Approaches for optimization and discovery of nanoparticles and uses thereof

Abstract

Disclosed herein are methods for rapidly screening polymer nanoparticles by exposing a cell to the polymer nanoparticle, the polymer nanoparticle comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; and sorting the exposed cell by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein via flow cytometry; and evaluating the polymer nanoparticle for its ability to deliver the mRNA to the cell and express the protein based on the presence or absence of the signals. Novel polymer nanoparticles, in particular, lipid nanoparticles are also provided herein.

Description

Attorney Docket No.5470.971.WO APPROACHES FOR OPTIMIZATION AND DISCOVERY OF NANOPARTICLES AND USES THEREOF REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No.63 / 556,581, filed February 22, 2024, the entire contents of which are incorporated by reference herein. FIELD OF INVENTION

[0002] The invention relates to methods of identifying polymer nanoparticles (e.g., lipid nanoparticles) with increased endosomal escape and / or improved delivery and translation of nucleic acids. The invention further relates to novel polymer nanoparticles for nucleic acid delivery to cells in vitro, ex vivo, and in vivo. BACKGROUND

[0003] Although delivery vectors have been developed to deliver molecules such as RNA into cells, compositions for delivery with improved efficacy, including compositions optimized for specific applications, for example, types of cells, disease, or delivery methods, are difficult to identify. There is a need in the art for methods to rapidly screen the activity of nanoparticles with varying chemistries and / or in different applications. SUMMARY OF THE INVENTION

[0004] The present invention is based, in part, on the development of a strategy for rapidly identifying molecular entities (RIME) to screen the activity of nanoparticles, allowing for rapid screening of the activity of nanoparticles with multicomponent variable chemistries, and allowing for optimization of nanoparticles for specific types of applications. The present invention also relates to optimized lipid nanoparticles identified by the screening methods.

[0005] In an aspect, a method of evaluating a polymer nanoparticle is provided, the method comprising: exposing a cell to the polymer nanoparticle, the polymer nanoparticle comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; sorting the exposed cell by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein;Attorney Docket No.5470.971.WO and evaluating the polymer nanoparticle for its ability to deliver the mRNA to the cell and express the protein based on the presence or absence of the signals.

[0006] In another aspect, a method of identifying a polymer nanoparticle with increased endosomal escape ability is provided, the method comprising: exposing a plurality of cells to a candidate polymer nanoparticle, the candidate polymer nanoparticle comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; sorting the exposed cells by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein; quantifying the cells comprising both the detectably labeled mRNA and the detectable protein; and assessing the quantification of the cells relative to cells exposed to a reference polymer nanoparticle, to thereby identify the polymer nanoparticle with increased endosomal escape ability.

[0007] In an aspect, a method of comparing the ability of polymer nanoparticles to deliver an mRNA and express a protein in a cell is provided, comprising: exposing a plurality of cells to the polymer nanoparticles, each polymer nanoparticles comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; sorting the exposed cells by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein of each cell; and comparing each polymer nanoparticles for its ability to deliver the mRNA to the cell and express the protein based on the present or absence of the signal.

[0008] In another aspect, a lipid nanoparticle (LNP) is provided, comprising an ionizable lipid, a phospholipid, a sterol, and / or a PEG lipid, and one or more excipients selected from octaarginine, cyclosporine, calcium phosphate, or TAT (48-57) peptide.

[0009] In an aspect, a LNP is provided comprising a detectably labeled mRNA, the mRNA encoding a detectable protein, and an mRNA encoding a protein of interest.

[0010] In an aspect, a LNP is provided comprising a detectably labeled mRNA, the mRNA encoding a detectable protein, and a nucleic acid barcode.

[0011] These and other aspects of the invention are set forth in more detail in the description of the invention below. BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG.1. Schematic illustration of an exemplary approach for rapidly identifying molecular entities (RIME) to screen the activity of LNPs.Attorney Docket No.5470.971.WO

[0013] FIG. 2. Schematical illustration of the formulation of mRNA encapsulated lipid nanoparticles (LNPs) via microfluidic chip.

[0014] FIGS. 3A-3B. Original gating to quantify Cy5-EGFP expression at 6 hours (FIG. 3A) and 24 hours (FIG.3B) for HepG2 cells.

[0015] FIGS.4A-4B. In vitro evaluation methods of mRNA LNPs. Cell viability (FIG.4A) and in vitro FLuc expression (FIG. 4B) of mRNA LNPs with Moderna control under 100 μL 500 ng / mL of total FLuc mRNA doses on HepG2 cells for 24 hours (Data are shown for triplicates).

