WO2024259158A2

WO2024259158A2 - Lipid nanoparticles for peptide delivery and methods of making and using the same

Info

Publication number: WO2024259158A2
Application number: PCT/US2024/033894
Authority: WO
Inventors: Christopher Akinleye ALABI; Souvik GHOSAL
Original assignee: Cornell University
Current assignee: Cornell University
Priority date: 2023-06-13
Filing date: 2024-06-13
Publication date: 2024-12-19
Anticipated expiration: 2025-12-13
Also published as: WO2024259158A3

Abstract

Lipid nanoparticles, compositions comprising lipid nanoparticles, and methods of making and using lipid nanoparticles. In various examples, a lipid nanoparticle: ionizable cationic amino lipid(s); PEG-lipid(s); phospholipid(s); and sterol(s); or a combination component comprising two or more groups having the functionality of two the aforementioned lipid components, which replaces those two aforementioned lipid components; and optionally, one or more cationic lipid component(s). In various examples, the lipid nanoparticle comprises one or more peptide(s), functionalized peptide(s), or the like, or any combination thereof, one or more of which may be therapeutic. In various examples, a composition, which may be a pharmaceutical composition, comprises a plurality of the lipid nanoparticles. In various examples, the lipid nanoparticles are used in method of delivering peptide(s), functionalized peptide(s), or the like, or any combination thereof. In various examples, the lipid nanoparticles are administered to an individual, which may be in need of treatment.

Description

LIPID NANOPARTICLES FOR PEPTIDE DELIVERY AND METHODS OF MAKING AND USING THE SAME

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63/472,771, filed June 13, 2023, and 63/563,839, filed March 11, 2024; the contents of the above-identified applications are hereby fully incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which is submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml file is named “018617 01778 Sequence Listing. xml”, was created on June 13, 2024, and is 14,621 bytes in size.

BACKGROUND OF THE DISCLOSURE

[0003] The past decade has seen a surge in the development of protein degrading molecules, especially those targeting various onco-proteins. PROTACs (PROteolysis TArgeting Chimeras) are heterobifunctional degraders that facilitate the selective degradation of a diverse array of intracellular proteins by hijacking the cell’s own naturally occurring protein degradation mechanism-the ubiquitin-proteasome system (UPS). By employing a ligand that recruits an E3 ubiquitin ligase and another that binds a protein of interest (POI), PROTACs induce an artificial interaction between an E3 and a POI. The ternary complex catalyzes selective ubiquitin-tagging of the POI, leading to its proteasome-mediated degradation. In contrast to the conventional stoichiometric binding mechanism employed by classic small molecule inhibitors, PROTACs exploit an event-driven approach that promotes the degradation of multiple POIs, ensuring a high turnover frequency and potent catalytic activity. However, a significant challenge remains, as many intracellular proteins are still considered “undruggable” due to the absence of a well- defined binding pocket.

[0004] As an alternative to small molecule-based approaches, peptide-based ligands possess large protein-protein interaction surfaces, making them suitable for targeting any POI. Coupled with the rapid development of structural biology techniques that provide detailed protein-protein structural information, mature directed-evolution technologies such as phage and yeast display, and emerging computational approaches for rapid discovery of synthetic binding peptides, peptide-based ligands are ideal for extending the scope of PROTACs to “undruggable” proteins. Several Peptide-based Proteolysis Targeting Chimeras (PepTACs) targeting oncoproteins and transcription factors make use of a cationic cell-penetrating peptides, cyclic peptides, or peptide stapling to facilitate cellular uptake. While promising, these approaches still suffer from poor cellular permeability, thus requiring very high doses in the tens of micromolar range for PepTAC activity. These examples are in stark contrast to small molecule PROTACs that routinely enable degradation in the sub-nanomolar range. Furthermore, PepTACs suffer from limited serum stability, preventing their wide-scale adoption in vivo.

[0005] The development of small molecule-based degraders against intracellular protein targets is a rapidly growing field that is hindered by the limited availability of high-quality small molecule ligands that bind to the target of interest. Despite the feasibility of designing peptide ligands against any protein target, peptide-based degraders still face significant obstacles such as, limited serum stability and poor cellular internalization.

SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides, inter alia, lipid nanoparticles. The present disclosure also provides methods of making and using lipid nanoparticles.

[0007] In an aspect, the present disclosure provides lipid nanoparticles. In various examples, a lipid nanoparticle comprises: i) one or more first lipid component(s), wherein the first lipid component(s) is/are independently ionizable cationic amino lipid(s); one or more second lipid component(s), wherein the second lipid component(s) is/are independently PEG-lipid(s); one or more third lipid component s) wherein the third lipid component(s) is/are independently phospholipid(s); one or more fourth lipid component(s), wherein the fourth lipid component(s) is/are independently sterol(s); optionally, one or more cationic lipid component(s), wherein the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof; or ii) one or more first combination lipid component(s), wherein the first combination lipid component s) independently comprise one or more ionizable cationic amino lipid group(s); and one or more phospholipid group(s); one or more second lipid component(s), wherein the second lipid component s) is/are independently PEG-lipid(s); one or more fourth lipid component(s) wherein the fourth lipid component(s) is/are independently sterol(s); optionally, one or more cationic lipid component(s), wherein the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof; o iii) one or more second combination lipid component(s), wherein the second combination lipid component(s) independently comprise one or more ionizable cationic amino lipid group(s); and one or more sterol group(s); one or more second lipid component(s), wherein the second lipid component(s) is/are independently PEG-lipid(s); one ormore third lipid component(s) wherein the third lipid component(s) is/are independently phospholipid(s); or optionally, one or more cationic lipid component(s), wherein the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof. In various examples, the cationic head group(s) independently comprise a quaternary ammonium group, guanidinium group, amidinium group, imidazolium group, pyridinium group, pyrrolidinium group, a salt thereof, or a structural and/or functional analog thereof and/or the degradable group(s) independently comprise an ester group, a phosphate group, a disulfide group, an imine group, an amide group, a thioester group, an acetal or ketal group, a hydrazone group, a cis-aconityl group, an orthoester group, or a structural and/or functional analog thereof. In various examples, the one or more peptide(s), the one or more functionalized peptide(s) is/are independently a therapeutic peptide or a therapeutic functionalized peptide. In various examples, the one or more peptide(s), the one or more functionalized peptide(s) is/are independently chosen from protein degrading peptides, amphiphilic proteolysis targeting peptides, PEptide-based Proteolysis TArgeting Chimeras (PEPTACs), and structural and/or functional analogs thereof. In various example, the lipid nanoparticle comprises a longest linear dimension of about 10 nanometers to about 1000 nanometers, including all 0.1 nanometer values and ranges therebetween.

[0008] In an aspect, the present disclosure provides compositions. In various examples, a composition comprises a plurality of lipid nanoparticles, wherein the lipid nanoparticles are independently a lipid nanoparticle of the present disclosure. In various examples, the composition is a solution or an aqueous dispersion. In various examples, the lipid nanoparticles exhibit or the composition exhibits an isoelectric point (or an apparent pKa) of about the composition comprises one or more pharmaceutical excipient(s). In various examples, the composition does not exhibit substantial or any observable lipid nanoparticle aggregation, a substantial change or any change in lipid nanoparticle size for at least one-week or more at a temperature of about 4 degrees Celsius (°C) or both.

[0009] In an aspect, the present disclosure provides methods of making lipid nanoparticles. In various examples, a method of making lipid nanoparticles (such as, for example, lipid nanoparticles of the present disclosure) comprises: contacting a first composition comprising, if present, the first lipid component(s), the second lipid component(s), the third lipid component(s), the fourth lipid component(s), the cationic lipid component(s), the first combination lipid component(s), and the second lipid components and one or more organic solvent(s) and a second composition comprising one or more peptide(s), one or more functionalized peptide(s), or any combination thereof, wherein the lipid nanoparticles are formed; optionally, removing residual organic solvent(s) after lipid nanoparticle formation; and dialyzing the lipid nanoparticle formulation against water or a pH buffered solution. In various examples, the method further comprising isolating the lipid nanoparticles.

[0010] In an aspect, the present disclosure provides uses of lipid nanoparticles. In various examples, lipid nanoparticles are used in peptide, functionalized peptide, or any combination thereof delivery and in kits.

[0011] In various examples, a method of peptide, functionalized peptide, or any combination thereof delivery comprises: contacting a population of cells or an individual, with a plurality of lipid nanoparticles, wherein the lipid nanoparticles are independently a lipid nanoparticle of the present disclosure. In various examples, at least a portion of or all of the lipid nanoparticles are delivered to the population of cells or the individual. In various examples, the contacting is in vitro or in vivo. In various examples, the method comprises treating, preventing, or any combination thereof, a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual. In various examples, the method comprises administration of the plurality of lipid nanoparticles to an individual. In various examples, at least a portion, substantially all, or all the lipid nanoparticles(s) is/are taken up by a cell or cells of the population of cells or the individual, and the lipid nanoparticles is/are is released within the cell or cells. In various examples, the peptide(s), the functionalized peptide(s), or any combination thereof after delivery independently retain/retains substantially all (or all) structural features and/or 50% or more, 70% or more, 90% or more, or 95% or more of its biological activity compared with the native peptide or the peptide or the functionalized peptide delivered without use of the lipid nanoparticle of the present disclosure. In various examples, the individual is diagnosed with and/or is in need of treatment for a disease, disease state, and the disease, the disease state is treatable, preventable, by the peptide(s), the functionalized peptide(s), or the combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0012] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0013] FIG. 1 shows encapsulation of PepTACs in LNPs allows for endosomal uptake, escape, and cytosolic delivery. Upon release in the cytoplasm, PepTACs form a ternary complex with the E3 ubiquitin ligase, which initiates ubiquitination and degradation of the POI by the proteosome.

[0014] FIG. 2 shows A - AlphaFold-Multimer prediction of CREPT CCT dimer (4NAD) with the 21-mer CREPT ligand. B - Flow cytometry data showing transfection of LNPs formulated at pH 3 with and without DOTAP in HeLa cells at 500 nM. C -MC3 LNPs formulated at different mol percentages of DOTAP transfected into HeLa cells with 500 nM ^CRPepTAC. D - Effect of different DOTAP derivatives (all at 10 mol %) on LNP-mediated ^CRPepTAC (200 nM) delivery into HeLa cells. E - Effect of pH, and F - ionizable lipid/^CRPepTAC wt/wt ratio on LNP-mediated ^CRPepTAC delivery. G - Delivery of ^CRPepTAC via MC3 LNP into different cell types, H - at different concentrations. I - Uptake of fluorescein- ^CRPepTAC into HeLa cells as a function of time. Data are displayed as mean ± SD by one-way ANOVA followed by Tukey correction for multiple comparisons. * p < 0.05, ** p < 0.01, ***p < 0.001, ****_p < 0.0001.

[0015] FIG. 3 shows A - Confocal imaging of LNPs with ^CRPepTAC transfected into HeLa cells. Nucleus stained with Hoechst400, endosome tracker for endosomal staining, fluorescein labelled ^CRPepTAC and microtubule tracker. Scale bar = 30 pm. B - Magnified image of a single cell showing endosomal and cytoplasmic staining of fluorescein-^CRPepTAC using LNPs formulated at pH 3. Scale bar = 10 pm.

[0016] FIG. 4 shows A - HeLa cellular uptake of LNPs formulated with ^CRPepTAC or peptide ligand alone at various concentrations. B - Encapsulation efficiency of LNPs formulated with ^CRPepTAC or peptide ligand alone. C - HeLa cell uptake of CREPT ligand (CL) lipopeptides (500 nM) with different alkyl tail lengths. D - Wnt signalling pathway and CREPT- initiated transcription. E -Western blots of CREPT protein degradation in different cells with ^CRPepTAC alone, formulated with iLNPs with and without the proteosome inhibitor, epoxymicin (Epox). Cells were treated for 24h and then harvested. F - Degradation of CREPT-Luc in HeLa cells treated with LNP- ^CRPepTAC for 24 h (h=hour(s)) with and without epoxymicin (Epox). The data was normalized to Renilla expression in the same cells. G -Wnt active DLD-1 cells and H -HepG2 cells were transfected with Topflash and Fopflash luciferase followed by different concentrations of LNP-^CRPepTAC. Luciferase reporter activity was assayed 24 hours after ^CRPepTAC treatment. I - Effect of LNP-^CRPepTAC treatments at different concentrations on Wnt-active DLD-1 cell viability at 24, 48 and 72h. Cell viability was assessed via an MTS assay. J - Effect of LNP-^CRPepTAC treatments at different concentrations on Wnt-active DLD-1 and HepG2, and non-Wnt active HeLa cell viability after 72 h. Cell viability was assessed via an MTS assay. Data are displayed as mean ± SD by one-way ANOVA. * p < 0.05, ** p < 0.01, ***p < 0.001, ****P < 0.0001.

[0017] FIG. 5 shows A - AlphaFol d-Multimer prediction of P-catenin (armadillo repeat region) with the BCL9 derived peptide ligand. B - Western blots of P-catenin protein degradation in different cells with ^|iC:itPepTAC alone, formulated with LNPs with and without the proteosome inhibitor, epoxymicin. Cells were treated for 24h and then harvested. C - Quantification of HeLa cell western blot (duplicates). D - Wnt active DLD-1 cells and E - HepG2 cells were transfected with Topflash and Fopflash luciferase followed by different concentrations of LNP-^pCatPepTAC. Luciferase reporter activity was assayed 24 hours after ^pCatPepTAC treatment. F - Effect of LNP- P^CatPepTAC treatments at different concentrations on Wnt-active DLD-1 cell viability at 24, 48 and 72h. Cell viability was assessed via an MTS assay. G - Effect of LNP-^pCatPepTAC treatments at different concentrations on Wnt-active DLD-1 and HepG2, and non-Wnt active HeLa cell viability after 72h. Cell viability was assessed via an MTS assay. Data are displayed as mean ± SD by one-way ANOVA. * * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.

[0018] FIG. 6 shows A - Whole animal fluorescence imaging of SKH-1 hairless mice after 1 hour and 24 hours. Top - LNP-^CRPepTAC, bottom ^CRPepTAC only (5 mg/kg, 50% labeled cargo). B - Ex vivo imaging and C -quantified fluorescence signal after 1 hour and 24 hours of Cy5.5 labeled PepTACs in major organs extracted from SKH-1 hairless mice IV injected with LNP-PepTAC, PepTAC alone and PBS. Confocal images of sectioned liver tissue that received Cy5.5 labeled LNP-PepTAC or PepTAC alone after D - 1 hour and E - 24 hours stained with nuclei stain (Hoechst, left panel). Cy5.5 fluorescence represented in the middle panel, and the overlay is shown on the right panel. F - Liver [3-catenin levels assessed via western blot from normal healthy Balb/c mice treated every 3 days for 10 days with saline or 5 mg/kg ^|iCalPepTAC- LNPs. Data are displayed as a violin plot and analyzed using an unpaired t-test with Welch’s correction. * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.

[0019] FIG. 7 shows dynamic light scattering of different iLNP:PepTAC formulations. A) LNP formulations (MC3:PepTAC 2w/w, pH formulation) with and without DOTAP. B) LNP formulations (MC3:PepTAC 2w/w, +DOTAP, ) at pH 3 and pH5, with and without a fluorophore on the PepTAC.

[0020] FIG. 8 shows zeta potential measurements of iLNP:PepTAC formulations at A) different pH’s, and B) with and without DOTAP.

[0021] FIG. 9 shows CryoTEM of iLNP:PepTAC formulation (pH 3, MC3:PepTAC 2w/w with DOTAP).

[0022] FIG. 10 shows Encapsulation efficiency of DOTAP LNP-PepTAC formulations (A) with and without DOTAP, and (B) with DOTAP at pH7, pH5, pH3.

[0023] FIG. 11 shows effects of different structural analogs of DOTAP in the cellular uptake of fluorescein-labeled PepTAC in HeLa cells depicted in the histograms obtained through flow cytometry. Careful observation of the subtle structural differences of different DOTAP-analogs shown in the right panel suggests that the permanently cationic nature of the 5^th lipid is probably the most critical structural feature in order to obtain a good cellular uptake of labeled PepTAC. [0024] FIG. 12 shows A) circular Dichroism of PepTACs at different pH values used in the formulation. B) Circular Dichroism of LNP -PepTACs at different formulation pH values relative to PepTACs alone.

[0025] FIG. 13 shows the effect of DOPE vs DSPC in the LNP-PepTAC formulation. HeLa cells were transfected with the LNP-PepTAC formulation for 24hrs.

[0026] FIG. 14 shows the effect of MC3: DSPC ratio in the LNP-PepTAC formulation on uptake in HeLa cells.

[0027] FIG. 15 shows dose dependent uptake of MC3 LNP -PepTACs formulated at pH5 in HeLa cells. [0028] FIG. 16 shows the effect of different ionizable lipids in A) SK-BR-3, and B) SKOV-3 cells.

[0029] FIG. 17 shows the effect of MC3:PepTAC wt/wt ratio on SK-BR-3 cellular uptake.

[0030] FIG. 18 shows the effect of MC3:PepTAC wt/wt ratio on SKOV-3 cellular uptake.

[0031] FIG. 19 shows the effect of pH on uptake in A) SKBR-3 and B) SKOV-3 cells.

[0032] FIG. 20 shows systematic dose-dependent cellular uptake of fluorescein-labeled

^CRPepTAC in DLD1 cells.

[0033] FIG. 21 shows systematic dose-dependent cellular uptake of fluorescein-labeled

^CRPepTAC in HepG2 cells.

[0034] FIG. 22 shows temperature dependent uptake of LNP-PepTACs in HeLa cells (MC3:PepTAC 2wt/wt, 2h incubation with 500 nM PepTAC).

[0035] FIG. 23 shows cellular uptake of lipid modified CL peptides of different lipid tail lengths (C6, CIO and Cl 2) formulated with LNPs and transfected into HeLa cells.

[0036] FIG. 24 shows the effect of DOTAP as a 5^th lipid in the LNP formulation of ^CRPepTAC. CREPT degradation was measured in HeLa cells transiently expressing Firefly luciferase-fused CREPT protein.

[0037] FIG. 25 shows the effect of LNP-^CRPepTAC treatments at different concentrations on Wnt-active HepG2 cell viability at 24, 48 and 72h. Cell viability was assessed via an MTS assay. [0038] FIG. 26 shows the effect of LNP-^pCatPepTAC treatments at different concentrations on Wnt-active HepG2 cell viability at 24, 48 and 72h. Cell viability was assessed via an MTS assay.

[0039] FIG. 27 shows Apparent pKa (using the TNS assay) of MC3 PepTAC -LNPs formulated at pH3 and pH5 (MC3: PepTAC 2w/w).

[0040] FIG. 28 shows representative Western Blot image showing P-Catenin expression level in mice treated with saline versus mice treated with ^pCatPepTAC encapsulation inside LNP. n = 5 per group.

[0041] FIG. 29 shows formulation optimization for lipid nanoparticles loaded with fluorescein-labeled ^CREPTPepTAC. Four parameters were investigated: A) Nanoparticle size, B) cellular uptake, characterized by the mean fluorescein intensity, C) encapsulation efficiency which captures information about the peptide loading efficiency, and D) serum stability characterized by % encapsulation efficiency in presence of serum (1 : 1 volume ratio of serum :buffer, 24 hr incubation at room temperature). The collective data suggests that -30% DOPE with 2-2.5% PEG lipid is optimal for in vivo translation.

[0042] FIG. 30 shows TOPFLash assay performed with a PepTAC designed against [3- catenin based on P-catenin-TCF4 protein-protein interaction surface, formulated with LNPs. The LNP -PepTAC was administered to DLD1 cells and shows -25% reduction in Wnt- transcriptional activity at doses of 200 nM or greater. The data demonstrates LNP -based delivery of a PepTAC with a negative overall net charge of -6 at physiological pH. The sequence of the peptide-based degrader (N to C) is: IYP(OH)AL-Ahx-DELISFKDEGEQEERDLADVKSSLVN- NH2 (SEQ ID NO: 3) where P(OH) is a hydroxylated proline. The peptide is amidated at the C- terminus.

