to a site accessible by a blood vessel. If administered via intravenous injection, the nucleic acid molecule or composition may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously, by peritoneal dialysis, or pump infusion).For parenteral administration, the LNP, pharmaceutical composition, or kit is preferably formulated in a sterilized pyrogen-free form.

[0113] As indicated above, an LNP, pharmaceutical composition, or kit as described herein may be in a form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic agent(s) (e.g., a therapeutically effective amount of CF10: LNP) is dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide, or a suitable buffer, 1,3 -butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution (D5W, 0.9% sterile saline). The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). In cases where the therapeutic agent(s) is only sparingly or slightly soluble in water, a dissolution-enhancing or solubilizing agent can be added, or the solvent may include 10-60% w / w of propylene glycol or the like. The LNP, pharmaceutical composition, or kit described herein may be administered to an individual (e g., rodents, humans, nonhuman primates, canines, felines, ovines, bovines) in any suitable formulation according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (21st ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, (2005) and Encyclopedia of Pharmaceutical Technology, (3rded.) eds. J. Swarbrick and J. C. Boylan, Marcel Dekker, CRC Press, New York (2006), a standard text in this field, and in USP / NF). A description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington: supra. Other substances may be added to the nucleic acid molecules and compositions to stabilize and / or preserve them.

[0114] The therapeutic methods described herein in general include administration of a therapeutically effective amount of the nucleic acid molecules and compositions described herein to an individual (e.g., human) in need thereof, particularly a human. Such treatment will be suitably administered to individuals, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof (e.g., cancer). Determination of those individuals "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider. Numerous prognostic markers or factors for categorizing CRC patients or individuals for the likely outcome of treatment are known. See, e.g., Lin PS & Semrad TJ, Molecular Testing for the Treatment of Advanced Colorectal Cancer: An Overview Methods Mol Biol. 2018 1765:281-297; and Zacharakis et al., Predictors of survival in stage IV metastatic colorectal cancer Anticancer Res, 201030(2): 653-660. In some embodiments, the individual in need of treatment is afflicted with a relapsed CRC (e.g., the individual was previously treated for CRC, then in partial or complete remission for CRC, and then the CRC has returned).

[0115] The disclosed nucleic acid molecule (e.g., CF“X”) encapsulated lipid nanoparticles have been shown to improve cell uptake and cytotoxicity relative to nucleic acid molecule (e.g., CF“X”) alone (free, non LNP encapsulated nucleic acid) and anticancer activity at less than 1 / 10 the administered dose. There is no toxicity, and a delivery schedule like lx week injection could be as or more effective than long infusions.

[0116] It will be appreciated by persons skilled in the art that the embodiments described herein are not limited to what has been particularly shown and described. Rather, the scope of the invention is defined by the claims which follow. It should further be understood that the above description is only representative of illustrative examples of embodiments. The description has not attempted to exhaustively enumerate all possible variations. The alternate embodiments may nothave been presented for a specific component of a nanocarrier, or a step of the method, and may result from a different combination of described constituents, or the un-described alternate embodiments may be available for a composition, kit, or method, and is not to be considered a disclaimer of those alternate embodiments. It will be appreciated that many of those un-described embodiments are within the literal scope of the following claims, and others are equivalent.EXAMPLESMaterials and Methods

[0117] CF10 and CF10 alkyne were obtained from ST Pharma, validated by high-resolution mass spectrometry, and dissolved in 0.9% sterile saline. Clinical-grade 5-FU (5-fluorouracil) (50 mg / mL) was purchased from the Baptist Hospital clinical pharmacy. Cy5.5 azide was purchased from BroadPharm (San Diego, CA). Thymidine 10 (T10) was purchased from Integrated DNA Technologies (Coralville, IA). Copper sulfate pentahydrate, sodium ascorbate, and tris(3-hydroxypropyltriazolylmethyl) amine (THTA) were bought from Sigma-Aldrich (St. Louis, MO). 1, l'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin- 1 -yl)ethyl)azanediyl)bis(dodecan-2-ol) (C 12-200) was purchased from Cayman Chemicals (Ann Arbor, Michigan). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 14:0 PEG2000 PE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000) (ammonium salt) were purchased from Avanti Research (Birmingham, AL). Microfluidic channels and accessories were bought from the Microfluidic Chip Shop. Tritium-labeled deoxyuridine (3H-dU) was purchased from Moravek Inc. (Brea, California). Rabbit polyclonal Anti-Topoisomerase 1 (ab28432), Rab5 (ab18211), and Lamp1 (ab24170) were bought from Abeam (Cambridge, MA). HRP-conjugated anti-rabbit secondary antibody was purchased from Cell Signaling (Danvers, MA). LumiTrackerLyso Green was bought from Lumiprobe (Cockeysville, Maryland). Anti-Topoisomerase T-DNA Covalent Complexes Antibody, clone 1.1 A was purchased from Sigma- Aldrich.EXAMPLE 1: Preparation of CF10-Cy5.5 conjugate