[0016] FIGS. 5A-5G. LNP formulation characterization. Size / diameter (FIG. 5A), PDI (FIG. 5B), zeta potentials (FIG. 5C), and mRNA encapsulation efficiency (FIG.5D) of mRNA LNPs (the data presented in FIGS. 5A-5D are shown as mean ± standard deviation, n = 3). FIG. 5E. Schematic diagram of stability and FLuc expression of mRNA LNPs with different excipients and possible interactions among mRNA, lipids, and various excipients. FIG. 5F, The stability of different mRNA LNPs after incubation in pH 4, 5, 6 and 7.4 of DMEM + 10% FBS + 1% penicillin–streptomycin (DMEM) or RPMI + 10% FBS + 1% penicillin–streptomycin (RPMI) solutions at either 4°C or 37°C for 5 days. Stability was indicated by the size change ratio of day 5 relative to day 0. The horizontal line represents a size change ratio of 1, indicating no size change after 5 days of incubation and, thus, demonstrating the mRNA LNPs are stable under these conditions. FIG.5G, Heat-map format of ratio of the size change of LNPs with different excipients after incubation in 100 mM urea, Tween® 20 and NaCl solution over 48 hours.

[0017] FIGS. 6A-6D. Cell viability (FIG. 6A) and in vitro FLuc expression (FIGS. 6B-6C). Normalized in vitro FLuc expression of mRNA LNPs with Moderna control under 100 μL 500 ng / mL of total FLuc mRNA doses on HepG2 cells over desired time (day 0, 7, 14 and 21) (FIG. 6B). The percentage of in vitro FLuc expression over desired time (7, 14 and 21) as compared to day 0 (Data are shown for triplicates) (FIG.6C). FIG.6D, FLuc expression of LNPs with different excipients under 50 ng, 100 ng and 200 ng FLuc doses across HepG2 cells for 24 hours. Octaarg, Octaarginine. Cyclo, cyclosporine.

[0018] FIGS. 7A-7C. In vitro evaluation methods of mRNA LNPs. Cell viability (FIG. 7A), FLuc expression (FIG.7B), and cell uptake (FIG.7C) of mRNA LNPs. HepG2 cells were treated with mRNA LNPs for 24 hours at 37°C (the data presented in FIGS.7A-7C are shown as mean ± standard deviation, n = 3).Attorney Docket No.5470.971.WO

[0019] FIG.8. Schematic illustration of overall goal and simple and fast exemplary approach to screen the activity of LNPs with flow cytometry.

[0020] FIGS. 9A-9D. Cell sorting and visualization of HepG2 cells treated with Cy5-EGFP mRNA LNPs. FIG.9A, Representative data of EGFP signals versus Cy5 signals on HepG2 cells incubated in the absence (control) or presence of mRNA LNPs for 24 hours at 37°C. Q1 represents the EGFP(+) and Cy5(+) signal (mRNA LNPs uptake and EGFP expression), Q2 represents the EGFP(+) and Cy5(−) signal (this is an empty gate, which serves as a control in which we should not see cells if the assay is working correctly), Q3 represents the EGFP(−) and Cy5(−) signal (no mRNA LNPs and no EGFP expression), Q4 represents the EGFP(−) and Cy5(+) signal (mRNA LNPs uptake and no EGFP expression). FIG.9B, A heat map summary of cell uptake and EGFP expression of HepG2 cells treated with Cy5-EGFP mRNA LNPs for 24 hours at 37°C (the data presented in FIG. 9B are shown as the mean ± standard deviation, n = 3). FIG. 9C, Schematic demonstrating confocal imaging. Cell Nuclei, Cy5 labeled mRNA encapsulated LNP and EGFP protein are indicated. FIG.9D, Representative confocal images of HepG2 cells treated with Cy5- EGFP mRNA LNPs for 24 hours at 37°C. Scale bars, 20 μm. The gating of the data in FIG.9A is shown in FIGS 3A-3B. Octaarg, Octaarginine. Cyclo, cyclosporine.

[0021] FIGS. 10A-10C. FIG. 10A, Representative data of EGFP signals vs. Cy5 signals on HepG2 cells incubated in the absence (control) or presence mRNA LNPs for 6 hours at 37°C. Q1 represents EGFP (+) and Cy5 (+) signal (mRNA LNPs uptake and EGFP expression), Q2 represents EGFP (+) and Cy5 (-) signal (false), Q3 represents EGFP (-) and Cy5 (-) signal (no mRNA LNPs and no EGFP expression), Q4 represents EGFP (-) and Cy5 (+) signal (mRNA LNPs uptake and no EGFP expression). FIG. 10B, Heat-map summary of cell uptake and EGFP expression of HepG2 cells treated with Cy5-EGFP mRNA LNPs for 6 hours at 37°C. FIG.10C, Representative data of EGFP signals vs. Cy5 signals on HepG2 cells incubated in the absence (control) or presence mRNA LNPs with Moderna control for 24 hours at 37°C. Octaarg, Octaarginine. Cyclo, cyclosporine.

[0022] FIG. 11. A schematic illustration of endosomal escape studies using a lysotracker confocal imaging assay.

[0023] FIGS.12A-12D. Mechanism investigation methods. FIG.12A, Representative confocal images of HepG2 cells treated with Atto-488-labeled mRNA LNPs. The endosomes are stained with Lysotracker Deep Red. Nuclei are stained with Hoechst 33342. Scale bars, 20 μm. FIG.12B,Attorney Docket No.5470.971.WO A PCC analysis of Atto-488-labeled mRNA LNPs treated with HepG2 cells (the data are presented as the mean ± standard deviation, and five representative cell images (>20 cells) were used to calculate the PCC value). FIG. 12C, A schematic illustration of endosomal escape mechanism studies using a calcein leakage confocal imaging assay with bafilomycin A1to test the ‘proton sponge effects.’ FIG.12D, Representative confocal images of HepG2 cells incubated with calcein or calcein and mRNA LNPs in the absence (top) and presence (bottom) of inhibitor bafilomycin A1for 4 hours at 37°C. Scale bars, 20 μm. Octaarg, Octaarginine. Cyclo, cyclosporine.