[0043] FIG. 31 shows TOPFlash assay performed with the same ^p-CatPepTAC sequence utilized in Example 1 and 2 where the sequence has been modified with acetyl group at the N- terminus. The PepTAC was formulated with LNPs and administered to DLD1 cells. LNP- PepTAC treated cells show nearly 50% reduction in Wnt-transcriptional activity starting from 50 nM dosage. This data highlights another example of a chemically modified peptide being delivered by LNPs and exerting functional activity.

[0044] FIG. 32 shows the effect of LNP-^CRPepTAC formulated using different buffers (10 mM citrate buffer at different pH’s) on the overall degradation of Luc-CREPT (Luciferase-fused CREPT protein) transiently expressed in HeLa cells. The data demonstrates that the LNP formulation obtained using a pH 3 buffer is the most active in vitro. However, the data also shows formulations in pH 5 and pH 7 buffer still maintain their activity.

[0045] FIG. 33 shows the effect of LNP-^CRPepTAC formulated with different ionizable lipids (MC3, ALC0315, SM102) on the overall degradation of Luc-CREPT (Luciferase-fused CREPT protein) transiently expressed in HeLa cells.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0046] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0047] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and/or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/- 0.1% or less of and from the specified value), insofar such variations in a variable and/or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0048] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about”, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0049] As used herein, unless otherwise indicated, the term “aliphatic group” is a branched or unbranched (linear) hydrocarbon group or a cyclic hydrocarbon (carbocyclic) group, optionally, comprising one or more degree(s) of unsaturation. An aliphatic group may be an alkyl group. Non-limiting examples of aliphatic groups with one or more degree(s) of unsaturation include alkenyl groups, alkynyl groups, aliphatic cyclic groups, and the like. In various examples, an aliphatic group is a C3 to Ceo aliphatic group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., a C3 to C20 aliphatic group). An aliphatic group may be unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, halide groups (-F, -Cl, -Br, -I, and the like), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, hydroxyl group, and the like, and combinations thereof. [0050] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like). Illustrative, non-limiting examples of groups include:

[0051] As used herein, unless otherwise stated, the term “structural analog” refers to any compound or portion thereof (such as, for example, a lipid component, a peptide, a functional peptide, an anion, or the like) if one atom or group of atoms, functional groups, or substructures is replaced with another atom or group of atoms, functional groups, substructures, or the like. In various examples, the term “structural analog” refers to any group that is derived from an original any compound or portion thereof (such as, for example, a lipid component, a peptide, a functional peptide, an anion, or the like) by a chemical reaction, where the compound is modified or partially substituted such that at least one structural feature of the compound or group is retained.

[0052] As used herein, unless otherwise stated, the term “functional analog” refers to any compound or portion thereof (such as, for example, a lipid component, a peptide, a functional peptide, an anion, or the like) that can be envisioned to arise from an original compound or portion thereof if one atom or group of atoms, functional groups, or substructures is replaced with another atom or group of atoms, functional groups, substructures, or the like and the compound or portion thereof exhibits substantially the same or the same function as the original compound. In various examples, the term “functional analog” refers to any group that is derived from an original compound or portion thereof (such as, for example, a lipid component, a peptide, a functional peptide, an anion, or the like, or the like) by a chemical reaction, where the compound is modified or partially substituted such that at least one structural feature of the compound or group is retained and the compound or portion thereof exhibits substantially the same or the same function as the original compound.

[0053] The present disclosure provides, inter alia, lipid nanoparticles. The present disclosure also provides compositions comprising lipid nanoparticles and methods of making and using lipid nanoparticles and compositions comprising lipid nanoparticles.

[0054] In an aspect, the present disclosure provides lipid nanoparticles (LNPs). In various examples, a lipid nanoparticle is made by a method of the present disclosure. Non-limiting examples of lipid nanoparticles are described herein.

[0055] In various examples, a lipid nanoparticle comprises one or more cationic lipid component(s) (which may be referred to as permanently cationic lipid component s)). In various examples, each cationic lipid component independently comprises a cationic head group, a degradable group, and a lipid group (such as, for example, a saturated or unsaturated lipid group) (which may be referred to as a tail(s)). In various examples, a lipid nanoparticle further comprises one or more or all of one or more first lipid component(s); one or more second lipid component(s); one or more third lipid component(s); or one or more fourth lipid component(s). In various examples, a lipid nanoparticle further comprises one or more peptide(s), one or more functionalized peptide(s), or any combination thereof. In various examples, two or more of the lipid components are replaced by a combination lipid component or component(s). In various examples, a lipid nanoparticle (e.g., an il.NP or the like) further comprises a first lipid component and second lipid component.

[0056] In various examples, a lipid nanoparticle comprises: i) one or more or all of: one or more first lipid component(s), where the first lipid component(s) is/are independently ionizable cationic lipid(s) (such as, for example, ionizable cationic amino lipid(s) or the like, or any combination thereof); one or more second lipid component(s), where the second lipid component(s) is/are independently PEG-lipid(s); one or more third lipid component(s) where the third lipid component(s) is/are independently phospholipid(s); one or more fourth lipid component(s), where the fourth lipid component(s) is/are independently sterol(s); optionally, one or more cationic lipid component(s); and optionally, one or more peptide(s), one or more functionalized peptide(s), or any combination thereof. In various examples, the cationic lipid component(s) independently comprise a cationic head group, a degradable group, and one or more lipid group(s).

[0057] A lipid nanoparticle can comprise various lipid components and combinations of lipid components. In various examples, a lipid component comprises an anionic head group, a neutral headgroup, a polymeric headgroup, or the like, a degradable group, and one or more lipid group(s) (tail(s)) (which may be saturated or unsaturated lipid group(s) (tail(s)). In various examples, a lipid component is a first lipid component, a second lipid component, a third lipid component, a fourth lipid component, a cationic lipid component, a combination lipid component or the like. In various examples, a lipid component (e.g., first lipid component(s), second lipid component(s), third lipid component(s), fourth lipid component(s), cationic lipid component(s), combination component(s) or the like) is/are independently a naturally-occurring lipid, a non- naturally-occurring lipid, a synthetic lipid, or the like. In various examples, a lipid component is a salt. In various examples, a lipid component further comprises one or more counter anion(s). Non-limiting examples of counter anions include chloride, bromide, iodide, and other halides, carboxylates (such as, for example, acetate and the like), nitrate, sulfate, sulfonates (such as, for example, trilfuoromethane sulfonate and the like), phosphate, phosphonates, and the like, structural and/or functional analogs thereof, and any combination thereof.

[0058] In various examples, a lipid nanoparticle comprises one or more ionizable cationic lipid(s) (e.g., first lipid component s)). Combinations of ionizable cationic lipids may be used. In various examples, an ionizable cationic lipid (which may be referred to as an ionizable lipid) is a lipid whose net charge is able to be changed (e g., to a positive charge) in response to its surroundings or environment (such as, for example, local environment or the like). In various examples, an ionizable cationic lipid is a pH-responsive lipid whose net charge changes in response to the pH of its surroundings or environment (such as, for example, local environment or the like). In various examples, an ionizable cationic lipid is neutral at physiological pH, but protonated at low pH, making them positively charged. Without intending to be bound by any particular theory, it is considered lipid nanoparticles comprising an ionizable cationic lipid can exhibit desirable and minimal positive charge density in the bloodstream. It is considered that this charge density can endow an ionizable cationic lipid with desirable biocompatibility, reduced off-target accumulation, and the like. Also, ionizable cationic lipid(s) may promote(s) self-assembly into nanoparticles via electrostatic interaction and aids in endosomal escape. In various examples, one or more or all of the ionizable cationic lipid(s) is/are ionizable cationic amino lipid(s), or the like, salts thereof, structural and/or functional analogs thereof.

[0059] Non-limiting examples of ionizable cationic lipids include (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA or MC3),

1.2-dioleyloxy-3 -dimethylaminopropane (DODMA), l,2-dioleoyl-3-dimethylammonium- propane (DODAP), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC Cholesterol), l,2-dipalmitoyl-3 -dimethylammonium -propane (16:0 DAP), l,2-distearyloxy-3- dimethylaminopropane (DSDMA), l,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA),

1.2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), l,2-bis(linoleoyloxy)-3- dimethylaminopropane (DLinDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolan (DLin-KC2-DMA), 2,2- dilinoleyl-4-dimethylaminopropyl-[l,3]-dioxolane (DLin-KC3-DMA), 2,2-dilinoleyl-4- dimethylaminobutyl-[l,3]-di oxolane (DLin-KC4-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), 9-[4-(dimethylamino)-l-oxobutoxy]- heptadecanedioic acid, l,17-di-(2Z)-2-nonen-l-yl ester (L319), 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol, Nl,N16-didodecyl-4,7,13-tris(3-(dodecylamino)- 3-oxopropyl)-4,7,10,13-tetraazahexadecanediamide (98ND12-5), l,l'-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]bis-2- dodecanol (C 12-200), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof. Non-limiting examples of ionizable cationic lipids are shown in Table 1.

[0060] Table 1.

[0061] In various examples, a lipid nanoparticle comprises one or more polyethylene (PEG)- lipid(s) (e.g., second lipid components). Combinations of PEG-lipids may be used. A polyethylene group of a PEG lipid can have various sizes. In various examples, a PEG lipid comprises one or more PEG group(s), such as, for example, PEG group(s) independently comprising a molecular weight (MW) of about 500 to about 20,000 g/mol, including all 0.1 g/mol values and ranges therebetween and/or about 10 to about 460 ethylene glycol (EG) groups, including all integer number of EG groups and ranges therebetween. Without intending to be bound by any particular theory, it is considered a PEG-lipid provides steric stabilization of a lipid nanoparticle from serum proteins and, along with cholesterol, enhances lipid nanoparticle stability in circulation.

[0062] Non-limiting examples of PEG-lipids include l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG-2000), di stearoyl -rac-glycerol-PEG2K, 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:0 PEG2000 PE), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (14:0 PEG2000 PE), N-palmitoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]}(C16 PEG2000 Ceramide), N-palmitoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)5000]](C 16 PEG5000 Ceramide), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[maleimide(poly ethylene glycol)-2000] (DSPE-PEG(2000) Maleimide), N-myristoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (PEG- CerC )), N-arachidoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (PEG- CerC2o), polyethyleneglycol-succinoyl-distearoylglycerol (PEG-S-DSG), polyethyleneglycol- succinoyl-dimyristolglycerol (PEG-S-DMG), N-[(methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-C-DMA), 1 ,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 ((m)PEG-DMG / (m)PEG-DMG-2000), polyethyleneglycol-distearoylglycerol (PEG-DSG), R-3-[(co-methoxy polyethylene glycol)2000)carbamoyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-DOMG / PEG-C-DOMG), alpha-(3' -{[l,2-di(myristyloxy)propanoxy] carbonylamino}propyl)-co-methoxy, polyoxyethylene (PEG-C-DMG-2000), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof. Non-limiting examples of PEG-lipids are shown in Table 2.

[0063] Table 2.

[0064] In various examples, a lipid nanoparticle comprises one or more phospholipid(s) (e.g., third lipid component(s)). In various examples, a phospholipid is a zwitterionic phospholipid or the like. Without intending to be bound by any particular theory, it is considered a phospholipid (such as, for example, a zwitterionic phospholipid or the like) can facilitate fusion of a lipid nanoparticle with an endosomal membrane upon endocytosis.

[0065] Non-limiting examples of phospholipids include (2R)-2,3- Bis(octadecanoyloxy)propyl 2-(trimethylazaniumyl)ethyl phosphate (DSPC), 1 -palmitoyl -2- oleoyl-glycero-3 -phosphocholine (16:0-18: 1 PC (POPC)), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- distearoyl-sn-glycero-3 -phosphoethanolamine (10:0 PE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), N-(dodecanoyl)-sphing-4-enine-l -phosphocholine (SM), 1,2- diphytanoyl-sn-glycero-3 -phosphatidylethanolamine (DPyPE), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof. Non-limiting examples of phospholipids are shown in Table 3.

[0066] Table 3.

[0067] In various examples, a lipid nanoparticle comprises one or more sterol(s) (e.g., fourth lipid component s)). Combinations of sterols may be used. Without intending to be bound by any particular theory, it is considered a sterol stabilizes a lipid nanoparticle (e.g., enhancing serum stability or the like, and/or enables cell fusion upon endocytosis or the like.

[0068] Non-limiting examples of sterols include cholesterol (cholest-5-en-3P-ol or

(!R,3aS,3bS,7S,9aR,9bS,l laR)-9a,l la-Dimethyl-l-[(2R)-6-methylheptan-2-yl]-

2, 3, 3 a, 3b, 4, 6, 7, 8, 9, 9a, 9b, 10, 11,1 la-tetradecahydro-lH-cyclopenta[a]phenanthren-7-ol), cholestanol, 7-dehydrocholesterol, cholestan-3-one, cholesteryl oleate stigmasterol, glucosyl stigmasterol, sitosterol, lanosterol, zymosterol, and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof. Non-limiting examples of sterols shown in Table 4.

[0069] Table 4.

[0070] A lipid nanoparticle may comprise various combination lipid components. In various examples, a combination lipid component comprises two or more functionalities and/ or structures (or a group formed therefrom) of two or more of any combination of lipid components (e.g., first lipid component(s), second lipid component(s), third lipid component s), fourth lipid component(s), cationic lipid component(s), or the like, or a group formed therefrom). In various examples, a combination component is present in an amount corresponding to the lipid component(s) replaced by combination component. In various examples, a combination lipid component comprises functionality of a cationic lipid component (or a group formed therefrom) and a first lipid component (e.g., ionizable cationic amino lipid) (or a group formed therefrom) in a single component, and replaces a cationic lipid component and a first lipid component. In various examples, a combination lipid component comprises functionality of a cationic lipid component and a first lipid component (e.g., ionizable cationic amino lipid) (or a group formed therefrom) and a third lipid component (e.g., a phospholipid or the like) (or a group formed therefrom) in a single component and replaces a cationic lipid component and a first component and a third component in a single component, and replaces a cationic lipid component, a first lipid component and a third lipid component. In various examples, a combination lipid component comprises functionality of a first lipid component (e.g., ionizable cationic amino lipid) (or a group formed therefrom) and a third lipid component (e.g., a phospholipid or the like) (or a group formed therefrom) in a single component and replaces a first lipid component and a third lipid component. In various examples, a combination lipid component comprises functionality of a first lipid component (e.g., ionizable cationic amino lipid) (or a group formed therefrom) and a fourth lipid component (e.g., a sterol or the like) (or a group formed therefrom) in a single component and replaces a first lipid component and a fourth lipid component. Nonlimiting examples of combination lipid components include, ionizable phospholipids, and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof. [0071] In various examples, a combination lipid component (e.g., a first combination lipid component) comprises an amino group and a phospholipid group. Non-limiting examples of combination lipid components (e g., a first combination lipid component or components) include aminophospholipids, salts thereof, structural and/or functional analogs thereof, and any combination thereof. Non-limiting examples of aminophospholipids are shown in Table 5.

[0072] Table 5.

[0073] In various examples, a combination lipid component (e.g., a second combination lipid component) comprises an amino group and a sterol group. Non-limiting examples of combination lipid components (e.g., a second combination lipid component or components) include 313-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC Cholesterol), BHEM- Cholesterol, [3-(lH-imidazol-l-yl)propyl]carbamate, cholest-5-en-3P-ol, bis( 3 S, 8 S,9 S, 1 OR, 13R, 14 S, 17R)- 10, 13 -dimethyl- 17-((R)-6-methylheptan-2-y 1)- 2,3,4,7,8,9,10, 1 l,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl) (3- (dimethylamino)propyl) phosphate, and the like, salts thereof, structural and/or functional analogs thereof. Non-limiting examples of combination lipid components are shown in Table 7. [0074] Table 6.

[0075] A lipid nanoparticle may comprise various cationic lipid components. In various examples, a cationic lipid component comprises a cationic head group and a degradable group. In various examples, a cationic head group comprises a static/permanent positive charge. In various examples, a cationic group is not an ionizable cationic head group (such as, for example, an ionizable cationic head group that is cationic based on pH (e g., as synthesized or based on the local environment pH, or the like)). In various examples, a cationic head group comprises a static/permanent positive charge that is not pH dependent. In various examples, a cationic head group is not a pH dependent ionizable group. In various examples, a cationic head group comprises (or is) a quaternary ammonium group, a sulfonium group, a phosphonium group, guanidinium group, amidinium group, imidazolium group, pyridinium group, pyrrolidinium group, or the like, a salt thereof, or a structural analog thereof. In various examples, a degradable group comprises (or is) an ester group, a phosphate group, a disulfide group, an imine group, an amide group, a thioester group, an acetal or ketal group, a hydrazone group, a cis-aconityl group, an orthoester group, or the like, or a structural analog thereof. In various examples, a lipid group comprises (or is) one or more aliphatic group(s) (such as, for example, a C4 to Cso group or groups, including all integer number of carbons and ranges therebetween), or a structural analog thereof. In various examples, the number of lipid group(s) (tail(s) of a single lipid component vary in number from 1 to 10 (dendritic), including all integer number of lipid groups and ranges therebetween (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 lipid groups). In various examples, a lipid group is fully saturated or comprises one or more unsaturation(s). In various examples, the number of unsaturation(s) in a lipid group is 1 to 10 aliphatic groups, which may depend on the length of the lipid group (tail). In various examples, each lipid group (tail) independently comprises (or is) a C3 to C20 aliphatic group, including all integer number of carbons and ranges therebetween.

[0076] In various examples, a cationic lipid component is a salt, in various examples, a cationic lipid further comprises a counter anion. Non-limiting examples of counter anions include chloride, bromide, iodide, and other halides, carboxylates (such as, for example, acetate and the like), nitrate, sulfate, sulfonates (such as, for example, trilfuoromethane sulfonate and the like), phosphate, phosphonates, and the like, structural and/or functional analogs thereof, and any combination thereof.

[0077] Nonlimiting examples of cationic lipid components include l,2-dioleoyl-3- trimethyl ammonium propane (DOTAP), didecyldimethylammonium bromide (DDAB), 1,2-di- O-octadecenyl-3-trimethylammonium propane (DOTMA), l,2-dioleoyl-3-dimethylammonium- propane (DODAP), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium bromide (DORI), O,O’-ditetradecanoyl-N-(a-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), l,2-stearoyl-3-trimethylammonium-propane (chloride salt) (18:0 TAP), 1,2- dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), l,2-dimyristoyl-3-trimethylammonium- propane (14:0 TAP), l,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (18:1 EPC), 2,3-dioleyloxy- N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

[0078] A lipid nanoparticle can comprise various amounts of lipid component(s) and, if present, peptide(s), functionalized peptide(s), or the like, or any combination thereof. In various examples, a lipid nanoparticle comprises, if present, a first lipid component or components (e.g., ionizable cationic lipid(s)) is/are present at about 5 mol% (based on the total moles of lipid components) to about 90 mol%, including all 0.1 mol% values and ranges therebetween (e.g., about 20 mol°/o to about 70 mol%); and/or, if present, a second lipid component or components (e.g., PEG-lipid(s)) is/are present at about 0.1 mol% to about 20 mol%, including all 0.1 mol% values and ranges therebetween (about 0.2 mol% to about 5 mol% or about 0.3 mol% to about 5 mol%); and/or, if present, a third lipid component or components (e.g., phospholipid(s)) is/are present at about 1 mol% to about 60 mol%, including all 0.1 mol% values and ranges therebetween (about 1 mol% to about 50 mol%, about 5 mol% to about 40 mol%, or about 5 mol% to about 40 mol%); and/or, if present, a fourth lipid component or components (e.g., sterol(s)) is/are present at about 5 mol% to about 80 mol%, including all 0.1 mol% values and ranges therebetween (e.g., about 10 mol% to about 60 mol%); and/or, if present, a cationic lipid component or components is/are present at about 0.5 mol% to about 20 mol%, including all 0.1 mol% values and ranges therebetween (e.g., about 2 mol% to about 10 mol% or about 5 mol% to about 10 mol%). In various examples, the mol% of the lipid component(s) and peptide(s), functional peptide(s), or the like, or a combination thereof equals 100 mol%. The amount of lipid component(s) and/or peptide(s), functionalized peptide(s), or the like, or any combination thereof can be determined by methods known in the art. In various examples, the amount of lipid component(s) and/or peptide(s), functionalized peptide(s), or the like, or any combination thereof is determined by high performance liquid chromatography (HPLC), LC-MS (liquid chromatography, such as, for example, HPLC or the like) combined with mass spectrometry (MS), or the like, or any combination thereof.