[0118] Alkyne-modified CF10 (CF10 alkyne) was tagged as demonstrated in the Scheme in FIG. 2 with the near-infrared fluorescent probe cyanine 5.5 (Cy5.5) using click chemistry. 2.3 mg of CF10 alkyne (1 eq.) was dissolved in 0.2 mL of ultrapure water in a glass vial with a small magnetic stir bar. 71 pL of 10 mM Cy5.5 (1 eq.) was added to the solution and purged with Argon. Then, 210 pL of 5 mM sodium ascorbate and 210 pL of 10 mM CuSO4·5H2O-THTA (1:2) complex were added under argon, and the mixture was stirred for 24 hours. The blue solution was collected in a 1000 MWCO dialysis tube and dialyzed overnight in DNAase-free water to remove inorganic components and unreacted materials. The solution was collected as the CF10-Cy5.5 conjugate and quantified using UV spectroscopy. The presence of absorbance peaks at 260 nm and 680 nm, corresponding to CF10 and Cy5.5, respectively, confirmed conjugate formation. ESI-MS further verified the product.EXAMPLE 2: Preparation of CF10-Cy5.5: LNP

[0119] CF10-Cy5.5: LNP was prepared by mixing CF10-Cy5.5 of Example 1 in C12-200 ionizable (cationic) lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000) as PEG-lipid, and cholesterol. The C12-200, cholesterol, DOPE, and C14-PEG2000 were taken at a mass concentration ratio of 2.0: 0.28: 0.52: 0.13 mg / ml in ethanol.

[0120] CF10-Cy5.5 was dissolved in 10 mM citrate (pH 3.0) buffer at a concentration of 0.4 mg / ml. A 3:1 volume ratio of CF10-Cy5.5 solution to lipid mixture was used to prepare lipidnanoparticles encapsulating CF10-Cy5.5 through chaotic mixing. Two 100 pL glass syringes, one filled with lipid solution and the other with CF10-Cy5.5 solution, were positioned side by side and connected to a microfluidic staggered herringbone mixture using tubing. The outlet of the microfluidic channel was connected to an Eppendorf tube filled with 40 pL of phosphate-buffered saline (PBS) solution for the dilution of the prepared nanoparticles. To generate CF10-Cy5.5: LNPs, the solutions were manually pushed simultaneously with an estimated flow rate close to 300 pL / min. The LNPs were transferred to 3.5 kDa MWCO dialysis cassettes and dialyzed at 4°C against lx PBS solution for 2h first and then overnight after replacing with a fresh PBS solution. As a control, blank LNPs were prepared without using any oligonucleotide. The LNPs were stored at 4°C until further use.EXAMPLE 3: Cellular Uptake of base CF10 and CF10: LNP.

[0121] HCT116 (RRID: CVCL 0291) and LS174T (RRID: CVCL 1384) colorectal cancer (CRC) cells and the HIEC-6 (CVCL 6C21) immortalized intestinal cell line were obtained from ATCC and were cultured using recommended media, validated by short tandem repeat analysis, and regularly confirmed to be negative for Mycoplasma. Cells were cultured as per the ATCC protocol for the respective cell lines.

[0122] Cells were seeded at 5000 cells / well in a 96-well plate. After 24 hours, cells were treated with CF 10-Cy5.5 (from Example 1) and CF10-Cy5.5: LNP (from Example 2) and incubated for the respective time points. Cells were washed with PBS twice and kept in PBS. Fluorescence intensities were recorded in a plate reader following the emission maxima of Cy5.5.