[0024] FIGS.13A-13B. Representative confocal images of HepG2 cells incubated with calcein or calcein and mRNA LNPs with nuclei and cytoskeletons staining in the absence (FIG.13A) or presence (FIG.13B) of inhibitor bafilomycin A1for 4 hours at 37°C are shown. Nuclei are stained with Hoechst 33342 and cytoskeletons (Cyto) are stained with CellMask™ Deep Red Actin Tracking Stain. Scale bars are 20 μm. Octaarg, Octaarginine. Cyclo, cyclosporine.

[0025] FIGS.14A-14H. In vivo evaluation methods of mRNA LNPs. mRNA LNPs at a dose of 0.65 mg / kg total mRNA were intravenously injected into mice (6–8 weeks old, weight 18-21 g). After 24 hours, the organs were imaged by IVIS imaging. The blood was collected to run the blood toxicity. The organs were collected to run histology. The weight was monitored to quantify the acute toxicity. FIG. 14A, Bioluminescence images of mRNA LNPs after 24 hours in various organs ex vivo for each group via IV administration. The mice injected with naked FLuc mRNA and only PBS served as controls. FIG.14B, Bioluminescence images of mRNA Moderna LNPs after 24 hours in various organs ex vivo for each group via I.V. administration. Mice injected with naked FLuc mRNA and only PBS served as controls. FIGS.14C-14D, Total luminescence (FIG. 14C) and associated percent of bioluminescence (FIG. 14D) of mRNA LNPs across various organs including the pancreas, spleen, liver, kidneys, uterus / ovaries, lung and heart. FIG. 14E, Human EPO concentrations after the injection of EPO mRNA LNPs for 24 hours. The mice injected with naked EPO mRNA and only PBS served as control. The concentration of human EPO was characterized by human EPO ELISA kits following the manufacturer’s protocol. FIG. 14F, Representative histology images of the liver, spleen and lung of mice after treatment with RNA LNPs via IV injection. Scale bars, 100 μm. FIG.14G, ALP, ALT, AST, BUN and creatinine (CREAT) blood testing results after the IV injection of mRNA LNPs. (The data presented in FIGS. 14B-14E and FIG.14G are shown as the mean ± standard deviation, n = 3). FIG.14H, PrincipalAttorney Docket No.5470.971.WO Component Analysis Plots for representative formulations. Octaarg, Octaarginine. Cyclo, cyclosporine.

[0026] FIGS. 15A-15B. Percent weight gain reported as mean ± SD 24 hours after respective intravenous dose of FLuc mRNA LNPs (FIG. 15A) or EPO mRNA (FIG. 15B) encapsulated LNPs into mice (data are shown for triplicates).

[0027] FIGS. 16A-16J. FIG. 16A, EGFP signals vs. Cy5 signals on DC2.4 cells incubated in the absence (control) or presence of LNPs with various excipients for 24 hours at 37°C. Q1 represents EGFP (+) and Cy5 (+) signal (LNPs were taken up and express EGFP), Q2 represents EGFP (+) and Cy5 (-) signal (false), Q3 represents EGFP (-) and Cy5 (-) signal (LNPs were not taken up and do not express EGFP), Q4 represents EGFP (-) and Cy5 (+) signal (LNPs were taken up but do not express EGFP). FIG.16B, Heat-map format of cell uptake, EGFP expression and expression ratio of DC2.4 cells treated with Cy5-EGFP mRNA LNPs with various excipients for 24 hours at 37°C. (All data presented as mean ± SD, n = 3). FIG.16C, Representative confocal images of DC2.4 cells treated with Cy5-EGFP mRNA LNPs with various excipients for 24 hours at 37°C. Scale bars are 20 μm. FIG.16D, FLuc expression of LNPs with different excipients under 50 ng, 100 ng and 200 ng FLuc doses across DC2.4 cells for 24 hours. Cellular association (FIG. 16E) and geometric mean fluorescence intensity (GMFI; FIG. 16F) of DC2.4 cells treated with 1000 ng / mL of LNPs with different excipients for 24 hours at 37°C. (All data presented as mean ± SD, n = 3). FIG.16G, Pearson Correlation Coefficient (PCC) analysis of ATTO-488 labelled LNPs with different excipients treated with DC2.4 cells. (Data presented as the mean ± SD, N > 50, ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 with 95% of confidence level from unpaired t-test). FIG. 16H, Representative confocal images of DC2.4 cells treated with 1000 ng / mL of ATTO-488 labelled LNPs with different excipients. Endo / lysosomes were stained with LysoTracker™ Deep Red. Nuclei were stained with Hoechst 33342. Scale bars are 10 µm. FIG. 16I, Representative luminescence biodistribution of LNPs, TAT LNPs and ATP LNPs encapsulated with FLuc mRNA ex vivo (n = 3) for each group via intramuscular (I.M.) administration. Mice injected with only PBS served as negative control. FIG. 16J, Associated percent of bioluminescence of LNPs, TAT LNPs and ATP LNPs encapsulated with FLuc mRNA across various organs including pancreas, spleen, liver, kidneys, uterus / ovaries, lung, and heart. Octaarg, Octaarginine. Cyclo, cyclosporine.Attorney Docket No.5470.971.WO DETAILED DESCRIPTION