[0079] A lipid nanoparticle may comprise various peptide(s), functionalized peptide(s), or the like, or any combination thereof. In various examples, a peptide comprises canonical aminoacids, non-canonical amino acids, amino acids on peptides with modifications (such as, for example, methylation, phosphorylation, or the like or any combination thereof), or the like, or any combination thereof. In various examples, a peptide is a naturally-occurring peptide, a non- naturally occurring peptides, a synthetic peptide, or the like. In various examples, one or more or all of the peptide(s), the functionalized peptide(s), or the like is/are independently a naturally occurring or synthetic polypeptide. In various examples, one or more or all of the peptide(s), the functionalized peptide(s), or the like independently comprise(s) a molecular weight less than about 10 kD and/or greater than about 50 amino acid residues. In various examples, a peptide or a functional peptide is a therapeutic (such as, for example, a drug or the like). In various examples, the peptide is not a structural component of the lipid nanoparticle. Non-limiting examples of peptides and functional peptides include peptide mimetics, D-peptides, P-peptides, gamma peptides, sulfonyl-gamma peptides, alpha/p peptides, cyclic peptides, peptoids, peptide nucleic acids (PNAs), stapled peptides, structural and/or functional analogs thereof, and the like, and any combination thereof. In various examples, a peptide or a functional peptide is not amenable to (or not amenable to efficient) intracellular delivery (e.g., is not able to be delivered (such as, for example, effectively delivered) intracellularly by itself). In various examples, a peptide is not a protein.

[0080] In various examples, a peptide, a functionalized peptide, or the like is a cationic (such as, for example, statically cationic) peptide, a cationic (such as, for example, statically cationic) functionalized peptide, an anionic (such as, for example, statically cationic) peptide, an anionic (such as, for example, statically anionic) functionalized peptide, a net neutral peptide, or a net neutral functionalized peptide, or the like. In various examples, a lipid nanoparticle does not comprise a negatively charged peptide, a negatively charged functionalized peptide, or the like. [0081] In various examples, a peptide or a functional peptide comprises one or more hydrophobic domain(s). In various examples, a hydrophobic domain is inherent or functionalized. In various examples, a peptide or a functional peptide comprises i) a hydrophilic peptide sequence/sequences or group/groups and ii) a hydrophobic alkyl linker group or the like) and/or a hydrophobic peptide sequence/sequences or group/groups (such as, for example, a lipid group (tail), a sequence comprising hydrophobic amino acids (such as, for example, leucine, valine, phenyl alanine, or the like, or any combination thereof (e.g., an E3-ligand sequence, or the like)). In various examples, a peptide or functional peptide comprises i) a hydrophilic peptide sequence/sequences or group/groups and ii) a hydrophobic alkyl group or aryl group (such as, for example, a naphthyl group or the like) and/or a hydrophobic peptide sequence/sequences or group/groups; or i) a hydrophobic peptide sequence/sequences or group/groups and ii) a hydrophobic group and/or a hydrophobic peptide sequence/sequences or group/groups; or i) a peptide sequence/sequences or group/groups and ii) a cleavable hydrophobic group and/or a hydrophobic peptide sequence/sequences or group/groups; or i) an amphiphilic peptide sequence/sequences or group/groups. In various examples, a peptide or a functional peptide comprises a hydrophilic peptide sequence and a hydrophobic peptide sequence (e.g., a lipid group/groups, a sequence/sequences of hydrophobic amino acids (such as, for example, leucine, valine, phenyl alanine, or the like, or any combination thereof).

[0082] In various examples, at least a portion, substantially, or all of the peptide(s), functionalized peptide(s), or the like is/are independently protein degrading peptides (which may be referred to as peptide degraders or peptide-based degraders), amphiphilic proteolysis targeting peptides, PEptide-based Proteolysis TArgeting Chimeras (PEPTACs), peptides that modify other proteins through the formation of a tertiary complex, peptides that bind to a protein, peptides that bind to a modified protein (lipoprotein, phosphorylated protein, methylated protein, glycosylated protein and the like), peptides that bind to nucleic acid, peptides that bind to sugars, and the like, structural and/or functional analogs thereof, and any combination thereof. In various examples, a protein degrading peptide can degrade a post-translation modified protein or the like.

[0083] Non-limiting examples of peptides and functionalized peptides (peptide sequence (N to C), which may be protein degraders (e.g., PEPTACs or the like) include IYP^OHAL-Ahx- KDVLSEKEKKLEEYKQKLARV (SEQ ID NO: 1) (CREPT degrader), IYP^OHAL-Ahx- SQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 2) (P-catenin degrader), IYP^OHAL-Ahx- DELISFKDEGEQEERDLADVKSSLVN (SEQ ID NO: 3) (P-catenin degrader), PIYPALA- GSGS-QLLRHLILH (SEQ ID NO: 4) (ERalpha degrader), IYP^OHAL-Ahx-QLLRHLILH (SEQ ID NO: 5) (ERalpha degrader), where P^OH stands for hydroxylated proline (such as, for example 4-hydroxyproline, 3 -hydroxyproline, and the like) and Ahx is 6-aminohexanoic acid (linker), and structural and/or functional analogs thereof. Without intending to be bound by any particular theory, it is considered the IYP^OHAL segment binds to the E3 protein, while the sequence to the right of the Ahx linker binds to the protein of interest, i.e., the protein to be degraded) and the protein of interest is degraded. In various examples, a protein degrader targets a specific protein and at least partially, substantially, or completely degrades the specific protein.

[0084] In various examples, a functional peptide comprises one or more hydrophobic tag(s). In various examples, a functional peptide is a peptide described herein that further comprises one or more hydrophobic tag(s). Non-limiting examples of hydrophobic tags include alkyl chains (e.g., C3 to C20 alkyl chains, including all integer numbers of carbons and ranges therebetween (e.g., a C10 alkyl chain), hydrophobic amino acid(s) (such as, for example, sequence(s) of hydrophobic amino acids), or the like, or any combination thereof. In various examples, the hydrophobic tag(s) is/are independently connected (e g., covalently bonded or the like) to a peptide via a degradable linker.

[0085] In various examples, the peptide(s), functionalized peptide(s), or the like are independently disposed at least partially or completely within a lipid nanoparticle or disposed on a surface of a lipid nanoparticle. In various examples, the peptide(s), functionalized peptide(s), or the like are independently encapsulated, sequestered, embedded, or the like by the lipid component(s) of a lipid nanoparticle. Without intending to be bound by any particular theory, it is considered the lipid nanoparticle at least partially or completely protects the peptide(s), functionalized peptide(s), or the like from an extracellular environment (such as, for example, proteases (e.g., serum proteases and the like) and the like).

[0086] A lipid nanoparticle can comprise various amounts (mass ratio, volume ratio, or the like) of lipid component(s) and/or peptide(s), functionalized peptide(s), or the like, or any combination thereof. In various examples, the mass ratio of the lipid components (e.g., the first lipid component s), the second lipid component(s), the third lipid component(s), the fourth lipid component(s), if present, and the cationic lipid component(s)) to peptide(s), functionalized peptide(s), or the like, or any combination thereof is about 0.5 to about 50 wt/wt, including all 0.1 mass ratio values and ranges therebetween

[0087] In various examples, a lipid nanoparticle is a solid lipid nanoparticle. In various examples, a lipid nanoparticle comprises a core (such as, for example, a solid core or the like) and a shell (such as for example, a membrane (e.g., a bilayer membrane or the like) disposed on at least a portion or all of an outer surface of the core. In various examples, a lipid nanoparticle comprises of small miniature nanoparticles within a bigger nanoparticle, giving it a solid-like interior. In various examples, a lipid nanoparticle comprises a lamellar (e.g., multilamellar or the like) structure/morphology, or the like. In various examples, a lipid nanoparticle is spherical, substantially spherical, or the like. In various examples, a lipid nanoparticle comprises undulations or hairiness on at least a portion or substantially all or all of its surface(s). In various examples, a lipid nanoparticle has a cup-like morphology.

[0088] A lipid nanoparticle can have various sizes and/or size distributions. In various examples, a lipid nanoparticle comprises or exhibits a longest linear dimension (such as, for example, a diameter or the like) of from about 10 nanometers to about 1000 nanometers, including all 0.1 nanometer values and ranges therebetween.

[0089] In various examples, a lipid nanoparticle is a stable lipid nanoparticle. In various examples, a stable lipid nanoparticle does not exhibit substantial or any aggregation (observable aggregation (e.g., by optical microscopy, electron microscopy, light scattering, or the like, or any combination thereof) or the like), a substantial change or any change (such as, for example, an observable change (e.g., by optical microscopy, electron microscopy, light scattering, or the like, or any combination thereof)) in size (such as, for example, a longest linear dimension (such as, for example, a diameter or the like) for at least one-week or more (e.g., at least two-weeks or more, at least three-weeks or more, at least four-weeks or more) at a temperature of about 4 degrees Celsius (°C), or both.

[0090] In an aspect, the present disclosure provides compositions. In various examples, a composition comprises a plurality of lipid nanoparticles of the present disclosure. In various examples, a composition is a pharmaceutical composition. In various examples, a composition is made by a method of the present disclosure. Non-limiting examples of compositions are described herein.

[0091] A composition can comprise various lipid nanoparticles. A composition can comprise combinations of lipid nanoparticles. In various examples, all of the lipid nanoparticles in the composition comprise substantially the same composition and/or substantially the same size. In various examples, one or more or all of the lipid nanoparticles comprise a different composition (such as, for example, different lipid component(s), different peptide(s) and/or different functionalized lipid nanoparticle(s), or the like) and/or size than the other lipid nanoparticles of the composition.

[0092] A composition can have various forms. In various examples, a composition is a solution (such as, for example, a saline solution or the like), an aqueous dispersion, or the like. [0093] A composition can have various amounts of lipid nanoparticles. In various examples, lipid nanoparticles are present at about a 0.2 to about a 40 lipid:peptide wt/wt ratio (based on the total weight of lipid component(s) and total weight of peptide(s), functional peptide(s), and the like, and any combination thereof), including all 0.1 lipid : peptide wt/wt ratio values and ranges therebetween (e.g., about 1 to about a 20 lipid:peptide wt/wt ratio). In various examples, the total amount of peptide(s), functional peptide(s), or the like, or any combination thereof delivered is the total amount of peptide.

[0094] In various examples, the lipid nanoparticles of a composition comprise or exhibit an average longest linear dimension (such as, for example, an average diameter or the like) of about 10 nanometers (nm) to about 1000 nanometers, including all 0.1 nanometer values and ranges therebetween. In various examples, about 90% or more, about 95% or more, about 98% or more, about 99% or more, about 99.9% or more, or about 100% of the lipid nanoparticles in a composition comprise or exhibit an average size (e.g., an average longest linear dimension (such as, for example, an average diameter or the like) or the like of about 10 nm to about 1000 microns, including all 0.1 nm values and ranges therebetween. In various examples, the average size (such as, for example, the longest linear dimension or the like) is determined by optical microscopy, electron microscopy, light scattering, or the like, or any combination thereof.

[0095] In various examples, the lipid nanoparticles in a composition or a composition comprise(s) or exhibit(s) an isoelectric point, which may be referred to as apparent pKa, of about pH 4 to about pH 10, including all 0.1 pH values and ranges therebetween. In various examples the lipid nanoparticles in a composition or a composition comprise(s) or exhibit(s) an apparent pKa of about pH 5 to about pH 9. The apparent pKa is the experimentally determined pH at which the number of ionized (protonated) and deionized groups (typically at the nanoparticle surface) are equal.

[0096] A composition may comprise free peptide(s), free functional peptide(s), or the like, or any combination thereof. In various examples, a composition further comprises free peptide(s), free functional peptide(s), or the like, or any combination thereof.

[0097] In various examples, a composition further comprises one or more additional component(s). In various examples, one or more or all of the additional component s) are pharmaceutically acceptable components.

[0098] As used herein, unless otherwise indicated, the term “pharmaceutically acceptable” refers to those components and dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of individuals (such as, for example, humans or other animals) without undesirable or excessive toxicity, irritation, or other problem or complication, which may be commensurate with a reasonable benefit/risk ratio.

[0099] Some non-limiting examples of materials which can be used as additional component(s) in a composition include sugars, such as, for example, lactose, glucose, sucrose, and the like; starches, such as, for example, corn starch, potato starch, and the like; cellulose, and its derivatives, such as, for example, sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, and the like; powdered tragacanth; malt; gelatin; talc; excipients, such as, for example, cocoa butter, suppository waxes, and the like; oils, such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, soybean oil, and the like; glycols, such as, for example, propylene glycol and the like; polyols, such as, for example, glycerin, sorbitol, mannitol, polyethylene glycol, and the like; esters, such as, for example, ethyl oleate, ethyl laurate, and the like; agar; buffering agents, such as, for example, magnesium hydroxide, aluminum hydroxide, and the like; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. (See, e.g., REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975) and Remington: The Science and Practice of Pharmacy (2012) 22nd Edition, Philadelphia, PA. Lippincott Williams & Wilkins).

[0100] In various examples, a composition is a stable composition. In various examples, a stable composition does not exhibit substantial or any lipid nanoparticle aggregation (observable aggregation (e.g., by optical microscopy, electron microscopy, light scattering, or the like, or any combination thereof) or the like), a substantial change or any change (observable change (e g., by optical microscopy, electron microscopy, light scattering, or the like, or any combination thereof) in lipid nanoparticle size (such as, for example, average of the lipid nanoparticle size) (such as, for example, a longest linear dimension (such as, for example, a diameter or the like) for at least one-week or more (e.g., at least two-weeks or more, at least three-weeks or more, at least four- weeks or more) at a temperature of about 4 degrees Celsius (°C) or both. In various examples, a stable composition does not exhibit a change (observable change (e.g., by optical microscopy, electron microscopy, light scattering, or the like, or any combination thereof) in lipid nanoparticle size (such as, for example, average of the lipid nanoparticle size) (such as, for example, a longest linear dimension (such as, for example, a diameter or the like) of greater than about 5%, greater than about 4%, greater than about 3%, greater than about 2%, greater than about 1%, greater than about 0.5%, or greater than about 0.1% ,or greater, 2% or greater, of the lipid nanoparticle for at least one-week or more (e.g., at least two-weeks or more, at least three- weeks or more, at least four-weeks or more) at a temperature of about 4 degrees Celsius (°C) or both.

[0101] In an aspect, the present disclosure provides methods of making lipid nanoparticles. In various examples, a method makes a lipid nanoparticle or a composition of the present disclosure. Non-limiting examples of methods of making a lipid nanoparticle or a composition of the present disclosures of the present disclosure are described herein.

[0102] In various examples, a method of making lipid nanoparticles (or a lipid nanoparticle composition) comprises providing a first composition (e.g., a lipid solution or the like) comprising the lipid component(s) (e.g., if present, first lipid component(s), second lipid component(s), third lipid component(s), fourth lipid component(s), cationic lipid component(s), combination lipid component(s), or the like, or a combination thereof) and one or more organic solvent(s); and a second composition comprising one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof and water (such as, for example, an aqueous buffer or the like); and contacting the first composition and the second composition, wherein the lipid nanoparticles are formed. In various examples, the lipid components are first lipid component(s), second lipid component(s), third lipid component(s), fourth lipid component(s), optionally, cationic lipid component(s), optionally, combination lipid component(s), which may replace two or more other lipid components, or the like.

[0103] In various examples, a method of making lipid nanoparticles (or a lipid nanoparticle composition) comprises contacting, which may be rapidly contacting, a first composition (e.g., a lipid solution or the like) comprising the lipid component(s) (e.g., first lipid component s), second lipid component(s), third lipid component(s), fourth lipid component(s), cationic lipid component(s), or the like) and one or more organic solvent(s); and a second composition comprising one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof and water (such as, for example, an aqueous buffer, a pH buffered solution, or the like), where the lipid nanoparticles are formed; and optionally, removing residual organic solvent(s) (such as, for example, by dialyzing the lipid nanoparticles (such as, for example, against and aqueous solvent (e.g., water or the like) or a pH buffered solution (e.g., buffered at or about a physiological pH or the like), or the like (which may be carried out directly (e.g., immediately) after lipid nanoparticle formation. In various examples, the lipid components are first lipid component(s), second lipid component s), third lipid component s), fourth lipid component(s), optionally, cationic lipid component(s), optionally, combination lipid component(s), which may replace two or more other lipid components, or the like).

[0104] In various examples, contacting a first composition and a second composition comprises mixing (e.g., stirring or the like), which may be rapid, or the like. In various examples, a first composition and a second composition are contacted in a fluidic device (such as, for example, a microfluidic device or the like) or the like.

[0105] Various amounts of first composition(s) and/or second composition(s) can be used. In various examples, the volume ratio of a first composition to a second composition is about 0.02 to about 2, including all 0.01 volume ratio values and ranges therebetween, and/or the mass ratio of first composition to second composition is about 0.1 to about 40, including all 0.1 mass ratio values and ranges therebetween (e.g., about 0.1 to about 1 or about 2 to about 40). [0106] Contacting can be performed under various conditions. A method (e g., a contacting) can comprise one or more steps and each step can be performed under the same or different conditions as other steps. Contacting can be carried out at various temperatures. In various examples, a reaction is carried out at room temperature (e.g., from about 20 °C to about 22 °C, including all 0.1 °C values and ranges therebetween), below room temperature (e.g., at about 0°C or below, such as for example, from about -200°C to about 0°C, including all 0.1 °C values and ranges therebetween) (e.g., about -10°C, about -50°C, about -100°C, about -150°C, or about - 200°C), above room temperature (e.g., at a temperature up to or about a boiling point of the solvent(s), if present) (e.g., at about 100°C or above, e.g. from about 100°C to about 400°C, including all 0.1 °C values and ranges therebetween) (e.g., about 100°C, about 200°C, about 300°C, about 400°C, or about 500°C), or any combination thereof (e.g., where each step is performed at a different temperature as other steps).

[0107] Contacting can be carried out at various pressures. In various examples, a contacting is carried out at atmospheric pressure (e.g., 1 standard atmosphere (atm) at sea level), at greater than atmospheric pressure (e.g. heating in a sealed pressurized reaction vessel and the like), at below atmospheric pressure (e.g., under vacuum (e.g., from about 1 mTorr or less to about 100 mTorr or less, including all 0.1 mTorr values and ranges therebetween) (e.g., about 100 mTorr or less, about 50 mTorr or less, about 10 mTorr or less, or about 1 mTorr or less) and the like), or any combination thereof (e.g., where each step is performed at a different pressure as other steps).

[0108] Contacting can be carried out for various times. The contacting time can depend on factors such as, for example, temperature, pressure, presence and/or efficiency of a catalyst, presence and/or intensity of an applied energy source, stirring, or the like, or a combination thereof. In various examples, contacting times, which may be referred to as mixing times, range from about milliseconds (e.g., 1 milliseconds) (e.g., using a microfluidic device or the like) to greater than about 1 hour, including all integer second values and ranges therebetween (e.g., from about 1 minute to about 150 hours, including all integer second values and ranges therebetween) (e.g., about 10 minutes, about 1 hour, about 12 hours, about 24 hours, about 120 hours, or about 150 hours), or any combination thereof (e.g., where each step is performed at a different time as other steps). Typically, the shorter the mixing time the smaller the lipid nanoparticles. [0109] In various examples, a method further comprises isolating or the like at least a portion, substantially all, or all the lipid nanoparticles. Suitable isolation methods, processes, etc. are known in the art. Combinations of isolation methods, processes, etc. can be used. In various examples, at least a portion, substantially all, or all of the lipid nanoparticles are isolated by centrifugation, filtration (such as, ultrafiltration or the like), dialysis, or the like, or any combination thereof.