[0123] As seen in FIGS. 3Aand 3B, cellular uptake of CF10-Cy5.5: LNPinLS174T CRC cell exhibited almost 2-fold concentration of 500 nM or higher compared to base CF10-Cy5.5.Similar increased cellular uptake was observed in HCT116 CRC cells, indicating lipid nanoparticles as an effective and efficient carrier / vehicle for CF10 for cellular delivery.EXAMPLE 4: Cellular Toxicity comparison between CF10, CF10: LNP, and 5-FU

[0124] Cells were plated at 3500 cells / well in 96-well plates. After 24 hours, the cells were treated with the CF10, CF10: LNP, and 5-FU and incubated for 72 hours. Cellular cytotoxicity was monitored using the MTS assay and recorded through a plate reader. As seen in FIGS. 4A and 4B, in HCT116 cells, treatment with CF10 and CF10: LNPs resulted in a surprisingly increase in potency in growth inhibition relative to 5-FU chemotherapy drug. Similar results were obtained in LS174T cells, with greater toxicity to CRC at higher dosage concentrations.EXAMPLE 5: Thymidylate Synthase (TS) Inhibition Assay

[0125] Cells were plated at 1×106cells / well in 6-well plates. After 24 hours treated with CF10, CF10: LNPs, or 5FU for 24h or 48h. The media was aspirated, and cells were washed with PBS 2x before incubating with3H-dU in FBS-free media. After 2 hours of incubation, 0.5 ml of the media was collected from each well to 1 ml of charcoal mix in a 1.5 ml centrifuge tube, vortexed briefly, and centrifuged at 4400 xg for 30 min at 4°C. Supernatants were collected from each tube and added to the scintillation liquid. The radioactivity of each vial was measured using a scintillation counter. As seen in FIG. 5, treatment with CF10 and CF10: LNPs resulted in TS inhibition at 10-fold lower dosage compared to 5-FU.EXAMPLE 6: In vivo Reduction in Tumor Volume

[0126] In vivo study was conducted where MC38 cells were injected into the spleen to travel through the portal vein to the liver in order to grow liver metastasis. After one week, as the tumor grew, treatments were started by using a control vehicle (e.g., sterile saline), 5-FU at 100 mg / kg, CF10 at 100 mg / kg, and CF10: LNP at 8 mg / kg. The tumor volume was monitored byinjecting RGD-peptide and imaging through IVIS. The relative radiant efficiency is directly proportional to the tumor volume.

[0127] As seen in FIG. 6, both CF10 and CF10: LNP exhibited a significant reduction in tumor size at 14 days and 21 days post-surgery. Additionally, CF10: LNP at more than 10-fold reduced dosage (8 mg / kg) exhibited a comparable reduction in tumor compared to CF10 at 100 mg / kg.

[0128] The experimental results described herein demonstrate that CF10, when formulated into a lipid nanoparticle formulation, is highly potent to CRC cells and provides a significant survival advantage, as a result of it being highly effective at reducing progression of tumor burden, relative to other chemotherapeutic drugs, including 5-FU and vehicle, in an orthotopic model of CRC. The orthotopic model used replicates key aspects of human disease in terms of invasion and metastasis, and CF10: LNP was effective at reducing the occurrence of distant metastases in this model. At a molecular level, CF10 potency correlates with enhanced thymidylate synthase (TS) inhibition, consistent with efficacy resulting from increased DNA-directed effects. Moreover, when CF10 is encapsulated in the lipid nanoparticle of the present disclosure, it demonstrates effective tumor reduction at a 1 / 10 dosage administration compared to the base CF10 formulation. There is no toxicity, and a delivery schedule like lx week injection could be as or more effective than long infusions.

[0129] The foregoing merely illustrates the principles of the disclosure. Any examples set forth in this specification are not intended to be limiting and merely set forth some of the many possible embodiments for the appended claims. Those skilled in the art will readily recognize various modifications and changes that may be made without following the example embodimentsand applications illustrated and described herein, and without departing from the true spirit and scope of the following claims.

[0130] All references cited and / or discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference.

[0131] Without excluding further possible embodiments, certain example embodiments are summarized in the following clauses:

[0132] Clause 1: A lipid nanoparticle comprising: a nucleic acid molecule comprising FdUMP having a polyethylene glycol (PEG) spacer appended to the 5 ’-terminus and a moiety appended to the 3 ’-terminus, wherein the moiety is not recognized by 3 ’-exonucleases; an ionizable lipid; a phospholipid; a sterol-derived lipid; and a pegylated lipid.

[0133] Clause 2: The lipid nanoparticle of clause 1, wherein the moiety is

[0134] Clause 3: The lipid nanoparticle of clause 1 or clause 2, wherein the moiety is selected from the group consisting of:

[0135] Clause 4: The lipid nanoparticle of clause 1, wherein the nucleic acid molecule iswherein X is 1 to 10, Y is 1 to 5.

[0136] Clause 5: The lipid nanoparticle of clause 4, wherein X is 10 and Y is 5.