[0028] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0030] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0031] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0032] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0033] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0034] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.Attorney Docket No.5470.971.WO

[0035] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0036] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0037] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0038] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0039] The term “consists essentially of” (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C-terminal ends of the recited sequence or additional nucleotides on the 5’ and / or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g., chaperone and / or isomerase activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0040] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0041] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. TheAttorney Docket No.5470.971.WO following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized), synthetic RNA of any sequence (e.g., chemically synthesized), nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acid molecules that have altered base-pairing abilities or increased resistance to nucleases.

[0042] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0043] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0044] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0045] The term “contact” or grammatical variations thereof refers to bringing two or more substances in sufficiently close proximity to each other for one to exert a biological effect on the other.

[0046] It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. TheAttorney Docket No.5470.971.WO permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Non-limiting examples of optional substituents as referred to herein include halogen, alkyl, aralkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, amido, nitro, cyano, amido, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aryl, and heteroaryl.

[0047] As used herein, the term “alkyl”, used either alone or in compound words such as “haloalkyl” includes straight-chain or branched C1-C20alkyl, such as methyl, ethyl, n-propyl, i- propyl, or the different butyl, pentyl or hexyl isomers, etc.

[0048] As used herein, the term “alkenyl”, used either alone or in compound words such as “haloalkenyl” includes straight-chain or branched C1-C20alkyl containing at least one double bond.

[0049] As used herein, the term “alkynyl”, used either alone or in compound words such as “haloalkynyl” includes straight-chain or branched C1-C20alkyl containing at least one triple bond.

[0050] As used herein, “unsaturated” refers to compounds or structures having at least one degree of unsaturation (e.g., at least one double or triple bond).

[0051] Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring can also be designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring, and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis / trans.”

[0052] All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless specific stereochemistry or isomeric form is specifically indicated. All processes used to prepare compounds and intermediates made therein are encompassed by the present disclosure. All tautomers of shown or described compounds are also encompassed by the present disclosure.

[0053] When any variable (e.g., Ri) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with one or more Ri moieties,Attorney Docket No.5470.971.WO then Ri at each occurrence is selected independently from the Markush group recited for Ri. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds within a designated atom’s normal valency.

[0054] A “subject” may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, lagomorph (e.g., rabbit), or rodent (e.g., mouse, rat). In some embodiments a human subject is a neonate, child, adult, or geriatric subject.

[0055] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.Attorney Docket No.5470.971.WO

[0056] “Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome, or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered, e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0057] Sorting cells as used herein can include filtering, gating, analyzing, targeting, isolating and / or separating cells, including cell populations and / or subpopulations. Sorting may be based, for example, upon signal characteristics and / or measurements, e.g., detectable characteristics such as light scattering, fluorescence, including measurement of correlated data, distribution of measurements within cellular population and / or subpopulation and interrelationships of the measurements and / or parameters to, for example, separate and / or group cells based on characteristics such as size, volume, surface markers, granularity, emission of fluorescence or other detectable markers.

[0058] The present invention relates to a method of evaluating a polymer nanoparticle, the method comprising: exposing a cell to the polymer nanoparticle, the polymer nanoparticle comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; sorting theAttorney Docket No.5470.971.WO exposed cell by the presence or absence of a signal from the detectably labeled mRNA (e.g., a first signal) and the detectable protein (e.g., a second signal); and evaluating the polymer nanoparticle for its ability to deliver the mRNA to the cell and express the protein based on the presence or absence of the signals (i.e., the first signal and second signal). Advantageously, presence of a signal from the detectably labeled mRNA indicates uptake of the nanoparticle by the cell. Presence of a signal of an expressed protein encoded by the mRNA indicates cells in which the polymer nanoparticle has escaped the endosomes and resulted in protein expression from the mRNA. Accordingly, presence of a both the positive signal of the detectably labeled mRNA and the positive signal of the detectable protein indicates both LNP uptake and protein expression, allowing measurement by signal of the functionality of the nanoparticle, e.g., highest cellular uptake and protein expression.

[0059] While the invention is described in terms of using flow cytometry, one of skill in the art understands that alternative signal measurement techniques may be used as long as they are capable of detecting the detectably labeled mRNA and the detectable protein. In some embodiments, confirmation of the uptake and expression can be performed, for example, by confocal microscopy or other confirmatory imaging.

[0060] Exemplary flow cytometry methods include, but are not limited to, flow cytometry with a cell sorter that allows collection of samples for further analyses, Fluorescence-Activated Cell Sorting (FACS), imaging flow cytometry, mass cytometry, and acoustic focusing cytometry.