[0110] In an aspect, the present disclosure provides uses of a lipid nanoparticle or lipid nanoparticles or a composition of the present disclosure. Non-limiting examples of uses of a lipid nanoparticle or lipid nanoparticles or a composition of the present disclosure are described herein.

[0111] In various examples, a method comprises delivery (e.g., in vivo delivery, in vitro delivery, ex vivo delivery, or the like) of a lipid nanoparticle or lipid nanoparticles or a composition and/or one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof of a lipid nanoparticle or lipid nanoparticles, which may be present in a composition, to a cell or a population of cells, or the like. In various examples, a method comprises delivery of a lipid nanoparticle or lipid nanoparticles and/or one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof of a lipid nanoparticle or lipid nanoparticles (which may be an effective amount thereof), which may be present in a composition, to an individual. In various examples, a method comprises intracellular delivery of a lipid nanoparticle or lipid nanoparticles and/or one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof of a lipid nanoparticle or lipid nanoparticles. In various examples, a contacting is in vitro, in vivo, ex vivo, or the like. In various examples, delivery is intracellular delivery or the like. In various examples, at least a portion, substantially all, or all the lipid nanoparticle(s) is/are taken up by a cell or cells of the population of cells or the individual, and the lipid nanoparticles is/are is released within the cell or cells.

[0112] In various examples, the functionalized peptide(s), or the like, or any combination thereof and the peptide(s), the functionalized peptide(s), or the like, or any combination thereof that are contacted with the cell(s) or administered to an individual independently or in the aggregate retain/retains (e.g., after delivery) substantially all (or all) structural features and/or 50% or more, 70% or more, 90% or more, or 95% or more biological activity compared with the native peptide or the peptide delivered without use of a lipid nanoparticle (a lipid nanoparticle of the present disclosure).

[0113] A lipid nanoparticle or lipid nanoparticles or a composition can be delivered to various individuals. In various examples, an individual is diagnosed with and/or is in need of treatment for a disease, disease state, or the like, and the disease, the disease state, or the like is treatable, preventable, or the like, by the peptide(s), the functionalized peptide(s), or the like, or any combination thereof.

[0114] In various examples, a lipid nanoparticle or lipid nanoparticles and/or a composition or compositions is suitable for (or the administration is) intravenous, subcutaneous, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavemous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebral, intraci sternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavemosum, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intraovarian, intrapericardial, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, subarachnoid, subconjunctival, sublingual, submucosal, transplacental, transtympanic, or the like, or any combination thereof administration.

[0115] An individual (e g., an individual in need of treatment or the like) may be a human or other animal (which may be a non-human mammal). In various examples, an individual is a mammal. Non-limiting examples of individuals include humans and non-human animals. Nonlimiting examples of non-human animals (which may be mammals) include cows, pigs, goats, rabbits, and other agricultural animals, mice, rats, pets (such as, for example, dogs, cats, and the like), service animals, and the like.

[0116] In various examples a method comprises treating, preventing, or the like, or any combination thereof, a current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof, in the individual. In various examples, administration of the lipid nanoparticles (such as, for example, administration of the peptide(s), the functionalized peptide(s), or the like, or any combination thereof) results in treating, preventing, or the like, or any combination thereof, a current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof, in the individual. As used herein, “treatment” is not limited to treatment and encompasses alleviation of one or more or all of the symptom(s) of a disease, disease state, condition, disorder, side effect, or the like.

[0117] In various examples, a method comprises targeting, diagnosing, treating, preventing, or the like, or any combination thereof, a current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof, in an individual by administration of a lipid nanoparticle or lipid nanoparticles and/or one or more functionalized peptide(s), or the like, or any combination thereof and/or one or more composition(s) to the individual. In various examples, the current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof is chosen from infections, cancers, neurological conditions/diseases, neurodegenerative diseases, psychological conditions/diseases, inflammatory conditions/diseases, cardio-vascular diseases, and the like, and any combination thereof. In various examples, it is considered the current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof, is any current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof, that is targetable, diagnosable, treatable, preventable, or the like, or any combination thereof, by one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof of a lipid nanoparticle or lipid nanoparticles.

[0118] In various examples, a current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof is one that is treatable, preventable or the like by protein degrading peptides (which may be referred to as peptide degraders or peptide-based degraders), amphiphilic proteolysis targeting peptides, peptides that modify other proteins through the formation of a tertiary complex, peptides that bind to a protein, peptides that bind to a modified protein (lipoprotein, phosphorylated protein, methylated protein, glycosylated protein and the like), peptides that bind to nucleic acid, peptides that bind to sugars, and the like, or the like, or any combination thereof. Non-limiting examples of current or potential diseases, disease states, conditions, disorders, side effects, or the like, or any combination thereof are provided herein. In various examples, a current or potential disease/diseases, a disease state/disease states, a condition/conditions, a disorder/disorders, a side effect/side effects, or the like, or any combination thereof include autoimmune diseases, cancers, infections (such as, for example, microbial infections, viral infections, and the like, and any combination thereof), and the like, and any combination thereof.

[0119] “Treating” or “treatment” of any disease or disorder refers, in various examples, to ameliorating (e.g., arresting, reversing, alleviating, or the like) the disease, disease state, condition, disorder, side effect, or the like, or a combination thereof the disease, disease state, condition, disorder, side effect, or the like, or a combination thereof, or reducing the manifestation, extent or severity of one or more clinical symptom(s) thereof, or the like). In various other examples, “treating” or “treatment” refers to ameliorating (e.g., arresting, reversing, alleviating, or the like) one or more physical parameter(s), which, independently, may or may not be discernible by the individual. In yet other examples, “treating” or “treatment” refers to modulating disease, disease state, condition, disorder, side effect, or the like, or a combination thereof, either physically (e.g., stabilization of one or more discernible symptom(s), or the like), physiologically (e.g., stabilization of one or more physical parameter(s), or the like), or both. In yet other examples, treating” or “treatment” relates to slowing the progression of the disease, disease state, condition, disorder, side effect, or the like, or a combination thereof.

[0120] As used herein, unless otherwise indicated, the term “effective amount” means that amount of lipid nanoparticles comprising peptide(s), functionalized peptide(s), or the like, or any combination thereof will (or is expected to) elicit he biological or medical response of an individual (or a tissue, system, or the like, thereof) that is being sought, for instance, by a researcher, clinician, or the like. An effective amount may be a therapeutically effective amount. The term “therapeutically effective amount” includes any amount which, as compared to a corresponding individual who has not received such amount, results in improved treatment, healing, prevention, or amelioration (e.g., arresting, reversing, alleviating, or the like) of a disease, disease state, condition, disorder, side effect, or the like or a decrease in the rate of advancement of a disease, disease state, condition, disorder, or the like, or the like. The term also includes within its scope amounts effective to enhance normal physiological function. In various examples, the individual is considered effectively treated if the treated individual is not thereafter diagnosed with the disease or disease state, or one or more symptom(s), one or more indication(s), or the like of condition, disorder, disease, or disease state, or the like is at least partially or completely prevented, inhibited, alleviated, or the like). [0121 ] In various examples, an effective amount results in prophylaxis or the like of a disease, disease state, condition, disorder, side effect, or the like. The term “prophylaxis” includes prevention and refers to a measure or procedure which is to prevent rather than cure or treat a disease. Preventing may refer to a reduction in risk of acquiring or developing a disease, disease state, condition, disorder, side effect, or the like causing one or more clinical symptom(s) the disease, disease state, condition, disorder, side effect, or the like not to develop in an individual that may be exposed to a disease causing agent or an individual predisposed to the disease in advance of disease outset.

[0122] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the lipid nanoparticle(s) and/or composition(s) required. A selected dosage level can depend upon a variety of factors including, but not limited to, the activity of the particular composition employed, the time of administration, the rate of excretion or metabolism of the particular composition being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. For example, a physician or veterinarian could start doses of a lipid nanoparticle or lipid nanoparticles and/or a composition or compositions employed at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0123] In an aspect, the present disclosure provides kits. Non-limiting examples of kits are provided herein.

[0124] In various examples, a kit comprises a lipid nanoparticle or lipid nanoparticles and/or composition(s) of the present disclosure and/or one or more starting material(s) for any of same. In various examples, a kit includes a closed or sealed package that comprises the lipid nanoparticle or lipid nanoparticles and/or the composition(s). In various examples, the package comprises one or more closed or sealed vials, bottles, blister (bubble) packs, or any other suitable packaging for the sale, distribution, or use of the lipid nanoparticles(s) and/or the composition(s) and/or starting material(s). The printed material may include printed information. The printed information may be provided on a label, on a paper insert, printed on a packaging material, or the like. The printed information may include information that identifies the lipid nanoparti cl es(s) and/or the composition(s) and/or starting material(s) in the package, the amounts and types of other active and/or inactive ingredients in the lipid nanoparticles (s) and/or the composition(s) and/or starting material(s), and instructions for taking (e.g., administration or the like) the lipid nanoparticles (s) and/or the composition(s) and/or starting material(s). The instructions may include information, such as, for example, the number of doses to take over a given period of time, and/or information directed to a pharmacist and/or another health care provider, such as, for example, a physician or the like, or a patient. The printed material may include an indication or indications that the lipid nanoparticles (s) and/or the composition(s) and/or starting material(s) and/or any other agent provided therein is for treatment of an individual. In various examples, the kit includes a label describing the contents of the kit and providing indications and/or instructions regarding use of the contents of the kit to treat an individual.

[0125] The following table lists peptides and nucleotides disclosed in the present disclosure:

[0126] The following Statements describe various examples of lipid nanoparticles, compositions, methods of making lipid nanoparticles, and uses of lipid nanoparticles of the present disclosure and are not intended to be in any way limiting:

Statement 1. A lipid nanoparticle comprising: one or more cationic lipid component(s) comprising a cationic head group, a degradable group, and one or more lipid group(s) (such as, for example, a saturated or unsaturated lipid group) (which may be referred to as a tail(s)). Statement 2. A lipid nanoparticle according to Statement 1, where the cationic lipid component(s) is/are independently chosen from l,2-dioleoyl-3-trimethylammonium propane (DOTAP), didecyldimethylammonium bromide (DDAB), l,2-di-O-octadecenyl-3- trimethyl ammonium propane (DOTMA), l,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium bromide (DORI), O,O’-ditetradecanoyl-N-(a-trimethylammonioacetyl)diethanolamine chloride (DC-6- 14), l,2-stearoyl-3-trimethylammonium-propane (chloride salt) (18:0 TAP), l,2-dipalmitoyl-3- trimethylammonium-propane (16:0 TAP), l,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), l,2-dioleoyl-sn-glycero-3 -ethylphosphocholine (18:1 EPC), 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 3. A lipid nanoparticle according to Statement 1 or 2, further comprising one or more or all of: one or more first lipid component s); one or more second lipid component(s); one or more third lipid component(s); or one or more fourth lipid component(s).

Statement 4. A lipid nanoparticle according to Statement 3, where the first lipid component(s) is/are present at about 5 mol% (based on the total moles of lipid components) to about 90 mol%, including all 0.1 mol% values and ranges therebetween (e.g., about 20 mol% to about 70 mol%); and/or the second lipid component s) is/are present at about 0.1 mol% to about 20 mol%, including all 0.1 mol% values and ranges therebetween (about 0.3 mol% to about 5 mol%); and/or the third lipid component(s) is/are present at about 1 mol% to about 50 mol%, including all 0.1 mol% values and ranges therebetween (about 5 mol% to about 40 mol%); and/or the fourth lipid component(s) is/are present at about 5 mol% to about 80 mol%, including all 0.1 mol% values and ranges therebetween (e.g., about 10 mol% to about 60 mol%); and/or the cationic lipid component(s) is/are present at about 0.5 mol% to about 20 mol%, including all 0.1 mol% values and ranges therebetween (e.g., about 5 mol% to about 10 mol%). Statement 5. A lipid nanoparticle according to any one of the preceding Statements, where the lipid nanoparticle further comprises one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof.

Statement 6. A lipid nanoparticle according to Statement 5, where the peptide(s), functionalized peptide(s), or the like is/are chosen from protein degrading peptides (which may be referred to as peptide degraders or peptide-based degraders), amphiphilic proteolysis targeting peptides, peptides that modify other proteins through the formation of a tertiary complex, peptides that bind to a protein, peptides that bind to a modified protein (lipoprotein, phosphorylated protein, methylated protein, glycosylated protein and the like), peptides that bind to nucleic acid, peptides that bind to sugars, and the like, structural and/or functional analogs thereof, and any combination thereof.

Statement 7. A lipid nanoparticle according to Statement 5 or 6, where the mass ratio of the lipid components (the first lipid component(s), the second lipid component(s), the third lipid component(s), the fourth lipid component s), if present, and the cationic lipid component s)) to peptide(s), functionalized peptide(s), or the like, or any combination thereof is about 0.5 to about 50 wt/wt, including all 0.1 mass ratio values and ranges therebetween.

Statement 8. A lipid nanoparticle according to any one of the preceding Statements, where the lipid nanoparticle comprises or exhibits a longest linear dimension (such as, for example, a diameter or the like) of from about 10 nanometers to about 1000 nanometers, including all 0.1 nanometer values and ranges therebetween.

Statement 9. A lipid nanoparticle according to any one of the preceding Statements, where the lipid nanoparticle comprises or exhibits an isoelectric point of about pH 4 to about pH 10, including all 0.1 pH values and ranges therebetween (e.g., about pH 5 to about pH 9).

Statement 10. A lipid nanoparticle according to any one of the preceding Statements, where the lipid nanoparticle is stable.

Statement 11. A composition comprising a plurality of lipid nanoparticles of the present disclosure (such as, for example, lipid nanoparticles according to any one of the preceding Statements).

Statement 12. A composition according to Statement 11, where the lipid nanoparticles comprise or exhibit an average longest linear dimension (such as, for example, an average diameter or the like) of about 10 nanometers to about 1000 nanometers, including all 0.1 nanometer values and ranges therebetween

Statement 13. A composition according to Statement 11 or 12, where the lipid nanoparticles comprise or exhibit an isoelectric point of about pH 4 to about pH 10, including all 0.1 pH values and ranges therebetween (e.g., about pH 5 to about pH 9).

Statement 14. A composition according to any one of Statements 11-13, where the composition comprises one or more pharmaceutical excipient(s) or the like.

Statement 15. A composition according to any one of Statements 11-14, where the composition is stable.

Statement 16. A method of making lipid nanoparticles (or a lipid nanoparticle composition) comprising: providing a first composition (e.g., a lipid solution or the like) comprising the lipid component(s) (e.g., first lipid component(s), second lipid component(s), third lipid component(s), fourth lipid component(s), cationic lipid component(s), or the like) and one or more organic solvent(s); and a second composition comprising one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof and water (such as, for example, an aqueous buffer or the like); and contacting the first composition and the second composition, where the lipid nanoparticles are formed; and optionally, removing residual organic solvent(s) (such as, for example, by dialyzing the lipid nanoparticles against and aqueous solvent or the like) (which may be carried out directly (e.g., immediately) after lipid nanoparticle formation.

Statement 17. A method according to Statement 16, where the mass ratio of first composition to second composition is about 0.1 to about 1, including all 0. 1 mass ratio values and ranges therebetween.

Statement 18. A method according to Statement 16 or 17, the method further comprising isolating the lipid nanoparticles.

Statement 19. A method of peptide, functionalized peptide, or the like, or any combination thereof delivery comprising: contacting a population of cells, an individual, or the like, with one or more lipid nanoparticle(s) and/or one or more composition(s), each composition comprising a plurality of lipid nanoparticles (e.g., as disclosed herein, such as, for example, lipid nanoparticle(s) according to any one of Statements 1-10, composition(s) according to any one of Statements 11-15, prepared by a method according to any one of Statements 16-18, or any combination thereof), where at least a portion or all of the lipid nanoparticles independently comprise one or more peptide(s), one or more functionalized peptide(s), or the like, or any combination thereof, where at least a portion of or all of the lipid nanoparticle(s) (and/or the peptide(s), the functionalized peptide(s), or the like, or any combination thereof) is/are delivered to the population of cells, the individual, or the like.

Statement 20. A method according to Statement 19, where the contacting comprises administration of the plurality of lipid nanoparticles to an individual.

Statement 21. A method according to Statement 19 or 20, where the lipid nanoparticles is/are taken up by a cell or cells of the population of cells or the individual, and the lipid nanoparticles is/are is released within the cell or cells.

Statement 22. A method according to any one of Statements 19-21, where at least a portion or all of the lipid nanoparticles comprises the peptide(s), the functionalized peptide(s), or the like, or any combination thereof and the peptide(s), the functionalized peptide(s), or the like, or any combination thereof independently retain/retains (e.g., after delivery) substantially all (or all) structural features and/or 50% or more, 70% or more, 90% or more, or 95% or more biological activity compared with the native peptide or functionalized peptide or the peptide or functionalized peptide delivered without use of a lipid nanoparticle (a lipid nanoparticle of the present disclosure).

Statement 23. A method according to any one of Statements 19-22, where the individual is diagnosed with and/or is in need of treatment for a disease, disease state, or the like, and the disease, the disease state, or the like is treatable, preventable, or the like, by the peptide(s), the functionalized peptide(s), or the like, or any combination thereof.

Statement 24. A lipid nanoparticle comprising: i) one or more first lipid component(s), where the first lipid component(s) is/are independently ionizable cationic amino lipid(s); one or more second lipid component(s), where the second lipid component s) is/are independently PEG- lipid(s); one or more third lipid component(s) where the third lipid component(s) is/are independently phospholipid(s); one or more fourth lipid component s), where the fourth lipid component(s) is/are independently sterol(s); optionally, one or more cationic lipid component(s), where the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof; or ii) one or more first combination lipid component(s), where the first combination lipid component(s) independently comprise one or more ionizable cationic amino lipid group(s); and one or more phospholipid group(s); one or more second lipid component(s), where the second lipid component(s) is/are independently PEG- lipid(s); one or more fourth lipid component(s) where the fourth lipid component(s) is/are independently sterol(s); optionally, one or more cationic lipid component(s), where the cationic lipid component s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof; or iii) one or more second combination lipid component(s), where the second combination lipid component(s) independently comprise one or more ionizable cationic amino lipid group(s); and one or more sterol group(s); one or more second lipid component(s), where the second lipid component(s) is/are independently PEG-lipid(s); one or more third lipid component(s) where the third lipid component(s) is/are independently phospholipid(s); or optionally, one or more cationic lipid component(s), where the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof.