[0137] Clause 6: The lipid nanoparticle of any one of clauses 1-5, wherein the ionizable lipid is selected from the group consisting of 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 2-[2,2-Di-[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N, N-dimethyl-2,3-bis[(9Z, 12Z)-octadeca-9, 12-dienoxy]propan-l -amine (HGT4003), (15Z, 18Z)-N, N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-15,18-dien-l-amine, (HGT5000), (15Z, 18Z)-N, N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-4, 15, 18- trien-1 -amine (HGT5001), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), di stearoylphosphatidylcholine (DSPC), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), and a combination thereof

[0138] Clause 7: The lipid nanoparticle of any one of clauses 1 -6, wherein the phospholipid is selected from the group consisting of Dilauroyl lecithin (DLPC), Dimyristoyl phosphatidylcholine (DMPC), Dioleoyl lecithin (DOPC), Dipalmitoyl phosphatidylcholine (DPPC), Di stearoyl phosphatidylcholine (DSPC), Dioleoyl phosphatidylcholine (DUPC), Palmitoyl oleoyl phosphatidylcholine (POPC), l,2-Di-O-octadecyl-sn-glycero-3 -phosphocholine,1 -Oleoyl-2-cholesteryldimethylsuccinoyl-sn-glycero-3-phosphocholine, 1 -Hexadecyl-sn-glycero-3 -phosphocholine, 1,2-Divinyl-sn-glycero-3 -phosphocholine, 1,2-Diarylacyl-sn-glycero-3 -phosphocholine, l,2-Dioleoyl-sn-glycero-3phosphorylethanolamine (DOPE), 1,2-Di-phytanoyl-sn-gly cero-3 -phosphoethanolamine, l,2-Distearoyl-sn-glycero-3 -phosphoethanolamine, 1,2-Diethenol-sn-glycero-3-phosphoethanolamine, 1,2-Divinyl-sn-gly cero-3 -phosphoethanolamine, l,2-Diaryl-sn-glycero-3-phosphoethanolamine, l,2-Dithiohexaenoate-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phosphate-(l-glycerol) sodium salt (DOPG), and a combination thereof.

[0139] Clause 8: The lipid nanoparticle of any one of clauses 1-7, wherein the sterol -derived lipid is selected from the group consisting of a cholesterol, coprosterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, bile acid, and a combination thereof.

[0140] Clause 9: The lipid nanoparticle of any one of clauses 1-8, wherein the pegylated lipid is selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000 (14:0 PEG2000 PE), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DOPE -PEG), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-gly cero-3 -phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-disteryl glycerol (DSG-PEG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (DAGPEG), PEG-dipalmitoyl phosphatidylethanolamine (DPPE-PEG), or PEG- 1,2-dimyristyloxlpropyl-3-amine (DMA-c-PEG), and a combination thereof.

[0141] Clause 10: The lipid nanoparticle of any one of clauses 1-9, wherein the ionizable lipid comprises 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200).

[0142] Clause 11: The lipid nanoparticle of any one of clauses 1-10, wherein the phospholipid comprises 1,2 Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0143] Clause 12: The lipid nanoparticle of any one of clauses 1-11, wherein the sterolderived lipid comprises cholesterol.

[0144] Clause 13: The lipid nanoparticle of any one of clauses 1-12, wherein the pegylated lipid comprises l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000.

[0145] Clause 14: The lipid nanoparticle of any one of clauses 1-13, comprising a mass ratio between the total lipid component and the nucleic acid molecule is from about 5 to about 30.

[0146] Clause 15: The lipid nanoparticle of any one of clauses 1-14, wherein the ionizable lipid is present in an amount ranging from about 5% to about 95%, by total weight of the lipid nanoparticle, the phospholipid is present in an amount ranging from about 1% to about 80% by total weight of the lipid nanoparticle, the sterol-derive lipid is present in an amount ranging from about 1% to about 80%, and the pegylated lipid is present in an amount ranging from about 1% to about 20%, by total weight of the lipid nanoparticle.

[0147] Clause 16: The lipid nanoparticle of any one of clauses 1-15, comprising a mass ratio between the nucleic acid molecule and the ionizable lipid is from about 1 / 4 to about 1 / 2.

[0148] Clause 17: The lipid nanoparticle of any one of clauses 1-16, comprising a mass ratio between the nucleic acid molecule and the phospholipid is from about 1 / 2 to about 1 / 1.