[0061] Polymer nanoparticles (PNPs) can comprise polymers generally formed by spontaneous self-assembly of surfactant directed polymers to form the PNPs. The size of PNPs is typically in the range of about 1 to about 1000 nm. PNPs can comprise natural polymers (e.g., alginate, albumin, gelatin), synthetic polymers including block copolymers and ionic polymers, and any combination thereof, include functionalized polymer-based materials such as a calcium phosphate silicate nanoparticle, a calcium phosphate nanoparticle, a silica nanoparticle, and poly(amido- amine), poly-beta amino-esters (PBAEs), and polyethylenimine (PEI). Hydrogels from proteins (e.g., collagen, gelatin, fibrin) and polysaccharides (e.g., alginate, chitosan, agarose, and hyaluronic acid) as well as synthetic hydrogels from PEG, poly(vinyl alcohol) and poly(acrylic acid) can be utilized in the present invention. See, e.g., Wan (2012) Polymers 4(2):1084-1108, incorporated herein by reference.Attorney Docket No.5470.971.WO

[0062] In an embodiment, the polymer nanoparticle is a lipid nanoparticle (LNP). LNPs comprise an ionizable lipid, a phospholipid, a sterol, and / or a PEG molecule, and optionally one or more excipients. Example LNPs include those disclosed in Schoenmaker et al. (2021) Int’l J. Pharma. 601:120586, and Hou et al. (2021) Nat. Rev. Mater. 6:1078-1094, each of which is incorporated herein by reference. Additional exemplary nanoparticles that can be used in the invention include those described, for example, in U.S. Provisional Application Nos.63 / 496,140 filed April 14, 2023, and 63 / 384,168 filed November 17, 2022.

[0063] In an embodiment, the LNP comprises one or more ionizable lipids, e.g., 1, 2, 3, 4, or 5 or more ionizable lipids. In an aspect, the ionizable lipid is an unsaturated, multi-tail, polymeric, biodegradable, or branched-tail ionizable lipid. See, Han et al. (2021) Nat. Comm. 12:7233, Figures 1 and 2, incorporated herein by reference. Example ionizable lipids include 1,2-dilinoleyl- N,N-dimethyl-3-aminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane (DLin-KC2-DMA), and dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3- DMA). The ionizable lipid can preferably be selected from 9-heptadecanyl 8-{(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102) and / or [(4- hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)(ALC-0315).

[0064] In an embodiment, the LNP comprises one or more phospholipids, e.g., 1, 2, 3, 4, or 5 or more phospholipids. In an aspect, the phospholipid is dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), or any combination thereof.

[0065] In an embodiment, the LNP comprises one or more PEG lipids, e.g., 1, 2, 3, 4, or 5 or more PEG lipids. The amount of PEG lipid can be tuned to adjust particle size, particle stability and aggregation, and zeta potential. Exemplary PEG lipids may comprise varying PEG lengths. In an aspect, the PEG is a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy- (polyethylene glycol)] of varying lengths, for example, 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy-(polyethylene glycol)-350] (ammonium salt) (C14-PEG350), C14-PEG1000, C14-PEG2000, C18-PEG2000, C14-PEG3000, or any combination thereof. In an aspect, the PEG lipid is C14-PEG-2000, i.e., 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy-(polyethylene glycol)-2000] (ammonium salt).

[0066] In some embodiments, the one or more excipients of the nanoparticle (e.g., polymer nanoparticle or LNP) is a small molecule, a biologic, or an inorganic compound.Attorney Docket No.5470.971.WO

[0067] Small molecules can include, but are not limited to, chemotherapeutic agents, anti- oncogenic agents, anti-microbial agents (e.g., antibiotics, antivirals, antifungals, and antiparasitics), non-peptidic hormones, other pharmaceutically active substances, and the like. Biologics, substances made in, extracted from, and / or synthesized in, in whole or in part, a living organism may also be used as an excipient of the nanoparticles. Example biologics include, but are not limited to, proteins (e.g., antibodies), sugars, and any combination thereof, or may be a living entity such as a cell or tissue. Inorganic compounds, include, for example, compounds comprising two or more non-carbon elements, for example, tricalcium phosphate, ammonia, metals.

[0068] In some embodiments, the one or more excipients of the nanoparticle (e.g., polymer nanoparticle or LNP) is a hydrophobic (e.g., non-polar, liophilic), hydrophilic (e.g., ionic and / or polar), or amphiphilic molecule.

[0069] In some embodiments, the LNP comprises about 15 mol% to about 95 mol% (e.g., about 20 mol% to about 70 mol%, about 25 mol% to about 60 mol%, about 30 mol% to about 50 mol%) ionizable lipid, about 5 mol% to about 30 mol% (e.g., about 5 mol% to about 25 mol%, about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 5 mol% to about 10 mol%) phospholipid, about 20 mol% to about 60 mol% (e.g., about 22 mol% to about 55 mol%, about 24 mol% to about 50 mol%, about 26 mol% to about 45 mol%) sterol, about 1 mol% to about 5 mol% (e.g., about 1 mol% to about 4 mol%, about 1 mol% to about 3 mol%, about 1 mol% to about 2 mol%) PEG lipid, and optionally an excipient at about 0.05 mol% to about 35 mol% (e.g., about 0.05 mol% to about 10 mol%, 0.2 mol% to about 10 mol%, about 2 mol% to about 35 mol %). In one embodiment, the LNP comprises ionizable lipid in the range of about 15 mol% to about 95 mol%, phospholipid in the range of about 5 mol% to about 30 mol%, sterol in the range of about 20 mol% to about 60 mol%, and / or a PEG lipid in the range of about 1 mol% to about 5 mol%.