Statement 25. A lipid nanoparticle according to Statement 24, where the first lipid component(s) is/are chosen from ionizable cationic amino lipids, and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 26. A lipid nanoparticle according to Statement 24 or 25, where the ionizable cationic amino lipid(s) is/are chosen from (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (D-Lin-MC3-DMA or MC3), l,2-dioleyloxy-3- dimethylaminopropane (DODMA), l,2-dioleoyl-3-dimethylammonium-propane (DODAP), 3B- [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC Cholesterol), l,2-dipalmitoyl-3- dimethylammonium-propane (16:0 DAP)), l,2-distearyloxy-3 -dimethylaminopropane (DSDMA), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-3- dimethylaminopropane (DLenDMA), l,2-bis(linoleoyloxy)-3 -dimethylaminopropane (DLinDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolan (DLin-KC2-DMA), 2,2-dilinoleyl-4- dimethylaminopropyl-[ 1 ,3 ]-di oxolane (DLin-KC3 -DMA), 2,2-dilinoleyl-4-dimethylaminobutyl- [l,3]-dioxolane (DLin-KC4-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), 9-[4-(dimethylamino)-l-oxobutoxy]-heptadecanedioic acid, l,17-di-(2Z)-2-nonen-l-yl ester (L319), 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, Nl, N16-di dodecyl - 4,7,13 -tri s( 3 -(dodecylamino)-3 -oxopropyl)-4, 7, 10,13 -tetraazahexadecanedi amide (98ND 12-5), 1 ,l'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l- piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 27. A lipid nanoparticle according to any one of preceding Statements, where one or more or all of the ionizable cationic lipid(s) is/are independently a pH-responsive lipid. Statement 28. A lipid nanoparticle according to any one of the preceding Statements, where the second lipid component(s) is/are chosen from l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG-2000), distearoyl-rac-glycerol-PEG2K, 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:0 PEG2000 PE), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (14:0 PEG2000 PE), N-palmitoyl-sphingosine-l-{succinyl[methoxy(poly ethylene glycol)2000]}(C16 PEG2000 Ceramide), N-palmitoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)5000]}(C16 PEG5000 Ceramide), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol )-2000] (DSPE-PEG(2000) Maleimide), N-myristoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (PEG- CerCi4)), N-arachidoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (PEG- CerC2o), polyethyleneglycol-succinoyl-distearoylglycerol (PEG-S-DSG), polyethyleneglycol- succinoyl-dimyristolglycerol (PEG-S-DMG), N-[(methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-C-DMA), 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 ((m)PEG-DMG / (m)PEG-DMG-2000), polyethyleneglycol-distearoylglycerol (PEG-DSG), R-3-[(co-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyristyloxlpropyl-3-amine (PEG-DOMG / PEG-C-DOMG), alpha-(3' -{[l,2-di(myristyloxy)propanoxy] carbonylamino} propyl)-co-methoxy, polyoxyethylene (PEG-C-DMG-2000), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 29. A lipid nanoparticle according to any one of the preceding Statements, where the third lipid component(s) is/are chosen from (2R)-2,3-Bis(octadecanoyloxy)propyl 2- (trimethylazaniumyl)ethyl phosphate (DSPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (16:0-18: 1 PC (POPC)), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-di oleoyl -sn- glycero-3-phosphoethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (10:0 PE), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), N-(dodecanoyl)-sphing-4- enine-1 -phosphocholine (SM), l,2-diphytanoyl-sn-glycero-3-phosphatidylethanolamine (DPyPE), salts thereof, structural and/or functional analogs thereof, and any combination thereof. Statement 30. A lipid nanoparticle according to any one of the preceding Statements, where the fourth lipid component(s) is/are chosen from cholesterol, cholestanol, 7-dehydrocholesterol, cholestan-3-one, cholesteryl oleate stigmasterol, glucosyl stigmasterol, sitosterol, lanosterol, zymosterol, and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 31. A lipid nanoparticle according to any one of the preceding Statements, where the first combination lipid component(s) is/are chosen from aminophospholipids, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 32. A lipid nanoparticle according to any one of the preceding Statements, where the second combination lipid component(s) is/are chosen from 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol (DC Cholesterol), BHEM-Cholesterol, [3-(lH-imidazol-l- yl)propyl]carbamate, cholest-5-en-3P-ol, />z ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,l 1,12,13,14,15,16,17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl) (3-(dimethylamino)propyl) phosphate, and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 33. A lipid nanoparticle according to any one of the preceding Statements, where the lipid nanoparticle is a solid lipid nanoparticle.

Statement 34. A lipid nanoparticle according to any one of the preceding Statements, the cationic head group(s) independently comprise a quaternary ammonium group, guanidinium group, amidinium group, imidazolium group, pyridinium group, pyrrolidinium group, or the like, a salt thereof, or a structural and/or functional analog thereof.

Statement 35. A lipid nanoparticle according to any one of the preceding Statements, where the degradable group(s) independently comprise an ester group, a phosphate group, a disulfide group, an imine group, an amide group, a thioester group, an acetal or ketal group, a hydrazone group, a cis-aconityl group, an orthoester group, or the like, or a structural and/or functional analog thereof. Statement 37. A lipid nanoparticle according to any one of the preceding Statements, where the lipid group(s) independently comprise one or more C-i to Ceo aliphatic group(s) or a structural and/or functional analog thereof.

Statement 38. A lipid nanoparticle according to claim 1, where the cationic lipid component(s) is/are independently chosen from l,2-dioleoyl-3-trimethylammonium propane (DOTAP), didecyldimethylammonium bromide (DDAB), l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), l,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(2- hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium bromide (DORI), 0,0’- ditetradecanoyl-N-(a-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 1,2-stearoyl- 3 -trimethylammonium -propane (chloride salt) (18:0 TAP), l,2-dipalmitoyl-3- trimethylammonium-propane (16:0 TAP), l,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18: 1 EPC), 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), and the like, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

Statement 39. A lipid nanoparticle according to any one of the preceding Statements, where the first lipid component(s) is/are present at about 5 mol% (based on the total moles of the lipid components) to about 90 mol%; and/or the second lipid component s) is/are present at about 0.1 mol% to about 20 mol% (based on the total moles of the lipid components); and/or the third lipid component(s) is/are present at about 5 mol% to about 60 mol% (based on the total moles of the lipid components); and/or the fourth lipid component(s) is/are present at about 5 mol% to about 80 mol% (based on the total moles of the lipid components); and/or the cationic lipid component(s), if present, is/are present at about 0.5 mol% to about 20 mol% (based on the total moles of the lipid components).

Statement 40. A lipid nanoparticle according to any one of the preceding Statements, where the one or more peptide(s), the one or more functionalized peptide(s) is/are independently a therapeutic peptide or a therapeutic functionalized peptide.

Statement 41. A lipid nanoparticle according to any one of the preceding Statements, where the one or more peptide(s), the one or more functionalized peptide(s), or any combination thereof independently comprise one or more hydrophobic domain(s).

Statement 42. A lipid nanoparticle according to any one of the preceding Statements, where the one or more peptide(s), the one or more functionalized peptide(s) independently comprise i) a hydrophilic peptide sequence/sequences or group/groups and ii) a hydrophobic alkyl group and/or a hydrophobic peptide sequence/sequences or group/groups; or i) a hydrophobic peptide sequence/sequences or group/groups and ii) a hydrophobic group and/or a hydrophobic peptide sequence/sequences or group/groups; or i) a peptide sequence/sequences or group/groups and ii) a cleavable hydrophobic group and/or a hydrophobic peptide sequence/sequences or group/groups; or an amphiphilic sequence/sequences or group/groups.

Statement 43. A lipid nanoparticle according to any one of the preceding Statements, where the one or more peptide(s), the one or more functionalized peptide(s) is/are independently a peptide mimetic, a D-peptide, 0-peptides, gamma peptides, sulfonyl-gamma peptides, alpha/0 peptides, a cyclic peptide, a peptoid, a peptide nucleic acid (PNA), or a stapled peptide.

Statement 44. A lipid nanoparticle according to any one of the preceding Statements, where one or more or all of the one or more peptide(s), the one or more functionalized peptide(s) is/are independently a cationic peptide, a cationic functionalized peptide, an anionic peptide, an anionic functionalized peptide, a net neutral peptide, or a net neutral functionalized peptide.

Statement 45. A lipid nanoparticle according to any one of the preceding Statements, where the one or more peptide(s), the one or more functionalized peptide(s) is/are independently chosen from protein degrading peptides, amphiphilic proteolysis targeting peptides, PEptide-based Proteolysis TArgeting Chimeras (PEPTACs), and the like, and structural and/or functional analogs thereof.

Statement 46. A lipid nanoparticle according to any one of the preceding Statements, where the one or more peptide(s), the one or more functionalized peptide(s) is/are independently comprises (or is): IYP^OHAL-Ahx-KDVLSEKEKKLEEYKQKLARV (SEQ ID NO: 1), IYP^OHAL-Ahx- SQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 2), IYP^OHAL-Ahx- DELISFKDEGEQEERDLADVKSSLVN (SEQ ID NO: 3), PIYPALA-GSGS-QLLRHLILH (SEQ ID NO: 4), IYP^OIIAL-Ahx-QLLRHLILH (SEQ ID NO: 5), where P^on stands for hydroxylated proline (such as, for example, 3 -hydroxyproline, 4-hydroxyproline, or the like) and Ahx is 6-aminohexanoic acid, or the like, or a structural and/or functional analog thereof.

Statement 47. A lipid nanoparticle according to any one of the preceding Statements, where the mass ratio of, if present, the first lipid component(s), the second lipid component(s), the third lipid component s), the fourth lipid component s), the cationic lipid component(s), the first combination lipid component(s), and the second lipid components to peptide(s), functionalized peptide(s), or any combination thereof is about 0.5 to about 50 % wt/wt, including all 0.1 % wt/wt values and ranges therebetween.

Statement 48. A lipid nanoparticle according to any one of the preceding Statements, where the lipid nanoparticle comprises a longest linear dimension of about 10 nanometers to about 1000 nanometers, including all integer nanometer values and ranges therebetween.

Statement 49. A composition comprising a plurality of lipid nanoparticles, where the lipid nanoparticles are independently a lipid nanoparticle of the present disclosure (e.g., a lipid nanoparticle according to any one of Statements 24 to 48).

Statement 50. A composition according to Statement 49, where the composition is a solution, or an aqueous dispersion.

Statement 51. A composition according to Statement 49 or 50, where the lipid nanoparticles are present at about 0.2 to about 40 lipid:peptide wt/wt ratio (based on the total weight of, if present, the first lipid component(s), the second lipid component(s), the third lipid component(s), the fourth lipid component(s), the cationic lipid component(s), the first combination lipid component(s), and the second combination lipid components and total weight of peptide(s), functionalized peptide(s), and any combination thereof), including all 0.1 wt/wt ratio values and ranges therebetween).

Statement 52. A composition according to any one of Statements 49 to 51, where the composition further comprises free peptide(s), free functionalized peptide(s), or any combination thereof.

Statement 53. A composition according to any one of Statements 49 to 52, where the lipid nanoparticles comprise an average longest linear dimension of about 10 nanometers to about 1000 nanometers, including all integer nanometer values and ranges therebetween.

Statement 54. A composition according to any one of Statements 49 to 53, where the lipid nanoparticles exhibit or the composition exhibits an isoelectric point (or apparent pKa) of about pH 4 to about pH 10.

Statement 55. A composition according to any one of Statements 49 to 54, where the composition comprises one or more pharmaceutical excipient(s).

Statement 56. A composition according to any one of Statements 49 to 55, where the composition does not exhibit substantial or any observable lipid nanoparticle aggregation, a substantial change or any change in lipid nanoparticle size for at least one-week or more at a temperature of about 4 degrees Celsius (°C) or both.

Statement 57. A method of making lipid nanoparticles (e.g., where the lipid nanoparticles are independently a lipid nanoparticle of the present disclosure (such as, for example, a lipid nanoparticle according to any one of Statements 24 to 48)) comprising: contacting a first composition comprising, if present, the first lipid component(s), the second lipid component(s), the third lipid component(s), the fourth lipid component(s), the cationic lipid component(s), the first combination lipid component s), and the second lipid components and one or more organic solvent(s) and a second composition comprising one or more peptide(s), one or more functionalized peptide(s), or any combination thereof, where the lipid nanoparticles are formed; optionally, removing residual organic solvent(s) after lipid nanoparticle formation; and dialyzing the lipid nanoparticle formulation against water or a pH buffered solution.

Statement 58. A method according to Statement 57, where the volume ratio of a first composition to a second composition is about 0.02 to about 2, including all 0.005 volume ratio values and ranges therebetween.

Statement 59. A method according to Statement 57 or 58, where the mass ratio of first composition to second composition is about 0.2 to about 40, including all 0.05 mass ratio values and ranges therebetween.

Statement 60. A method according to any one of Statements 57 to 59, the method further comprising isolating the lipid nanoparticles.

Statement 61. A method of peptide, functionalized peptide, or any combination thereof delivery comprising: contacting a population of cells or an individual, with a plurality of lipid nanoparticles of the present disclosure (e.g., where the lipid nanoparticles are independently a lipid nanoparticle of the present disclosure (such as, for example, a lipid nanoparticle according to any one of Statements 24 to 48)), where at least a portion of or all of the lipid nanoparticles are delivered to the population of cells or the individual.

Statement 62. A method according to Statement 61, where the contacting is in vitro or in vivo. Statement 63. A method according to Statement 61 or 62, where the method comprises treating, preventing, or any combination thereof, a current or potential disease, disease state, condition, disorder, side effect, or the like, or any combination thereof, in the individual. Statement 64. A method according to any one of Statements 61 to 63, where the contacting comprises administration of the plurality of lipid nanoparticles to an individual.

Statement 65. A method according to any one of Statements 61 to 64, where at least a portion, substantially all, or all the lipid nanoparticles(s) is/are taken up by a cell or cells of the population of cells or the individual, and the lipid nanoparticles is/are is released within the cell or cells.

Statement 66. A method according to any one of Statements 61 to 65, where the peptide(s), the functionalized peptide(s), or any combination thereof after delivery independently or in the aggregate retain/retains substantially all (or all) structural features and/or 50% or more, 70% or more, 90% or more, or 95% or more of its biological activity compared with the native peptide(s) or the peptide(s) or the functionalized peptide(s) delivered without use of a lipid nanoparticle the present disclosure (e.g., a lipid nanoparticle according to any one of Statements 24 to 48, a lipid nanoparticle of a composition of the present disclosure (such as, for example, a composition according to any one of Statements 49 to 56), a lipid nanoparticle made by a method of the present disclosure (such as, for example, a lipid nanoparticle according to any one Statements 57 to 60)).

Statement 67. A method according to any one of Statements 61 to 66, where the individual is diagnosed with and/or is in need of treatment for a disease, disease state, or the like, and the disease, the disease state, or the like, is treatable, preventable, by the peptide(s), the functionalized peptide(s), or the combination thereof.

[0127] The steps of the methods described in the various examples disclosed herein are sufficient to carry out the methods of the present disclosure. Thus, in various examples, a method consists essentially of a combination of the steps of the methods disclosed herein. In various other examples, a method consists of such steps.

[0128] The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any manner.

EXAMPLE 1

[0129] This example describes lipid nanoparticles of the present disclosure, and methods of making and using same. [0130] Nanoparticle-mediated delivery of peptide-based degraders enables targeted protein degradation. Lipid nanoparticle (LNP) formulations were used to facilitate the delivery of Peptide-based proteolysis TArgeting Chimeras (PepTACs). Our investigations reveal robust intracellular transport of PepTAC-LNPs across various clinically relevant human cell lines. Our studies also underscore the critical nature of the linker and hydrophobic E3 binding ligand for efficient LNP packaging and transport. We demonstrate the clinical utility of this strategy by engineering PepTACs targeting two critical transcription factors, P-catenin and CREPT (cellcycle-related and expression-elevated protein in tumor), involved in the Wnt-signalling pathway. The PepTACs induced target-specific protein degradation and led to a significant reduction in Wnt-driven gene expression and cancer cell proliferation. Mouse biodistribution studies revealed robust accumulation of PepTAC-LNPs in the spleen and liver, among other organs, and PepTACs designed against P-catenin and formulated in LNPs showed a reduction in P-catenin levels in the liver. Our findings demonstrate that LNPs can be formulated to encapsulate PepTACs, thus enabling robust delivery and potent intracellular protein degradation.

[0131] Given the superior attributes of peptide-based ligands over small molecules, our aim was to improve the extracellular stability and cell-membrane transport of PepTACs by encapsulating them in a nanoparticle carrier (FIG. 1). We envisioned a system in which nanoparticles shield the PepTAC cargo from extracellular proteases, promote cellular uptake, facilitate PepTAC escape from endosomes, and thereby enable PepTAC-mediated ubiquitination of target proteins followed by degradation through the UPS (FIG. 1). We used ionizable lipid nanoparticles (LNPs) as a carrier and a previously reported 27-mer PepTAC designed against a validated onco-target, CREPT (cell-cycle-related and expression-elevated protein in tumor) to test peptide delivery capabilities of reformulated LNPs. Our studies show that PepTAC amphiphilicity is essential for LNP loading and intracellular delivery irrespective of the choice of the ionizable lipid component. PepTAC -LNP formulations against CREPT were rapidly taken up and enabled potent targeted degradation of the CREPT protein at sub-nanomolar doses across various cell lines, rivalling traditional small molecule PROTACs. To broaden the scope of this work, we designed a new PepTAC against another oncoprotein, P-catenin, and showed similar potent in vitro degradation and downstream effects on transcriptional activity. Furthermore, we assessed their potential for clinical translation and observed that systemically administered LNPs increased PepTAC accumulation in the liver and spleen, enabling P-catenin degradation. Together, our findings emphasize the influence of lipid formulation and peptide amphiphilicity on uptake efficiency and open the door for the use of PepTACs as therapeutics as well as tools for chemical biology.

[0132] Results. Intracellular delivery of PepTACs via LNPs. We began our investigation with a PepTAC previously shown to degrade CREPT, also known as RPRD1B (regulation of nuclear pre-mRNA domain-containing protein IB). CREPT accelerates the cell cycle and promotes tumor growth. Aberrant expression has been found in numerous types of cancers and its expression has been shown to negatively correlate with patient outcomes. Studies have demonstrated that CREPT interacts with RNA polymerase II, which promotes chromatin loop formation and activates cyclin DI transcription, among others, in response to Wnt signaling. The PepTAC utilized consisted of a 21-mer CREPT-binding sequence connected to an E3 binding peptide via a short 6-aminohexanoic acid (Ahx) linker (FIG. 2A). This peptide-binding ligand was derived from a leucine-zipper-like motif located within the CREPT CCT (coiled-coil terminus) domain, specifically spanning from K266 to V286. Previous predictions using Schrodinger on CREPT CCT monomer determined that this peptide ligand binds to the same K266-V286 region, facilitated by three leucine residues. AlphaFold-Multimer predictions of the CREPT CCT dimer (PDB: 4NAD) with the peptide ligand confirmed the formation of a head-to- tail CREPT CCT homodimer and association of the peptide ligand with the leucine zipper motif at K266 to V286 (FIG. 2A). In the study by Ma et. al., a short cell-penetrating RRRRK (SEQ ID NO: 12) sequence was attached to the C-terminus of the PepTAC to enable its cellular internalization. This PepTAC, referred to as PRTC in the previous study, enabled the degradation of CREPT in PANC-1 cells at concentrations in excess of 10 pM. Using this as a starting point, we formulated the CREPT PepTAC (^CRPepTAC) using the standard LNP formulation consisting of an ionizable MC3 lipid, cholesterol, DSPC, and DMG-PEG2K lipids at a 50:38.5: 10:1.5 ratio. This formulation produced stable LNPs, as characterized by dynamic light scattering (FIG. 7). Although the addition of this PepTAC -LNP formulation to HeLa cells resulted in some cell transfection (FIG. 2B), we observed that the addition of 10 mol% DOTAP as a 5^th lipid to this formulation resulted in robust transfection of the entire cell population (FIG. 2B). This dependency on DOTAP persisted irrespective of the ionizable lipid composition. A similar trend was observed with and without DOTAP using the principal ionizable lipid in the Pfizer/BioNTech (ALC0315) and Moderna (SMI 02) mRNA vaccine formulations (FIG. 2B). MC3 demonstrated improved ^CRPepTAC delivery compared to ALC0315 and SMI 02 (FIG. 2B), making it the preferred choice for subsequent transfection studies. LNP formulations with DOTAP resulted in stable nanoparticles similar to those without DOTAP, as characterized by dynamic light scattering and zeta potential (FIGS. 7 and 8). The PepTAC-LNPs analyzed by cryo-electron microscopy exhibited a cup-shaped morphology (likely due to dehydration defects of the drying process) (FIG. 9). The impact of DOTAP on transfection efficiency is partially attributed to enhanced PepTAC encapsulation, exhibiting a 70% encapsulation with DOTAP versus 32% without DOTAP (FIG. 10).

[0133] Formulations containing varying amounts of DOTAP exhibited minimal differences in transfection efficiency within the range of 5-20% DOTAP (FIG. 2C). However, transfection efficiency declined at higher concentrations of DOTAP. The impact of DOTAP cannot be attributed to the increase in the fraction of cationic lipid relative to the other lipids in the formulation. This is evident as replacing DOTAP with an equivalent amount of MC3, or its ionizable equivalent, DODAP, led to a marked decrease in transfection efficiency relative to that seen with an equivalent amount of DOTAP (FIG. 2D).

[0134] To investigate the chemical and structural characteristics of DOTAP that contribute to enhanced transfection, we examined additional structural analogues such as DDAB (FIG. 11), which possesses a different saturated lipid tail but a similar cationic headgroup, and EPC (FIG.