[0149] Clause 18: The lipid nanoparticle of any one of clauses 1-17, comprising a mass ratio between the nucleic acid molecule and the sterol-derived lipid is from about 1 / 1 to about 5 / 1.

[0150] Clause 19: The lipid nanoparticle of any one of clauses 1-18, comprising a mass ratio between the nucleic acid molecule and the pegylated lipid is from about 1 / 1 to about 5 / 1.

[0151] Clause 20: The lipid nanoparticle of any one of clauses 1-19, further comprising an additional therapeutic agent.

[0152] Clause 21: The lipid nanoparticle of clause 20, wherein the additional therapeutic agent is encapsulated by the lipid nanoparticle.

[0153] Clause 22: The lipid nanoparticle of clause 20 or 21, wherein the additional therapeutic agent forms a non-covalent complex with the lipid nanoparticle.

[0154] Clause 23: The lipid nanoparticle of any one of clauses 20-22, wherein the additional therapeutic is selected from the group consisting of irinotecan, leucovorin, oxaliplatin, Ataxia-telangiectasia and Rad3-related (ATR) inhibitors, Weel inhibitors, SLFN11 mRNA, GEM-DEC or siRNA targeting TS, and a combination thereof.

[0155] Clause 24: The lipid nanoparticle of any one of clauses 20-23, wherein mass ratio between the lipid nanoparticle and the additional therapeutic agent is from about 5 / 1 to about 20 / 1.

[0156] Clause 25: The lipid nanoparticle of any one of clauses 1-24, wherein the lipid nanoparticle is modified at surface of the nanoparticle with a surface modifier.

[0157] Clause 26: The lipid nanoparticle of clause 25, wherein the surface modifier comprises cell surface receptors, antibodies, antibody fragments, peptide, aptamers, small molecules, cleavable linkers, cell -penetrating peptides, or a combination thereof.

[0158] Clause 26: The lipid nanoparticle of clause 25 or 26, wherein the nanoparticle surface is modified with folinic acid.

[0159] Clause 27: The lipid nanoparticle of any one of clauses 25-27, wherein mass ratio between the lipid nanoparticle and the surface modifier is from about: to about:.

[0160] Clause 28: A pharmaceutical composition comprising the lipid nanoparticle of any one of clauses 1-27.

[0161] Clause 30: A kit comprising the lipid nanoparticle or the pharmaceutical composition of any one of clauses 1-29.

[0162] Clause 31: A method of intracellular delivery of a therapeutic agent comprising, contacting a cell or administering to a subject an effective amount of the lipid nanoparticle of any one of clauses 1-27.

[0163] Clause 32: A method of treating cancer comprising administering to a subject an effective amount of the lipid nanoparticle of any one of clauses 1-27.

[0164] Clause 33: The method of clause 32, wherein the cancer is a colorectal cancer.

[0165] Clause 34: The method of clause 32 or clause 33, further comprising administering to the subject an additional therapy selected from the group consisting of immunotherapy, chemotherapy, radiotherapy, surgery, and a combination thereof.

[0166] Clause 35: The method of any one of clauses 32-34, wherein the cancer is resistant to prior treatment of 5 -fluorouracil (5-FU).

Claims

What is claimed is:

1. A lipid nanoparticle comprising:a nucleic acid molecule comprising FdUMP having a polyethylene glycol (PEG) spacer appended to a 5’-terminus and a moiety appended to a 3’-terminus, wherein the moiety is not recognized by 3 ’-exonucleases;an ionizable lipid;a phospholipid;a sterol-derived lipid; anda pegylated lipid.

2. The lipid nanoparticle of claim 1, wherein the moiety is,, wherein the PEG spacer has a molecular weight ranging from about 5,000 to about 40,000 daltons.