[0070] In some embodiments, the detectable label can include, but is not limited to, a fluorescent label, chemiluminescent label, electrochemiluminescent label, a bioluminescent label, an isotopic label, or radiolabel. Detectable labels suitable for use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Example radiolabels include, for example,3H,125I,35S,14C, or32P. Example fluorescent labels include, but are not limited to, a cyanine (e.g., Cy2m Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 Cy7),Attorney Docket No.5470.971.WO rhodamine, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Allophycocyanin (APC), Peridinin-Chlorophyll-Protein (PerCP), Alexa Fluor®, or DyLight® dye.

[0071] Example detectable proteins include, but are not limited to, enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), Venus, monomeric Infrared Fluorescent Protein (mIFP), Long Stokes Shift monomeric Orange (LssmOrange), Tag Red Fluorescent Protein 657 (TagRFP657), monomeric Apple (mApple), monomeric Orange2 (mOrange2), Sapphire, monomeric Tag Blue Fluorescent Protein (mTagBFP2), tdTomato, monomeric Cherry (mCherry), monomeric Ruby (mRuby), and monomeric Cerulean3 (mCerulean3).

[0072] In embodiments, the detectable protein is a protein that fluoresces when exposed to light. In some embodiments, the detectably labeled mRNA and the detectable protein emit signals at different wavelengths so the two signals (e.g., a first signal and a second signal) can be distinguished, for example at least a 10 nm difference in wavelength, 20 nm difference in wavelength, 30 nm difference in wavelength, 40 nm difference in wavelength, 50 nm difference in wavelength, or more. In some embodiments, the detectable label emits at a wavelength in the range of about 350 nm and about 800 nm, i.e., spectral range used in flow cytometry.

[0073] Advantageously, a plurality of LNPs can be evaluated. In some embodiments, each LNP has a different formulation. For example, the formulation may vary in one or more components of the composition, e.g., type of phospholipid, and / or may vary in amounts of the components of the composition. In some embodiments, a plurality of LNPs can be evaluated with a plurality of cells.

[0074] In some embodiments, the method comprises exposing a plurality of cells to a nanoparticle (e.g., LNP or polymer nanoparticle). In some embodiments, the plurality of cells comprise more than one cell type. In some embodiments, the methods can comprise evaluating the cell preference of polymer nanoparticles by exposing the nanoparticles to a variety of cell types. Cell types can include cells based on cell state, tissue type, cancer type, surface marker, or other category. In an embodiment, cells are sorted (e.g., gated) in the methods of the present invention. As used herein, cell sorting can be based on gating approaches used in cytometry, for example, sequential selection of populations of cells which may include factors such as characteristic size, granularity, and expression of various cell markers. In some embodiments, the method further comprises assaying viability of the cell, which may be performed by assays known in the art. In embodiments, the assaying viability of the cell comprises incubating a plurality of cells with PNPsAttorney Docket No.5470.971.WO comprising an mRNA encoding a detectable label, e.g., firefly luciferase, and measuring absorbance relative to untreated cells or other reference standard.

[0075] In some embodiments, methods can comprise evaluating a type of cell with a plurality of different types of polymer nanoparticles, e.g., candidate polymer nanoparticles, to determine cell preference of a polymer nanoparticle. In some embodiments a cell type is exposed to a plurality of polymer nanoparticles varied in composition of the nanoparticle and / or its contents. The same cell type can be seeded across wells of a multiwell plate (or injected into a subject for in vivo procedures), with each well (or subject) incubated with a different nanoparticle and subsequently evaluated for presence of both mRNA detectable label and expression of a detectable protein. The presence of both detectable signals as well as the quantification of the detectable signals can determine particles capable of effective delivery to the cell and / or cell preference for nanoparticles. Reference nanoparticles can be utilized for baseline measurements, with detectable signals compared relative to cells exposed to candidate polymer nanoparticles, to thereby identify one or more polymer nanoparticles with desirable characteristics in the cell.

[0076] Exposing cells to a nanoparticle, e.g., contacting a cell or plurality of cells with a nanoparticle, may occur in vivo, ex vivo, or in vitro. Exposing can comprise seeding cells on a solid surface substrate, for example, a multiwell plate, and delivering nanoparticles to the cells. In some embodiment, exposing can comprise injecting polymer nanoparticles into a subject, for example by administering in vivo (e.g., via intravenous, intramuscular, intraperitoneal injection). In some in vitro embodiments, the cells are seeded at 10, 102, 103, 104, 105, 106, or 107cells per well, for example, in the range of about 50 to about 1,500,000 cells per well. The nanoparticles can be delivered to the plurality of cells, for example, by adding an aliquot of nanoparticles to each well, and incubated prior to evaluation of the cells. In some embodiments, the exposed cells are sorted via high-throughput platforms by flow cytometry and / or other high-throughput screening platforms (e.g., high-content microscopy) allowing for rapid screening of multiple samples and identification of polymer nanoparticles with desirable characteristics in a particular cell type and / or cell environment. In some embodiments, a target cell is an insect, plant, animal, fungal (e.g., yeast) or bacterial cell. In some embodiments, the administration is to a cell in a subject.