11), an analogue that incorporates a phosphodiester in the headgroup. The collective results show that only structural analogues with a permanently cationic headgroup improved transfection efficiency (FIG. 2D). We next investigated the effect of formulation pH and lipid/ ^CRPepTAC ratio on transfection efficiency. Transfection efficiency improved with decreasing both pH and MC3/^CRPepTAC ratio (FIGS. 2E and 2F). The optimal conditions for maximum transfection efficiency were achieved with a formulation at pH3 and MC3/^CRPepTAC ratio of 2/1. The effect of pH on transfection efficiency can be attributed in part to the ^CRPepTAC encapsulation efficiency which showed modest encapsulation of 70% and 67% at pH 3 and 5 respectively, and poor encapsulation of only 22% at pH 7 (FIG. 10). The ^CRPepTAC maintained its alpha-helix structure at all pHs (FIG. 12). Changes to the type of phospholipid (DSPC vs. DOPE), or the phospholipid to ionizable lipid ratio, had minimal effect on cellular uptake (FIGS. 13 and 14). In addition to the studies performed in HeLa cells, the optimized ^CRPepTAC-LNP formulation also exhibits dose-dependent transfection across a diverse range of cell lines including human breast cancer (SK-BR-3), ovarian cancer (SKOV-3), colorectal adenocarcinoma (DLD-1), and hepatocellular carcinoma (HepG2) cell lines (FIGS. 2G, 2H, and 16-21). A similar dosedependent uptake was also observed when the LNPs were formulated at pH 5 (FIG. 15). Cellular uptake of ^CRPepTAC-LNP shows a rapid increase within the first 24 hours, followed by a decline in the subsequent 24 hours that may be attributed to peptide degradation and exocytosis of the free dye (FIG. 21). Cellular uptake was observed to be temperature-dependent, indicative of an energy dependent endocytic uptake mechanism (FIG. 22).

[0135] Subcellular localization of the delivered PepTACs was examined using confocal imaging (FIG. 3A). ^CRPepTAC-LNPs formulated at pH3 colocalized with an endosomal marker and showed diffuse staining indicative of cytosolic localization (FIGS. 3A and 3B). Conversely, the pH5 formulation displayed a higher degree of colocalization between ^CRPepTAC and endosomes (FIG. 3A). Notably, ^CRPepTAC alone showed no uptake or cellular association. This data suggests LNPs mediate PepTAC uptake via the endosomal route, escape the endosome and subsequently release PepTACs into the cytosol.

[0136] PepTAC amphiphilicity enables encapsulation and delivery via LNPs. The enhanced encapsulation and delivery observed at lower pH, as well as the need for an additional cationic lipid, initially indicated that electrostatic interactions played a role in LNP PepTAC encapsulation. However, the ^CRPepTAC sequence carries a net charge of +2 at pH 7 and a theoretical isoelectric point of 9.8. At the formulation pH of 3, all acidic residues on the ^CRPepTAC would be protonated, making it unlikely for encapsulation to solely rely on cationanion interactions. Upon closer scrutiny of the sequence, we observed that the Ahx linker and E3 binding ligand display a strong hydrophobic sequence patch, making the ^CRPepTAC sequence amphiphilic (FIG. 2A). Based on this observation, we hypothesized that amphiphilicity plays a pivotal role in LNP ^CRPepTAC encapsulation and delivery. To test this hypothesis, we prepared a fluorophore-labeled CREPT peptide-binding ligand (CL) without the linker and E3 domain and formulated it with the optimized LNP formulation. As depicted in FIG. 4A, delivery was severely impaired with the CL alone. Encapsulation studies confirmed low encapsulation efficiency led to poor cellular uptake (FIG. 4B). The data presented in FIGS. 4A and 4B strongly support the implication of peptide amphiphilicity as a crucial factor in PepTAC encapsulation and delivery. To showcase the generalizability of “amphiphilic-driven delivery”, we substituted the linker and E3 binding peptide with saturated alkyl chains of varying lengths (C6, CIO, Cl 2) in the ^CRPepTAC. Delivery of these lipopeptide constructs in LNPs resulted in robust transfection, with longer lipid tails exhibiting improved transfection efficiency (FIGS. 4C and 13).

[0137] ^CRPepTACs delivered by LNPs enable CREPT degradation. After establishing that LNPs enhance the cellular uptake of PepTACs, we next focused our investigations on assessing the protein degradation activity of the delivered ^CRPepTAC. CREPT is a highly tumorigenic protein overexpressed in Wnt-activated malignant cells and tissues. It functions as a crucial regulator of genes such as CCND1 that enhance cell proliferation and promote tumorigenesis (FIG. 4D). Considering CREPT's role in transcriptional activation, intracellular degradation of CREPT (i.e. RPRD1B) would disrupt the Wnt signaling pathway, directly impacting cell proliferation. To assess the functional delivery and efficacy of PepTAC-LNP formulations, we assayed the endogenous expression of CREPT in cells following ^CRPepTAC-LNP treatment. Our results demonstrated robust degradation of endogenous CREPT protein across various clinically relevant cell lines (FIG. 4E). Treatment with epoxomicin, a proteasome inhibitor, prevented protein degradation underscoring the role of the UPS in ^CRPepTAC -mediated protein degradation. Furthermore, we quantitatively evaluated CREPT protein degradation using a firefly luciferase-fused CREPT (Luc-CREPT) expressing plasmid. Cells were pre-transfected with Luc- CREPT and a Renilla luciferase as an internal control, then treated with varying amounts of ^CRPepTAC-LNP formulations. We observed dose-dependent degradation of Luc-CREPT protein (FIG. 4F). Again, degradation was inhibited in the presence of the epoxomicin. Degradation of Luc-CREPT slightly decreased at high concentrations (>200 nM) likely due to the "hook" effect, a phenomenon observed in many PROTACs. This occurs when PepTACs (including PROTACs) saturate binding to their protein target and E3 ligase, leading to the formation of binary complexes instead of the ternary complex required for ubiquitination and degradation. We observed 50% degradation (DCso) of CREPT at concentrations between 100-200 nM, representing one of the lowest reported DC50 among all PepTACs to the best of our knowledge. Finally, we assayed for CREPT degradation with and without DOTAP and observed diminished degradation when DOTAP was absent (FIG. 14).

[0138] We next carried out a TopFlash assay using two constitutive Wnt-activated cell lines, DLD-1 and HepG2, to assess whether ^CRPepTAC-LNP-mediated CREPT degradation could suppress Wnt signaling and transcriptional activity. The TopFlash construct is a T cell factor (TCF)/lymphoid enhancer-binding factor (LEF)-Firefly luciferase reporter vector that is activated in Wnt-active cells. The firefly luciferase gene in this reporter is controlled by the TCF/LEF responsive element, which is activated in the presence of CREPT, P-catenin and other transcriptional proteins. Therefore, degradation of CREPT should lead to a decrease in transcriptional activity and luciferase expression. As a control, we used a non-inducible luciferase vector called FopFlash, which is under the control of a minimal promoter without any response elements, thus providing a measure of background luciferase activity. In both Wnt- activated DLD-1 and HepG2 cells, we observed a significant concentration-dependent reduction in transcriptional activity following ^CRPepTAC-LNP treatment (FIGS. 4G and 4H). In DLD-1 cells, a 50% suppression of transcriptional activity was observed at concentrations as low as 10 nM (FIG. 4G). Consistent with previous observations, we observed the intrinsic "hook" effect at high concentrations, occurring beyond 200 nM in DLD-1 cells and beyond 500 nM in HepG2 cells (FIGS. 4G and 4H).

[0139] A decrease in the transcription of proliferative genes in Wnt-active cell lines should result in the suppression of cell growth. To assess this downstream effect, we conducted a timedependent MTS assay to examine the impact of the ^CRPepTAC-LNP formulation on cell proliferation and viability in Wnt-active DLD-1 and HepG2 cells, as well as non -Wnt-active HeLa cells. We observed a significant reduction in cell proliferation over time in Wnt-active DLD-1 (FIG. 41) and HepG2 cells (FIG. 25). This decrease in cell viability was specific to Wnt- active DLD-1 and HepG2 cells, as we observed minimal changes in the viability of non-Wnt- active HeLa cells 72 hours after treatment (FIG. 4J). These collective results demonstrate the ability of ^CRPepTAC-LNP to deliver PepTACs into the cytosol to degrade the target protein, CREPT. Protein degradation leads to a loss of function, as evidenced by the concurrent decrease in transcriptional activity and cell proliferation.

[0140] ^pCatPepTACs delivered by LNPs enable robust P-catenin degradation. To demonstrate the versatility of the LNP-mediated PepTAC delivery strategy, we designed a novel PepTAC targeting a well-studied oncoprotein, P-catenin. The design was based on a peptide derived from the BCL9 protein that binds to P-catenin as seen in the co-crystal structure and reproduced by AlphaFold (FIG. 5A). P-catenin was selected given its role in the Wnt-signaling pathway of tissue homeostasis and embryonic development, as well as in several types of human cancer, such as colorectal, breast, melanoma, and prostate, among several others. Like CREPT, P-catenin controls the expression of several key genes that regulate cell cycle, proliferation and tumorigenesis. Transcriptional activation of the Wnt/p-catenin pathway is dependent on formation of the P-catenin super complex involving BCL9 and TCF/LEF family of transcriptional factors. As such, molecules that degrade P-catenin can inhibit Wnt/p-catenin signal transduction and supress cell proliferation. To construct a PepTAC against P-catenin (^pCatPepTAC), we introduced the Ahx linker and the pentapeptide VHL-binder at the N-terminus of the P-catenin peptide binding sequence (SQEQLEHRERSLQTLRDIQRMLF) SEQ ID NO: 6). This N-terminal region is solvent exposed (FIG. 5A) and is expected to not interfere with ligand binding to its target.

[0141] Functional LNP-mediated delivery of P^CatPepTAC was assessed by measuring the endogenous cellular expression levels of P-catenin. Our results demonstrated robust degradation of P-catenin in clinically relevant cell lines (FIG. 5B). P-catenin degradation was inhibited by treatment with the proteasome inhibitor epoxomicin, indicating degradation through the UPS (FIGS. 5B and 5C). We next examined downstream effects of P-catenin degradation by conducting a TopFlash assay using two constitutive Wnt-activated cell lines, DLD-1 and HepG2. Degradation of P-catenin led to a significant concentration-dependent reduction in transcriptional activity following ^|iCatPepTAC-LNP treatment (FIGS. 5D and 5E). A 50% suppression of transcriptional activity was observed at concentrations as low as 20 nM in DLD-1 cells (FIG.

5D) and at 100 nM in HepG2 cells (FIG. 5E). The intrinsic "hook" effect isn’t observed in either of the Wnt-active cells and likely exists at concentrations higher than those tested.

[0142] Finally, the downstream effect of transcriptional suppression was assessed by performing time-dependent cell viability studies on Wnt-active DLD-1 and HepG2 cells, as well as non-Wnt-active HeLa cells. Similar to the studies with CREPT, we observed a concentrationdependent reduction in cell viability over time in Wnt-active DLD-1 (FIG. 5F) and HepG2 cells (FIG. 26). The effect on cell viability was specific to Wnt-active cells, as we observed minimal changes in the viability of non-Wnt-active HeLa cells 72 hours after treatment (FIG. 5G) despite evidence of P-catenin degradation in these cells (FIGS. 5B and 5C). These collective results demonstrate the delivery of ^pCalPepTACs by LNPs to the cytosol, enabling the targeted degradation of P-catenin. P-catenin degradation, like CREPT, led to a decrease in transcriptional activity and cell proliferation. [0143] LNPs extend PepTAC half-life in vivo, enable biodistribution to the liver and spleen, and facilitate P-catenin degradation in the liver. The current results demonstrate that LNPs facilitate PepTAC delivery into cells and that the delivered PepTACs retain their proteindegrading capabilities. To further explore their suitability for potential in vivo applications, we investigated the distribution of PepTAC -LNPs upon systemic administration. Recent studies highlight the significance of the apparent surface pKa of LNPs as a critical factor influencing the protein corona composition and LNP biodistribution upon systemic administration. Notably, LNPs with apparent pKa values ranging from 6 to 7 have been observed to accumulate primarily in the liver. Considering this link between pKa and biodistribution, we determined the apparent pKa of PepTAC -LNPs to be 6.5 using the 6-(p-toluidino)-2-naphthalenesulfonic acid (TNS) assay indicating preferential accumulation in the liver (FIG. 27). Indeed, biodistribution experiments in hairless SKH-1 mice using Cy5.5-labeled PepTAC -LNPs demonstrated a prominent accumulation of PepTAC -LNPs in the region of the liver (FIGS. 6A and 6B). Peak accumulation was observed at 1 hour post-injection (FIGS. 6A and 6B). Quantitative analyses demonstrated that LNPs significantly increased PepTAC accumulation in various organs, with a notable enhancement in the liver and spleen, compared to PepTAC alone (FIGS. 6B and 6C). Given their preferential distribution to the liver, we assessed cellular penetration of PepTAC- LNPs into liver cells. Confocal imaging of liver sections revealed significant intracellular accumulation of PepTAC -LNPs into liver cells at the 1 hour time point relative to PepTAC alone. After 24 hours, PepTAC signal from PepTAC-LNPs are still visible in the liver sections where no signal is observed with PepTACs alone. This data demonstrates that LNPs extend PepTAC accumulation in vivo relative to PepTAC alone.

[0144] Given their preferential distribution to the liver, we assessed the ability of the P^CatPepTACs encapsulated in LNPs to downregulate liver P-catenin in wild type mice. Mice were intravenously injected every 72 hours for ten days (four injections) with ^($CatPepTAC-LNPs. Following this treatment, liver tissue samples were harvested, digested, and analyzed for presence of P-catenin via Western Blot. P-catenin levels were significantly reduced in mice treated with ^pCatPepTAC-LNPs relative to saline control (FIGS. 6F and FIG. 28). In healthy mice, the expression of P-catenin in liver hepatocytes is relatively low, primarily due to the absence of Wnt signalling, which leads to its rapid endogenous degradation. Considering its natural low expression, the detection of -50% P-catenin degradation following ^|iCatPepTAC-LNP treatment is noteworthy and holds promise for other applications, especially in disease states characterized by the overexpression of P-catenin due to Wnt pathway activation.

[0145] Discussion. Poor cellular permeability and inherent instability in biological fluids are major obstacles that have impeded peptide-based drug development. To overcome these barriers, we repurposed the LNP formulation to enable efficient intracellular PepTAC delivery at nanomolar concentrations. LNPs can shield the PepTAC cargo from extracellular proteases, promote cellular uptake, facilitate PepTAC escape from endosomes, and enable PepTAC- mediated ubiquitination of target proteins followed by proteosome degradation. Unlike nucleic acids which load via electrostatic-driven assembly into LNPs, we show PepTAC amphiphilicity as a feature that enables encapsulation and delivery by LNPs (FIG. 4).

[0146] We formulated PepTACs against two transcription factors involved in the Wnt signaling pathway: CREPT and P-catenin, which is notably difficult to drug. Our results showed significant degradation of CREPT and P-catenin across various clinically relevant cell lines at concentrations as low as 100 nM, which is unprecedented for PepTACs. Critically, neither ^CRPepTAC alone nor ^CRPepTAC modified with a cell-penetrating peptide sequence induced degradation at nanomolar concentrations, (FIG. 4E) which is consistent with previous reports that show the onset of PepTAC-mediated degradation in micromolar range. Similar results were achieved with P^CatPepTAC-LNP formulations (FIG. 5) with robust P-catenin degradation at nanomolar ^pCatPepTAC concentrations. Considering their role in transcriptional activation, intracellular degradation of CREPT and P-catenin disrupted the Wnt-dependent transcriptional activity and directly impacted cell proliferation in Wnt-dependent cancer cells (DLD-1 and HepG2), with no discernible effect on Wnt-independent cells (HeLa).

[0147] Finally, we demonstrate the in vivo potential of these formulations by illustrating enhanced accumulation of LNP-delivered PepTACs in the liver and spleen and significant P- catenin degradation in the liver. Our current demonstration was limited to wild-type mice, as we aimed to de-risk this novel approach and establish the feasibility of PepTAC delivery in vivo. One potential application of this delivery system is in the development of therapeutics for hepatocellular carcinoma (HCC), given that hMet and P-catenin mutations account for approximately 10% of all HCC cases. Unlike siRNAs, PepTACs can induce rapid direct protein degradation, irrespective of protein half-life, and can recognize different protein states, such as mutant vs. wild type or active vs. inactive conformation, allowing for the depletion of specific protein subpopulations.

[0148] In cases where life-saving medicines are urgently needed (e.g., to contain an outbreak), drug discovery strategies that can be rapidly developed from existing protein structural data and widely deployed are of paramount importance. By overcoming the intracellular transport limitations of PepTACs, we have expanded their use broadly as novel chemical biology tools for dissecting protein networks and as potential therapeutic modalities for inhibiting new drug targets that have so far evaded pharmacological intervention.

[0149] Methods. Peptide synthesis. All peptide sequences were synthesized using Rink Amide MBHA resin (100-200 mesh) with an average loading efficiency of 0.93 mmol/g, FMOC- protected amino acids, DMF as the solvent, Diisopropylcarbodiimide (DIC) as the activator and oxyma as the activator base. Solid-phase peptide synthesis was carried out in a microwave- assisted automated solid-phase peptide synthesizer at a 0.1 mmol synthesis scale. The following peptide sequences were synthesized:

CREPT ligand (CL): KDVLSEKEKKLEEYKQKLARV (SEQ ID NO: 7)

^CRPepTAC: IYP^OHAL-Ahx-KDVLSEKEKKLEEYKQKLARV (SEQ ID NO: 1) ^pCatPepTAC: IYP^OHAL-Ahx-SQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 2) P^OH stands for hydroxylated proline and Ahx is 6-aminohexanoic acid. All sequences are written from '-terminus to C-terminus. Following peptide synthesis, the resins were cleaved using a cleavage cocktail consisting of 92.5% trifluoroacetic acid (TFA), 2.5% water, 2.5% triisopropylsilane (TIPS) and 2.5% phenol. Cold diethyl ether was added to the cleaved crude mixture which led to peptide precipitation as a white powder. Crude peptide was purified via reverse-phase HPLC (using water/acetonitrile with 0.1% trifluoroacetic acid as the solvent system and a 5-100% acetonitrile gradient in 20 minutes). The purified peptide fraction was characterized using MALDI-TOF mass spectroscopy and the purity was confirmed using an Agilent 1100 analytical HPLC. See Table 1.

[0150] Table 1. Mass Characterization for the peptide-based synthetic constructs obtained via MALDI-TOF spectrometer.