3. The lipid nanoparticle of claim 1, wherein the nucleic acid molecule is4. The lipid nanoparticle of any one of claims 1-3, wherein the ionizable lipid is selected from the group consisting of 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 2-[2,2-Di-[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N, N-dimethyl-2,3- bis[(9Z, 12Z)-octadeca-9, 12-dienoxy]propan-l -amine (HGT4003), (15Z, 18Z)-N, N- dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-15,18-dien-l-amine, (HGT5000), (15Z, 18Z)-N, N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa-4, 15, 18- trien-1 -amine (HGT5001), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), and a combination thereof5. The lipid nanoparticle of any one of claims 1-4, wherein the phospholipid is selected from the group consisting of Dilauroyl lecithin (DLPC), Dimyristoyl phosphatidylcholine (DMPC), Dioleoyl lecithin (DOPC), Dipalmitoyl phosphatidylcholine (DPPC), Distearoyl phosphatidylcholine (DSPC), Dioleoyl phosphatidylcholine (DUPC), Palmitoyl oleoyl phosphatidylcholine (POPC), l,2-Di-O-octadecyl-sn-glycero-3 -phosphocholine, l-Oleoyl-2- cholesteryldimethylsuccinoyl-sn-glycero-3-phosphocholine, l-Hexadecyl-sn-glycero-3- phosphocholine, l,2-Divinyl-sn-glycero-3 -phosphocholine, l,2-Diarylacyl-sn-glycero-3- phosphocholine, l,2-Dioleoyl-sn-glycero-3phosphorylethanolamine (DOPE), 1,2-Di- phytanoyl-sn-glycero-3-phosphoethanolamine,l,2-Distearoyl-sn-glycero-3- phosphoethanolamine, l,2-Diethenol-sn-glycero-3 -phosphoethanolamine, 1,2-Divinyl-sn- glycero-3-phosphoethanolamine, l,2-Diaryl-sn-glycero-3 -phosphoethanolamine, 1,2- Dithiohexaenoate-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phosphate- (1 -glycerol) sodium salt (DOPG), and a combination thereof.

6. The lipid nanoparticle of any one of claims 1-5, wherein the sterol -based lipid is selected from the group consisting of a cholesterol, coprosterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, bile acid, and a combination thereof.

7. The lipid nanoparticle of any one of claims 1-6, wherein the pegylated lipid is selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000 (14:0 PEG2000 PE), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DOPE -PEG), 1,2-dimyristoyl- sn-glycerol methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-disteryl glycerol (DSG-PEG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (DAGPEG), PEG-dipalmitoyl phosphatidylethanolamine (DPPE-PEG), or PEG- 1,2- dimyristyloxlpropyl-3-amine (DMA-c-PEG), and a combination thereof8. The lipid nanoparticle of any one of claims 1-7, whereinthe ionizable lipid comprises 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), the phospholipid comprises 1,2 Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), the sterol -derived lipid comprises cholesterol, andthe pegylated lipid comprises l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- methoxy (polyethylene glycol)-2000.

9. The lipid nanoparticle of any one of claims 1-8, comprising a mass ratio between total lipid component and the nucleic acid molecule is from about 5 to about 30.

10. The lipid nanoparticle of any one of claims 1-9, wherein the ionizable lipid is present in an amount ranging from about 5% to about 95%, by total weight of the lipid nanoparticle, the phospholipid is present in an amount ranging from about 1% to about 80% by total weight of the lipid nanoparticle, the sterol-derive lipid is present in an amount ranging from about 1% to about 80%, and the pegylated lipid is present in an amount ranging from about 1% to about 20%, by total weight of the lipid nanoparticle.

11. The lipid nanoparticle of any one of claims 1-10, further comprising an additional therapeutic agent, wherein mass ratio between the lipid nanoparticle and the additional therapeutic agent is from about 5 / 1 to about 20 / 1.

12. The lipid nanoparticle of claim 11, wherein the additional therapeutic agent forms a non- covalent complex with the lipid nanoparticle.

13. The lipid nanoparticle of claim 11 or 12, wherein the additional therapeutic is selected from the group consisting of irinotecan, leucovorin, oxaliplatin, Ataxia-telangiectasia and Rad3- related (ATR) inhibitors, Weel inhibitors, SLFN11 mRNA, GEM-DEC or siRNA targeting TS, and a combination thereof.

14. The lipid nanoparticle of any one of claims 1-13, wherein the lipid nanoparticle is modified at surface of the nanoparticle with a surface modifier selected from the group consisting of cell surface receptors, antibodies, antibody fragments, peptide, aptamers, small molecules, cleavable linkers, cell-penetrating peptides, and a combination thereof.

15. The lipid nanoparticle of claim 14, wherein the nanoparticle surface is modified with folinic acid.

16. A pharmaceutical composition comprising the lipid nanoparticle of any one of clauses 1-15.

17. A method of treating cancer comprising administering to a subject an effective amount of the lipid nanoparticle of any one of claims 1-16.

18. The method of claim 17, wherein the cancer is a colorectal cancer.

19. The method of claim 17 or claim 18, further comprising administering to the subject an additional therapy selected from the group consisting of immunotherapy, chemotherapy, radiotherapy, surgery, and a combination thereof.

20. The method of any one of claims 17-19, wherein the cancer is resistant to prior treatment of 5- fluorouracil (5-FU).

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