[0077] In another aspect, a method of identifying a polymer nanoparticle with increased endosomal escape ability is provided, the method comprising: exposing a plurality of cells to a candidate polymer nanoparticle, the candidate polymer nanoparticle comprising a detectablyAttorney Docket No.5470.971.WO labeled mRNA, the mRNA encoding a detectable protein; sorting the exposed cells by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein; quantifying the cells comprising both the detectably labeled mRNA and the detectable protein; and assessing the quantification of the cells relative to cells exposed to a reference polymer nanoparticle, to thereby identify the polymer nanoparticle with increased endosomal escape ability. In some embodiments, the exposed cells are sorted by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein via flow cytometry. A reference polymer nanoparticle can comprise, for example, a known polymer nanoparticle in the art, or polymer nanoparticle comprising the same components, but in different amounts, or without one or more excipients being evaluated.

[0078] In an aspect, a method of comparing the ability of polymer nanoparticles to deliver an mRNA and express a protein in a cell is provided, comprising: exposing a plurality of cells to the polymer nanoparticles, each polymer nanoparticle comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; sorting the exposed cells by the presence or absence of a signal from the detectably labeled mRNA (e.g., a first signal) and the detectable protein (e.g., a second signal) of each cell; and comparing each polymer nanoparticle for its ability to deliver the mRNA to the cell and express the protein based on the presence or absence of the signals (i.e., the first and second signals). In some embodiments, comparing the ability of polymer nanoparticles to deliver an mRNA and express a protein in a cell comprises sorting the exposed cells by the presence or absence of a signal from the detectably labeled mRNA (e.g., providing a first signal) and the detectable protein (e.g., providing a second signal) via flow cytometry.

[0079] In another aspect, a lipid nanoparticle (LNP) is provided, comprising an ionizable lipid, a phospholipid, a sterol, and / or a PEG lipid, and one or more excipients selected from octaarginine, cyclosporine, calcium phosphate, or TAT (48-57) peptide.

[0080] In an aspect, a LNP is provided comprising a detectably labeled mRNA, the mRNA encoding a detectable protein, and an mRNA encoding a protein of interest. In some embodiments, the mRNA encoding a protein of interest can be provided with the LNP to measure expression of the protein of interest relative to the detectable protein. The LNPs comprising an mRNA encoding a protein of interest can be evaluated using the methods described herein to identify nanoparticles, including evaluating a plurality of different LNPs comprising the mRNA encoding a protein of interest.Attorney Docket No.5470.971.WO

[0081] In an aspect, a LNP is provided comprising a detectably labeled mRNA, the mRNA encoding a detectable protein, and a nucleic acid barcode. The term “barcode” means an oligonucleotide present in a nucleic acid sequence in order to identify it, which can be identified through approaches known in the art, including, for example, sequencing. The barcode is preferably from 4 to 30 nucleotides long, for example 8 to 12 nucleotides long and can be included as a part of the detectably labeled mRNA or can be provided as a separate nucleic acid in the nanoparticle. A barcode may be included, for example, for identification of a cell that may be subject to further processing, assays, or evaluation, thereby allowing identification of the cell and / or its contents via the barcode sequence.

[0082] The methods herein can be used to identify particles having desirable characteristics, allowing a protein or functional RNA to be expressed in a cell, e.g., to provide a functional nucleic acid to a cell in vitro or in vivo. Accordingly, the methods described herein allow for identification of nanoparticles useful for the delivery of a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA. The methods are also useful in identifying and / or optimizing nanoparticles to deliver a nucleic acid of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo gene therapy or to screen polymer nanoparticles for delivery and production of a polypeptide in vitro or ex vivo.

[0083] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention. EXAMPLES Example 1: Overview of the Protocol