[0151] Non-specific peptide labeling with Fluorescein-NHS. Purified peptides were dissolved in DMSO to a concentration of 25 mg/mL. Fluorescein-NHS ester (5/6- carboxyfluorescein succinimidyl ester, 1.1 molar equivalents) and diisopropylethyl amine (DIPEA) (2.5 molar equivalents) were then combined and subsequently added to the peptide solution. The reaction mixture was stirred at room temperature for 2 hours and purified by RP- HPLC (using water/acetonitrile with 0.1% trifluoroacetic acid as the solvent system and a 5- 100% acetonitrile gradient in 20 minutes). The purified labeled peptides were lyophilized and characterized by MALDI-TOF MS. [0152] N-terminal labeling of peptides with fluorophore-NHS esters. The immobilized

^CRPepTAC peptide on a rink-amide resin (20mg) with a free N-terminus, all other amino acid side-chains protected, was swollen in 0.5 mL of DMF for 10 minutes. 0.4 mL of DMF was aspirated and 0.1 mL DMF with either fluorescein-NHS ester or Cy 5.5-NHS ester (5 molar equivalents) and DIPEA (10 molar equivalents) were added. The reaction mixture was stirred at room temperature for 12 hours. Subsequently, the resin was washed overnight with DMF and finally cleaved with 92.5% TFA, 2.5% water, 2.5% TIPS, and 2.5% phenol. The cleaved crude labeled peptide was dried with a stream of nitrogen, precipitated with the addition of pre-cooled ether, re-dissolved in water/acetonitrile (1 : 1 volume ratio), purified using RP-HPLC and characterized via MALDI-TOF MS. [0153] N-terminal lipidation and fluorescein modification of peptides. The immobilized CL peptide on a rink-amide resin (20mg) with a free N-terminus, all other amino acid side-chains protected, was swollen in 0.5 mL of DMF for 10 minutes. 0.4 mL of DMF was aspirated. Solutions of hexanoic, decanoic and dodecanoic acid (2.5 molar equivalents) were pre-incubated with HATU (2.5 molar equivalent) and DMAP (3 molar equivalents) dissolved in 0.1 mL DMF. The solutions were added to the swelled resin and stirred at room temperature for 12 hours. Subsequently, the resin was washed 3-5 times with both DMF and diethyl, and dried overnight. Cleavage from the resin was done with 92.5% TFA, 2.5% water, 2.5% TIPS, and 2.5% phenol. The cleaved, crude, lipid-modified CL-peptide was dried with a stream of nitrogen and precipitated via cold-ether precipitation at -80°C. The lipid-modified peptides were re-dissolved in water/acetonitrile (1 : 1 volume ratio), and purified using RP-HPLC followed by MALDI-TOF for characterization. Purified peptides were non-specifically labelled with fluorescein dye as previously described.

[0154] LNP Formulation for in vitro experiments. To make 100 pL of LNP formulation with 25 pg of peptides as the payload, D-Lin-MC3 (45 mol%, 5 pL of 10 mg/mL ethanol stock), DSPC (8.9 mol%, 6.15 pL of 2 mg/mL ethanol stock), cholesterol (34.7 mol%, 4.64 pL of 5 mg/mL ethanol stock), DMG-PEG-2000 (1.4 mol%, 2.93 pL of 2 mg/mL ethanol stock) and DOTAP (10 mol%, 2.42 pL of 5 mg/mL ethanol stock) were mixed with 3.9 pL of ethanol to make a 25 pL lipid-cocktail. Subsequently, 5 pL of 5 mg/mL (25 pg) PepTAC was diluted with 20 pL of 10 mM sodium citrate buffer solution (pH 3) to obtain a total of 25 pL aqueous PepTAC-solution. A micropipette was used to rapidly mix the lipid-cocktail with the aqueous PepTAC-solution. This mixture was then diluted with an equal volume of IxPBS buffer (pH 7.4, 50 pL) to make a formulation with a pre-dialysis volume 100 pL. This formulation was then dialyzed in a dialysis chamber (Slide-A-Lyzer™ MINI Dialysis Devices, 3.5K MWCO, Thermo Fisher) against sterile lx PBS, pH 7.4 for 2 hours. The formulation was stored at 4 °C.

[0155] To create LNP formulations at different pH values, a sodium citrate buffer of different pH values was used to prepare the 25 pL PepTAC-solution. Furthermore, 10 mol% of DDAB, DODAP, or EPC lipids were used in place of DOTAP for making formulations with varying compositions of the 5^th lipid. Similarly, varying molar percentages of DOTAP supplements (5-30%) were used to create different LNP formulations. [0156] LNP formulation for in vivo experiments. To make 100 pL of LNP formulation with 100 pg of peptides as the payload, D-Lin-MC3 (45 mol%, 8 L of 25 mg/mL ethanol stock), DSPC (8.9 mol%, 6.56 pL of 7.5 mg/mL ethanol stock), cholesterol (34.7 mol%, 4.64 pL of 20 mg/mL ethanol stock), DMG-PEG-2000 (1.4 mol%, 2.34 pL of 10 mg/mL ethanol stock) and DOTAP (10 mol%, 1.93 pL of 25 mg/mL ethanol stock) were mixed with 3.9 pL of ethanol to make a 25 pL lipid-cocktail. Next, 4 pL of 25 mg/mL (100 pg) of PepTAC was added to 21 pL of 10 mM sodium citrate buffer solution (pH 3) to make a total of 25 pL of aqueous PepTAC- solution. For biodistribution experiments, 25 pL of aqueous PepTAC-solution was made by mixing 19.4 pL of sodium citrate buffer, 2 pL of 5 mg/mL Cy 5.5-labeled ^CRPepTAC (to acheive 10% labeling) and 3.6 pL of 25 mg/mL unlabeled ^CRPepTAC peptide. A micropipette was used to rapidly mix the lipid-cocktail with the aqueous PepTAC-solution. This mixture was then rapidly diluted with an equal volume of IX PBS buffer (pH 7.4, 50 pL) to make a formulation with a pre-dialysis volume 100 pL. The formulation was dialyzed in a dialysis chamber (Slide- A- Lyzer™ MINI Dialysis Devices, 3.5K MWCO, Thermo Fisher) against sterile IX PBS, pH 7.4 for 4 hours. The formulation was stored at 4 °C.

[0157] Size and zeta potential measurements. PepTAC -LNP formulations were diluted 50- fold in IX PBS buffer. A total of 500 pL of the diluted formulations were placed in a polystyrene cuvette with a 10 mm path length and particle size measurements were made with a Malvern Zetasizer. The same formulation was placed in a Malvern capillary sample cell for zeta potential measurements.

[0158] Apparent pKa measurement via a TNS assay. A series of buffers with pH values between 2.5 and 8.5 were prepared by adjusting the pH of a solution containing 10 mM citrate, 10 mM phosphate, 10 mM borate, and 150 mM NaCl with 1 N HC1 and 5 N NaOH. 80 pL/well of each buffer solution was added to a black-wall 96-well plate. A 300 pM stock solution of TNS was prepared in DMSO and 2 pL of this solution was added to each of the buffer solutions in the 96-well plate. 20 pL of a particular LNP formulation was added to each well (with replicates). The fluorescence of the resulting solution was measured using a microplate reader (TEC AN Infinity M1000 Pro) using an excitation wavelength of 325 nm and an emission wavelength of 435 nm with 10 nm bandwidth. The emission intensity was plotted against pH and fit using a four-parameter sigmoidal logistic equation (GraphPad Prism) to obtain the apparent LNP surface pKa. [0159] Encapsulation efficiency of the LNPs. 15 pL of each LNP formulation (0.25 mg/ml with respect to labelled peptide) was placed in an Eppendorf tube and mixed with 5 pL of 5X Triton-X solution prepared with in PBS buffer followed by sonication for 10 minutes to lyse the particles. In a separate Eppendorf, another 15 pL of the same formulation was diluted with an equivalent 5 pL of IX PBS buffer. Both lysed and intact LNP solutions were mixed with 20 pL of the native gel loading buffer and loaded in a 16% Tri cine gel for a native gel electrophoresis. In-gel fluorescence of the non-encapsulated labeled payload from the lysed and intact LNP solutions was measured and quantified via ImageJ. Quantification using the relationship; [(J_lysed — I _intact) /I_lysed] X 100, gives a measurement of the % encapsulation efficiency.

[0160] Cloning for Firefly Luciferase-fused CREPT/RPRD1B expressing plasmid. Construction of pCMV-Luc-CREPT was performed using restriction cloning and propagated in Escherichia coli strain DH5a. Firefly luciferase was PCR amplified from pCDNA-Luciferase (Addgene, cat # 18964) using primers introducing a 5’ Kpnl site and a 3’ Nhel site, and CREPT was PRC amplified from pCMV-RPRDl (Sino Biological, cat # HG14027-NH) using primers introducing a 5’ Nhel site and a 3’ Xbal site. The two gene fragments were ligated using Nhel and then ligated into pCMV (Sino Biological) using Kpnl and Nhel. The primer sequences are given below:

Luc forward primer: 5’-GGTACCATGGAAGACGCCAAAAACATAAAGAAAGG-3’ (SEQ ID NO: 8)

Luc reverse primer: 5’-GCTAGCGCCTCCACCCACGGCGATCTTTCCGCC-3’ (SEQ ID NO: 9)

CREPT forward primer: 5’-GCTAGCTCCTCCTTCTCTGAGTCGGC-3’ (SEQ ID NO: 10) CREPT reverse primer: 5’-TCTAGATTAATGATGGTGGTGATGGTGGTCAGTTG AAAACAGGTCCCCAG-3’ (SEQ ID NO: 11).

[0161] Cell culture, imaging and cellular assays. HeLa and HepG2 cells were grown in DMEM media supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/ streptomycin, while DLDl-cells were grown in RPMI 1600 media with the same supplement. SK-OV-3 and SK-BR-3 cells were grown in McCoy 5A media with the same supplement. During cell-passaging, trypsin-EDTA (0.25%) with phenol red was used for the detachment of cells from the tissue-culture flasks. All cells were maintained at 37 °C, 5% CO2 and 90% relative humidity.

[0162] Cell-uptake assay via flow cytometry. 75,000 cells were seeded per well in 500 pL of DMEM media supplemented with 10% FBS and 1% penicillin/ streptomycin in a 24 well-plate. The plate was incubated for 18-20 hours at 37 °C with 5% CO2. Free fluorescein-labeled peptides and LNP-encapsulated fluorescein-labeled peptides in PBS buffer were added at various concentrations to the wells. In all LNP formulations, the PepTAC dose added to cells was based on the total amount of PepTAC added to the formulation. Thus, a PepTAC concentration of 0.25 mg/mL was used for the 100 uL formulations. After treatment, the plate was incubated for another 20-24 hours (kinetic studies were incubated for different time points). The media was aspirated, and cells were washed three times with lx PBS. Cells were then detached by pipetting 20-25 times with PBS. Detached cells were centrifuged (1200 x g for 6 mins), the supernatant aspirated, and pelleted cells were resuspended in fresh IX PBS for flow cytometry studies. Readings were taken on a FACSCalibur™ analyzer (Becton Dickinson) with fixed power and gain parameters. Flow cytometry data were analyzed using the FlowJo software. The fluorescence histogram and mean fluorescence intensity (MFI) data were obtained from 10,000- gated cells.

[0163] Confocal imaging. 70,000 HeLa cells/chamber were plated in a 4-chamber 35-mm glass-bottom microwell dish (MatTeK) and cultured at 37 °C for 20-24 hrs with 500 pL of DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. Each chamber was treated with different LNP formulations containing fluorescein-labeled peptides or free labeled peptides as controls. The chambers were incubated for 8 hours. Thereafter, media was aspirated, and each chamber was washed 2-3 times with IX sterile PBS. The chamber was then filled with 500 pL of dye-free Fluorbrite DMEM media pre-mixed with lx Hoechst (1 : 12000 dilution of Thermo Hoechst 33342), IX endosome-tracker 594 (1:1000 dilution of Biotium LysoView™ 594) and IX microtubule-tracker 647 (1 :1000 dilution of Biotium ViaFluor^R Live cell microtubule stain 647). After incubation for 10-15 minutes, the media was aspirated, and each chamber was washed 3-4 times with IX PBS and re-filled with dye-free Fluorbrite DMEM media. The chamber was then subjected to live cell imaging on a Zeiss i 880 inverted confocal microscope consisting of four LASER channels; the blue channel (405 nm excitation) to probe the Hoechst nuclei stain, the green channel (488 nm excitation) to probe the labeled PepTAC, the red channel (594 nm excitation) to probe endosomes and the far-red channel (647 nm, represented as grayscale) to probe cytosolic microtubules. The comparative image at different channels and the merged image qualitatively assesses the sub-cellular localization and distribution of the delivered PepTAC cargo.

[0164] Assessing intracellular target protein degradation via Western Blot. 150,000-200,000 cells were plated per well of a 12-well plate with 1 mL of DMEM media. The plate was then incubated for 18-20 hours. Free peptides or LNP-encapsulated peptides were subsequently added to individual wells in IX PBS buffer at various doses. In all LNP formulations, the PepTAC dose added to cells was based on the total amount of PepTAC added to the formulation. Thus, a PepTAC concentration of 0.25 mg/mL was used for the 100 pL formulations. After treatment, the plate was incubated for an additional 24 hours. Next, media was aspirated, and 75 pL of Pierce RIPA (Radio Immuno Precipitation Assay) lysis buffer, pre-mixed with IX Halt protease inhibitor cocktail and IX EDTA solution (5 mM final concentration), was added to each well at 0 °C (on an ice bath) and incubated for 10-15 minutes with occasional stirring. Cell lysates were collected in pre-cooled sterile Eppendorf tubes, and the water-insoluble/membrane-soluble fractions were separated by centrifuging the samples at 20,000 x g for 15 minutes. The supernatants were collected, and 15 pL of each lysate sample was used for total protein quantification using a standard BCA assay (5 pL per measurement, with three replicates).

[0165] In the CREPT and P-catenin knockdown experiment, 20 pg and 15 pg of cell lysates respectively were loaded into each well of a 4-12% bis-tris Nu-PAGE gel and IX MOPS running buffer was used to run the gels. Upon completion, the gel was transferred to a nitrocellulose membrane with a porosity of 0.45 pm using a wet-transfer apparatus and transfer buffer containing IX Tris-glycine with 20% methanol. The membrane was then treated with a blocking buffer consisting of IX TBST buffer with 5% milk powder for 2-3 hours. Following that treatment, the membrane was incubated overnight at 4 °C with the primary antibody solution, which was prepared by diluting the rabbit anti-CREPT antibody (1 :2500 dilution), mouse anti-0- Catenin antibody (1 : 1000 dilution), rabbit anti-P-tubulin antibody (1 :2500 dilution), and mouse anti-P-actin antibody (1 :2500 dilution) in the blocking buffer. The membrane was then washed 5- 6 times for 5 minutes with IX TBST buffer. Next, the membrane was treated with the secondary antibody solution, prepared by diluting the Starbright 700 Goat anti-rabbit IgG antibody (1 :5000 dilution) and Starbright 700 Goat anti-mouse IgG antibody (1 :5000 dilution) in the blocking buffer, for 1 hour on a rocking platform. Following another wash with IX TBST buffer, repeated 5-6 times for 5 minutes each, the blot was imaged using a BioRad Gel Dock system to measure the expression levels of the target proteins relative to the housekeeping genes.

[0166] TopFlash-FopFlash assay. 10,000-15,000 cells per well were seeded in a whitebottom 96-well plate using 0.1 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The plate was then incubated at 37°C with 5% CO2 for 18-20 hours. For transfection, the TOPFlash plasmid (90 ng/well) or the FOPFlash plasmid (90 ng/well) as a negative control, along with the Renilla-luciferase plasmid (12 ng/well) as a normalization control were introduced to cells. Lipofectamine 2000 transfection reagent (0.5 pL/well) and serum-reduced Opti-MEM media were used for the transfection process. The lipofectamine and plasmid stocks were diluted in Opti-MEM to make a total volume of 20 pL of the plasmid- lipoplex solution that was added to each well (10 pL for each plasmid-lipoplex). The posttransfection total volume per well was approximately 120 pL. After transfection, cells were incubated for 6-8 hours. Subsequently, 80 pL of media was aspirated from each well and replaced with 50 pL of fresh DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. Next, the cells were treated with 10 pL of LNP formulations, appropriately diluted in IX PBS to achieve different concentrations. The plate was incubated for an additional 24 hours. Following LNP treatment, media was aspirated, and the cells were washed twice with IX PBS. Then, 20 pL of IX passive lysis buffer (Promega Dual Gio kit) was added to each well and allowed to incubate for 2 minutes. For firefly luciferase measurement, the Firefly luciferase substrate (LAR II reagent, 100 pL/well, Promega Dual Gio kit, cat # El 960) was added to each well, and luminescence was measured using a microplate reader (TEC AN Infinite M1000 Pro). Finally, the IX Renilla-luciferase substrate mixed with a Stop & Gio buffer (100 pL/well, Promega Dual Gio kit, cat # E1960) was added, and the Renilla-Luminescence was measured to normalize the TOPFlash-Firefly luminescence.

[0167] MTS cell viability assay. 10,000 cells/well were seeded in a round-bottom transparent 96-well plate using 100 pL of DMEM/well supplemented with 10% FBS and 1% penicillin/streptomycin. The plate was incubated for 24 hours. Following this, 60 pL of media was removed from each well and 50 pL of fresh media was added along with 10 pL of LNP formulation, appropriately diluted to achieve the desired concentration. The plate was incubated again for different durations, specifically 24 hours, 48 hours, and 72 hours. After the specified incubation period, media was aspirated, and each well was washed twice with sterile IX PBS. Next, 100 pL of dye-free Fluorbrite DMEM media containing 5% (5 pL) MTS reagent premixed in it was added to each well, followed by another 3 hour incubation. The absorbance was measured using a microplate reader (TEC AN Infinite Ml 000 Pro) at a wavelength of 490 nm to assess cell viability and proliferation.

[0168] In Vivo Biodistribution of LNP-encapsulated PepTACs. All animal experiments were conducted in accordance with an approved IACUC protocol (#2019-0063). Twenty-eight female SHK1 hairless mice (vendor - Charles River Laboratories, Elite strain 477) aged 8-11 weeks were housed in a clean facility with 12-hour light/dark cycles and fed alfalfa-free chow ad libitum.

[0169] Biodistribution studies were conducted using an adaptation of a previously published method. For biodistribution studies, mice (n=5 per group) were administered either 5 mg/kg free PepTAC labeled with 50% Cy5.5 or 5 mg/kg LNP-encapsulated PepTAC labeled with 50% Cy5.5 via tail vein injection (approximately 100 pL injection using a 26 G needle). IX PBS (n=3, 100 pL injection) was used as a negative control. Mice were imaged using the IVIS Spectrum In Vivo Imaging Platform (Perkin Elmer, USA). To do this, mice were anesthetized using 3.5% isoflurane before transfer to the imaging stage of the IVIS Spectrum (Perkin Elmer, USA) maintained at 37 °C. During imaging, mice were maintained at 2% isoflurane anaesthesia. Mice were imaged in both supine and prone orientations at 1 and 24 hours. Images were obtained using a spectral unmixing sequence defined for Cy5.5. Following the final timepoint, mice were euthanized using 3.5% CO2 followed by cervical dislocation. The liver, spleen, kidneys, heart, lungs, brain, and GI tract were harvested and imaged ex vivo using the IVIS Spectrum (Perkin Elmer, USA).

[0170] To determine cellular-level biodistribution to the liver, 3 x 5 mm biopsy punches were obtained from separate lobes of the liver (excluding the caudate lobe). Samples were placed in histology cassettes and fixed in 10% (vol/vol) neutral buffered formalin for 48 hours prior to transfer to IX PBS. Fixed samples were dehydrated and embedded in paraffin, and 4 pm sections were prepared for microscopy analysis. For confocal imaging, the slides were treated with Hoechst for nuclei-staining, and representative images from specific z-sections were captured using an oil-immersion 40x lens. In the captured images, blue channel emission shows the spatial distribution of the nuclei at different cells in the tissue sections while the red channel emission shows the Cy 5.5-labeled payloads.

[0171] To compare the organ distribution seen at 24 hours to that observed at 1 hour, female SKH1 mice (n=5 per group) were administered either 5 mg/kg free PepTAC labeled with 50% Cy5.5 or 5 mg/kg LNP-encapsulated PepTAC labeled with 50% Cy5.5 via tail vein injection (approximately 100 pL injection using a 26 G needle). After 1 hour, mice were sacrificed and harvested as described above. Ex vivo imaging of the organs was performed, and liver samples processed for histology. Samples were fixed in 10% (vol/vol) neutral buffered formalin for 48 hours followed by transfer to IX PBS. Samples were prepared for microscopy as described above. To analyze the relative biodistribution of free PepTAC and LNP-encapsulated PepTAC to major organs, the Living Image Software (Perkin Elmer, USA) was used to draw custom regions of interest (ROIs) around images of organs taken ex vivo. The total radiant efficiency of each ROI was normalized to the pixel area of the ROI to obtain the average radiant efficiency.

[0172] Assessment of in vivo protein degradation via LNP-encapsulated PepTACs. Thirty female BALB/c mice aged 6-8 weeks were housed in a clean facility with 12-hour light/dark cycles and fed ad libitum. Animals were identified via ear punch. To assess the activity of the PepTAC mice (n=5 per group) were administered LNP-encapsulated PepTACs (against 0- catenin) at a dose of 5 mg/kg via tail vein injection (approximately 100 pL injection using a 26 G needle), one dose at every three days for 10 days (four doses) On the 11th day, mice were euthanized using 3.5% CO2 followed by cervical dislocation. The livers were immediately excised and rinsed with ice-cold sterile PBS buffer, and 3 x 5 mm biopsy punches were obtained from separate lobes of the livers (excluding the caudate lobe). Samples were placed in histology cassettes and fixed in 10% (vol/vol) neutral buffered formalin for 48 hours prior to transfer to IX PBS. The remaining liver tissue was snap frozen in liquid nitrogen and stored at -80 °C. These liver samples were homogenized with RIPA lysis buffer premixed with IX protease and phosphatase-inhibitor cocktail and lx EDTA (to prevent non-specific protease, metalloprotease, or phosphatase-mediated digestion) using a benchtop homogenizer (VWR VDI 12 Homogenizer). Samples were centrifuged at 4000 rpm for 10 minutes at 4 °C, and the supernatants were passed through a 40 pm cell strainer to remove remaining tissue particulate. A standard BCA assay was performed to quantify the total protein concentration for each tissue homogenate. Western Blot was performed (loading: 20 pg total protein/well) with the tissue homogenates to assess the expression level of endogenous P-catenin as well as the housekeeping gene, P-tubulin.

EXAMPLE 2

[0173] This example describes lipid nanoparticles of the present disclosure, and methods of making and using same.

[0174] A lipid nanoparticle formulation was formed using nanoparticles loaded with fluorescein-labeled ^CREPTPepTAC. The size of the particle (smaller nanoparticles are favored for improved pharmacokinetic profile), cellular uptake (characterized by the mean fluorescein intensity obtained from flow cytometric analysis), % encapsulation efficiency (captures information about the cargo loading efficiency), and serum stability (characterized by % encapsulation efficiency in presence of serum, a volume ratio of serum formulation = 1 : 1) were all determined. The darker colors in Fig. 29 indicate a favorable property as indicated by the color scale. The collective data in Fig. 29A-D suggests that 30% DOPE with 2-2.5% PEG lipid has the optimal combination of properties for systemic administration. Each experiment in Fig. 29 was conducted and the data obtained and analyzed according the methods described in EXAMPLE 1.

[0175] To further demonstrate the delivery capabilities of the LNP formulation, a new PepTAC designed against P-catenin based on P-catenin-TCF4 protein-protein interaction surface, was synthesized according to the standard peptide synthesis protocol, purified, and formulated with LNPs. The LNP -PepTAC was administered to DLD1 cells and a TOPFlash assay was performed as previously described. This assay shows -25% reduction in Wnt-transcriptional activity at doses of 200 nM or greater (FIG. 30). The data demonstrates LNP-based delivery of a PepTAC with a negative overall net charge of -6 at physiological pH. The sequence of the peptide-based degrader (N to C) used in this study is: IYP(OH)AL-Ahx- DELISFKDEGEQEERDLADVKSSLVN-NH2 (SEQ ID NO: 3) where P(OH) is a hydroxylated proline and Ahx is 6-aminohexanoic acid. The peptide was amidated at the C-terminus. These experiments were conducted and the data obtained and analyzed according the methods and teachings described in EXAMPLE 1.

[0176] Peptides that are acetylated at the N-terminus have been shown to display greater stability in vivo. To determine the effect of N-terminal acetylation on the biological function of the PepTACs, a TOPFlash assay performed with a ^|i-talPepTAC sequence: IYP^OHAL-Ahx- SQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 2), where the sequence has been modified with acetyl group at the N-terminus. The PepTAC was formulated with LNPs and administered to DLD1 cells. LNP -PepTAC treated cells show nearly 50% reduction in Wnt-transcriptional activity starting from 50 nM dosage (FIG 31). This data highlights another example of a chemically modified peptide being delivered by LNPs and exerting functional activity. These experiments were conducted and the data obtained and analyzed according the methods and teachings described in EXAMPLE 1.

[0177] Previous examples herein showed the effect of formulation pH on peptide uptake into cells. Here, we evaluated the effect of formulation pH on LNP-^CRPepTAC on the overall degradation of Luc-CREPT (Luciferase-fused CREPT protein) transiently expressed in HeLa cells. The data in FIG 32 demonstrates that the LNP formulation obtained using a pH 3 buffer is the most active in vitro. However, the data also shows formulations in pH 5 and pH 7 buffer still show activity. These experiments were conducted and the data obtained and analyzed according the methods and teachings described in EXAMPLE 1.

[0178] Previous examples herein showed the effect of different ionizable lipids on uptake efficiency of peptides. Here, we evaluated the effect of LNP-^CRPepTACs formulated with different ionizable lipids (MC3, ALC0315, SM102) on the overall degradation of Luc-CREPT (Luciferase-fused CREPT protein) transiently expressed in HeLa cells. The data in FIG. 33 shows that MC3, ALC-0315 and SM-102 ionizable lipids all show similar levels of Luc-CREPT protein degradation (-50%) across a broad concentration range. These experiments were conducted and the data obtained and analyzed according the methods described and teachings in EXAMPLE 1.

[0179] Although the present disclosure has been described with respect to one or more particular example(s), it will be understood that other examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A lipid nanoparticle comprising: i) one or more first lipid component(s), wherein the first lipid component(s) is/are independently ionizable cationic amino lipid(s); one or more second lipid component(s), wherein the second lipid component(s) is/are independently PEG-lipid(s); one or more third lipid component s) wherein the third lipid component(s) is/are independently phospholipid(s); one or more fourth lipid component(s), wherein the fourth lipid component(s) is/are independently sterol(s); optionally, one or more cationic lipid component(s), wherein the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof; or ii) one or more first combination lipid component(s), wherein the first combination lipid component(s) independently comprise one or more ionizable cationic amino lipid group(s); and one or more phospholipid group(s); one or more second lipid component(s), wherein the second lipid component(s) is/are independently PEG-lipid(s); one or more fourth lipid component(s) wherein the fourth lipid component s) is/are independently sterol(s); optionally, one or more cationic lipid component(s), wherein the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof; or iii) one or more second combination lipid component(s), wherein the second combination lipid component(s) independently comprise one or more ionizable cationic amino lipid group(s); and one or more sterol group(s); one or more second lipid component(s), wherein the second lipid component(s) is/are independently PEG-lipid(s); one or more third lipid component(s) wherein the third lipid component(s) is/are independently phospholipid(s); optionally, one or more cationic lipid component(s), wherein the cationic lipid component(s) independently comprises a cationic head group, a degradable group, and one or more lipid group(s); and one or more peptide(s), one or more functionalized peptide(s), or any combination thereof.

2. The lipid nanoparticle of claim 1, wherein the first lipid component(s) is/are chosen from ionizable cationic amino lipids, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

3. The lipid nanoparticle of claim 2, wherein the ionizable cationic amino lipid(s) is/are chosen from (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (D- Lin-MC3-DMA or MC3), l,2-dioleyloxy-3 -dimethylaminopropane (DODMA), l,2-dioleoyl-3- dimethylammonium-propane (DODAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol (DC Cholesterol), l,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP)), l,2-distearyloxy-3-dimethylaminopropane (DSDMA), l,2-dilinoleyloxy-3- dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-3 -dimethylaminopropane (DLenDMA), l,2-bis(linoleoyloxy)-3-dimethylaminopropane (DLinDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolan (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminopropyl-[l,3]-dioxolane (DLin- KC3-DMA), 2, 2-dilinoleyl-4-dimethylaminobutyl-[l,3]-di oxolane (DLin-KC4-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), 9-[4-(dimethylamino)-l- oxobutoxy]-heptadecanedioic acid, l,17-di-(2Z)-2-nonen-l-yl ester (L319), 3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol, Nl,N16-didodecyl-4,7,13-tris(3-(dodecylamino)- 3-oxopropyl)-4,7,10,13-tetraazahexadecanediamide (98ND12-5), l,l'-[[2-[4-[2-[[2-[bis(2- hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]bis-2- dodecanol (C12-200), salts thereof, structural and/or functional analogs thereof, and any combination thereof.

4. The lipid nanoparticle of claim 2, wherein one or more or all of the ionizable cationic lipid(s) is/are independently a pH-responsive lipid.

5. The lipid nanoparticle of claim 1, wherein the second lipid component(s) is/are chosen from l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (DMG-PEG-2000), distearoyl- rac-glycerol-PEG2K, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (18:0 PEG2000 PE), l,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (14:0 PEG2000 PE), N- palmitoyl-sphingosine- 1 - { succinyl [methoxy(polyethylene glycol)2000] } (C 16 PEG2000 Ceramide), N-palmitoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)5000]}(C16 PEG5000 Ceramide), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000] (DSPE-PEG(2000) Maleimide), N-myristoyl- sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (PEG-CerCi4)), N-arachidoyl- sphingosine-l-{ succinyl [methoxy(poly ethylene glycol)2000]} (PEG-CerC2o), polyethyleneglycol-succinoyl-distearoylglycerol (PEG-S-DSG), polyethyleneglycol-succinoyl- dimyristolglycerol (PEG-S-DMG), N-[(methoxy polyethylene glycol)2000)carbamoyl]-l,2- dimyristyloxlpropyl-3-amine (PEG-C-DMA), 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 ((m)PEG-DMG / (m)PEG-DMG-2000), polyethyleneglycoldi stearoylglycerol (PEG-DSG), R-3-[((o-m ethoxy polyethylene glycol)2000)carbamoyl]-l,2- dimyristyloxlpropyl-3-amine (PEG-DOMG / PEG-C-DOMG), alpha-(3' -{[1,2- di(myristyloxy)propanoxy] carbonylamino}propyl)-co-methoxy, polyoxyethylene (PEG-C- DMG-2000), salts thereof, structural and/or functional analogs thereof, and any combination thereof.

6. The lipid nanoparticle of claim 1, wherein the third lipid component(s) is/are chosen from (2R)-2,3-Bis(octadecanoyloxy)propyl 2-(trimethylazaniumyl)ethyl phosphate (DSPC), 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (16:0-18: 1 PC (POPC)), 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (10:0 PE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), N-(dodecanoyl)-sphing-4-enine-l -phosphocholine (SM), 1,2- diphytanoyl-sn-glycero-3-phosphatidylethanolamine (DPyPE), salts thereof, structural and/or functional analogs thereof, and any combination thereof.

7. The lipid nanoparticle of claim 1, wherein the fourth lipid component(s) is/are chosen from cholesterol, cholestanol, 7-dehydrocholesterol, cholestan-3-one, cholesteryl oleate stigmasterol, glucosyl stigmasterol, sitosterol, lanosterol, zymosterol, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

8. The lipid nanoparticle of claim 1, wherein the first combination lipid component(s) is/are chosen from aminophospholipids, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

9. The lipid nanoparticle of claim 1, wherein the second combination lipid component s) is/are chosen from 3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC Cholesterol), BHEM-Cholesterol, [3-(lH-imidazol-l-yl)propyl]carbamate, cholest-5-en-3p-ol, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)- 2,3,4,7,8,9,10,11,12, 13, 14, 15, 16,17-tetradecahydro-lH-cy cl openta[a]phenanthren-3-yl) (3- (dimethylamino)propyl) phosphate, salts thereof, structural and/or functional analogs thereof, and any combination thereof.

10. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle is a solid lipid nanoparticle.

11. The lipid nanoparticle of claim 1, the cationic head group(s) independently comprise a quaternary ammonium group, guanidinium group, amidinium group, imidazolium group, pyridinium group, pyrrolidinium group, a salt thereof, or a structural and/or functional analog thereof.

12. The lipid nanoparticle of claim 1, wherein the degradable group(s) independently comprise an ester group, a phosphate group, a disulfide group, an imine group, an amide group, a thioester group, an acetal or ketal group, a hydrazone group, a cis-aconityl group, an orthoester group, or a structural and/or functional analog thereof.

13. The lipid nanoparticle of claim 1, wherein the lipid group(s) independently comprise one or more C4 to Ceo aliphatic group(s) or a structural and/or functional analog thereof.

14. A lipid nanoparticle according to claim 1, wherein the cationic lipid component(s) is/are independently chosen from l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), didecyldimethylammonium bromide (DDAB), l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA), l,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(2- hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium bromide (DORI), 0,0’- ditetradecanoyl-N-(a-trimethylammonioacetyl)di ethanolamine chloride (DC-6-14), 1,2-stearoyl- 3-trimethylammonium-propane (chloride salt) (18:0 TAP), l,2-dipalmitoyl-3- trimethyl ammonium -propane (16:0 TAP), l,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18: 1 EPC), 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), salts thereof, structural and/or functional analogs thereof, and any combination thereof.

15. The lipid nanoparticle of claim 1, wherein the first lipid component s) is/are present at about 5 mol% (based on the total moles of the lipid components) to about 90 mol%; and/or the second lipid component(s) is/are present at about 0.1 mol% to about 20 mol% (based on the total moles of the lipid components); and/or the third lipid component(s) is/are present at about 5 mol% to about 60 mol% (based on the total moles of the lipid components); and/or the fourth lipid component(s) is/are present at about 5 mol% to about 80 mol% (based on the total moles of the lipid components); and/or the cationic lipid component(s), if present, is/are present at about 0.5 mol% to about 20 mol% (based on the total moles of the lipid components).

16. The lipid nanoparticle of claim 1, wherein the one or more peptide(s), the one or more functionalized peptide(s) is/are independently a therapeutic peptide or a therapeutic functionalized peptide.

17. The lipid nanoparticle of claim 1, wherein the one or more peptide(s), the one or more functionalized peptide(s), or any combination thereof independently comprise one or more hydrophobic domain(s).

18. The lipid nanoparticle of claim 1, wherein the one or more peptide(s), the one or more functionalized peptide(s) independently comprise i) a hydrophilic peptide sequence/sequences or group/groups and ii) a hydrophobic alkyl group and/or a hydrophobic peptide sequence/sequences or group/groups; or i) a hydrophobic peptide sequence/sequences or group/groups and ii) a hydrophobic group and/or a hydrophobic peptide sequence/sequences or group/groups; or i) a peptide sequence/sequences or group/groups and ii) a cleavable hydrophobic group and/or a hydrophobic peptide sequence/sequences or group/groups; or an amphiphilic sequence/sequences or group/groups.

19. The lipid nanoparticle of claim 1, wherein the one or more peptide(s), the one or more functionalized peptide(s) is/are independently a peptide mimetic, a D-peptide, P-peptides, gamma peptides, alpha/p peptides, sulfonyl-gamma peptides, a cyclic peptide, a peptoid, a peptide nucleic acid (PNA), or a stapled peptide.

20. The lipid nanoparticle of claim 1, wherein one or more or all of the one or more peptide(s), the one or more functionalized peptide(s) is/are independently a cationic peptide, a cationic functionalized peptide, an anionic peptide, an anionic functionalized peptide, a net neutral peptide, or a net neutral functionalized peptide.

21. The lipid nanoparticle of claim 1, wherein the one or more peptide(s), the one or more functionalized peptide(s) is/are independently chosen from protein degrading peptides, amphiphilic proteolysis targeting peptides, PEptide-based Proteolysis TArgeting Chimeras (PEPTACs), and structural and/or functional analogs thereof.

22. The lipid nanoparticle of claim 1, wherein the one or more peptide(s), the one or more functionalized peptide(s) is/are independently comprises: IYP^OHAL-Ahx- KDVLSEKEKKLEEYKQKLARV (SEQ ID NO: 1), IYP^OHAL-Ahx- SQEQLEHRERSLQTLRDIQRMLF (SEQ ID NO: 2), IYP^OHAL-Ahx- DELISFKDEGEQEERDLADVKSSLVN (SEQ ID NO: 3), PIYPALA-GSGS-QLLRHLILH (SEQ ID NO: 4), IYP^OHAL-Ahx-QLLRHLILH (SEQ ID NO: 5), wherein P^OH stands for hydroxylated proline and Ahx is 6-aminohexanoic acid, or a structural and/or functional analog thereof.

23. The lipid nanoparticle of claim 1, wherein the mass ratio of, if present, the first lipid component(s), the second lipid component(s), the third lipid component(s), the fourth lipid component(s), the cationic lipid component s), the first combination lipid component s), and the second lipid components to peptide(s), functionalized peptide(s), or any combination thereof is about 0.5 to about 50 wt/wt.

24. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle comprises a longest linear dimension of about 10 nanometers to about 1000 nanometers.

25. A composition comprising a plurality of lipid nanoparticles, wherein the lipid nanoparticles are independently a lipid nanoparticle of claim 1.

26. The composition of claim 25, wherein the composition is a solution or an aqueous dispersion.

27. The composition of claim 25, wherein the lipid nanoparticles are present at about 0.2 to about 40 lipid:peptide wt/wt ratio (based on the total weight of, if present, the first lipid component(s), the second lipid component(s), the third lipid component(s), the fourth lipid component(s), the cationic lipid component(s), the first combination lipid component(s), and the second combination lipid components and total weight of peptide(s), functionalized peptide(s), and any combination thereof).

28. The composition of claim 25, wherein the composition further comprises free peptide(s), free functionalized peptide(s), or any combination thereof.

29. The composition of claim 25, wherein the lipid nanoparticles comprise an average longest linear dimension of about 10 nanometers to about 1000 nanometers.

30. The composition of claim 25, wherein the lipid nanoparticles exhibit or the composition exhibits an isoelectric point of about pH 4 to about pH 10.

31. The composition of claim 25, wherein the composition comprises one or more pharmaceutical excipient(s).

32. The composition of claim 25, wherein the composition does not exhibit substantial or any observable lipid nanoparticle aggregation, a substantial change or any change in lipid nanoparticle size for at least one-week or more at a temperature of about 4 degrees Celsius (°C) or both.

33. A method of making lipid nanoparticles of claim 1 comprising: contacting a first composition comprising, if present, the first lipid component(s), the second lipid component(s), the third lipid component(s), the fourth lipid component s), the cationic lipid component(s), the first combination lipid component(s), and the second lipid components and one or more organic solvent(s) and a second composition comprising one or more peptide(s), one or more functionalized peptide(s), or any combination thereof, wherein the lipid nanoparticles are formed; optionally, removing residual organic solvent(s) after lipid nanoparticle formation; and dialyzing the lipid nanoparticle formulation against water or a pH buffered solution.

34. The method of claim 33, wherein the volume ratio of a first composition to a second composition is about 0.02 to about 2.

35. The method of claim 33, wherein the mass ratio of first composition to second composition is about 0.2 to about 40.

36. The method of claim 33, the method further comprising isolating the lipid nanoparticles.

37. A method of peptide, functionalized peptide, or any combination thereof delivery comprising: contacting a population of cells or an individual, with a plurality of lipid nanoparticles, wherein the lipid nanoparticles are independently a lipid nanoparticle of claim 1, wherein at least a portion of or all of the lipid nanoparticles are delivered to the population of cells or the individual.

38. The method of claim 37, wherein the contacting is in vitro or in vivo.

39. The method of claim 37, wherein the method comprises treating, preventing, or any combination thereof, a current or potential disease, disease state, condition, disorder, side effect, or any combination thereof, in the individual.

40. The method of claim 37, wherein the contacting comprises administration of the plurality of lipid nanoparticles to an individual.

41 . The method of claim 37, wherein at least a portion, substantially all, or all the lipid nanoparticles(s) is/are taken up by a cell or cells of the population of cells or the individual, and the lipid nanoparticles is/are is released within the cell or cells.

42. The method of claim 37, wherein the peptide(s), the functionalized peptide(s), or any combination thereof after delivery independently retain/retains substantially all (or all) structural features and/or 50% or more, 70% or more, 90% or more, or 95% or more of its biological activity compared with the native peptide(s) or the peptide(s) or the functionalized peptide(s) delivered without use of the lipid nanoparticle of claim 1.

43. The method of claim 37, wherein the individual is diagnosed with and/or is in need of treatment for a disease, disease state, and the disease, the disease state is treatable, preventable, by the peptide(s), the functionalized peptide(s), or the combination thereof.