[0084] Toward the goal of establishing RIME technology that can screen the activity of multiple types of mRNA LNPs, an approach for screening the activity of LNPs with excipients was developed. (FIG. 1). We first prepare representative mRNA LNPs by microfluidic mixing of a complete lipid mix solution in the organic phase (Example 3) and mRNA (with optional excipients) in the aqueous phase (Example 4, Steps 1-10)(FIG.2). This method was selected due to its ability to provide batch-to-batch consistency, achieved using a perfusion pump to mix mRNA LNP components at a tunable rate. We use the commercially available ionizable lipid SM-102 due to its usage in the Moderna COVID-19 mRNA LNP vaccine formulation (Baden et al. (2021) N.Attorney Docket No.5470.971.WO Engl. J. Med.384:403–416; Andresen & Fenton (2021) MRS Bull.46:832-839; Callaway (2020) Nature 587:337-338; Webb et al. (2022) Mol. Pharm. 19:1047-1058). The base representative mRNA LNP formulation – that is, without any additional excipients – is prepared by microfluidic mixing of the organic phase containing ionizable lipid (SM-102), phospholipid (DOPE; Zuhorn et al. (2005) Mol. Ther. 11:801-810), cholesterol (Lu et al. (2009) Mol. Pharm. 6:763-771; Narasipura et al. (2023) Bioconjug. Chem.34:1177-1197; Allen & Cullis (2013) Adv. Drug Deliv. Rev.65:36-48; Fenton et al. (2018) Angew. Chem. Int. Ed 57:13582-13586), and PEG-lipid (C14- PEG-2000; Mui et al. (2013) Mol. Ther. Nucleic Acids 2:e139) at a molar ratio of 48:10:40:2, and the aqueous phase containing mRNA. To demonstrate that our protocol can be applied to prepare and evaluate various mRNA LNP formulations, we also formulate mRNA LNPs with optional excipients, as summarized in Table 1. Table 1. Chemical structures of representative molecular excipients within mRNA LNPs used herein.Attorney Docket No.5470.971.WO C14-PEG-2000 (PEG Lipid)

[0085] Secondly, we characterize the obtained mRNA LNPs to ensure their effectiveness for further in vitro and in vivo experiments. This involves assessing key parameters such as size, PDI,Attorney Docket No.5470.971.WO zeta potential, mRNA encapsulation efficiency, and stability (Example 4, Steps 11A-D). This assessment is important since size and PDI are key parameters that impact the cellular delivery and immunogenicity of the mRNA LNPs (Malburet et al. (2022) Anal. Chem. 94:4677-4685; Hassett et al. (2021) J. Control. Release 335:237-246; Cullis & Hope (2017) Mol. Ther.25:1467- 1475; Yanez Arteta et al. (2018) Proc. Natl. Acad. Sci. USA 115:E3351-E3360); zeta potential can influence the biodistribution of mRNA LNPs (Guéguen et al. (2024) Eur. J. Pharm. Biopharm. 195:114077; Carrasco et al. (2021) Commun. Biol.4:956); mRNA encapsulation efficiency affects the effectiveness of LNPs for delivering mRNA drugs (Zhang et al. (2024) ACS Nano 18:7825- 7836; Tenchov et al. (2021) ACS Nano 15:16982-17015); and stability enhances the structural integrity of mRNA LNPs for storage and transportation, preventing their aggregation and maintaining the integrity of encapsulated mRNA drugs to enhance bioavailability and minimize the risk of immunogenicity (Zhang et al. (2023) npj Vaccines 8:156; Rohner et al. (2022) Nat. Biotechnol.40:1586-1600).

[0086] Thirdly, we quantitatively evaluate the in vitro protein expression and cell uptake of LNP formulations (Example 4, Steps 12-45) using reporter genes, such as Firefly luciferase (FLuc) and Cy5-EGFP (the Cy5 signal indicates the presence of the mRNA within cells, suggesting cell uptake, while the EGFP signal reflects the production of the target protein). Both fluorescent signals are quantifiable via flow cytometry. Incorporating Atto-488 labeled DOPE molecules (green) into an mRNA LNP formulation also enables the assessment of cell uptake via flow cytometry. In addition, we also qualitatively visualize the protein expression and cell uptake by confocal microscopy.

[0087] Fourthly, we investigate the mechanism of mRNA LNPs by confocal microscopy (Example 4, Steps 46-66). Specifically, we are interested in two key aspects: the proportion of LNPs escaping from endosomes upon cell uptake and the mechanism driving LNP release from endosomes. Upon endocytosis, mRNA LNPs become temporarily trapped in endosomes (Rui et al. (2022) Sci. Adv. 8:p.eabk2855; Pei & Buyanova (2019) Bioconjug. Chem. 30:273-283). The release of mRNA from these endosomes is imperative for protein translation, however only a small fraction (< 2%) of mRNA LNPs can successfully escape the endosome, representing a substantial bottleneck (Gilleron et al. (2013) Nat. Biotechnol.31:638-646; Herrera et al. (2021) Biomater. Sci. 9:4289-4300). Understanding the factors influencing this escape can provide insight into why certain mRNA LNPs are more effective than others. Moreover, it is also important to understandAttorney Docket No.5470.971.WO the underlying mechanisms behind endosome escape. In one proposed mechanism, referred to as the ‘proton sponge effect,’ LNPs “absorb” protons like a sponge in an acidic endosomal environment, triggering osmotic swelling and eventual rupture of the endosome (Boussif et al. (1995) Proc. Natl. Acad. Sci. USA 92:7297-7301; Vermeulen et al. (2018) Eur. J. Pharm. Biopharm.129:184-190; Behr (1997) CHIMIA 51:34). In our protocol, we provide an approach to quantify endosomal escape by utilizing Atto-488 labeled mRNA LNPs (green) and LysotrackerTMDeep Red to stain the endosome (red). The overlay of green and red (yielding yellow) indicates colocalization. Moreover, the endosomal escape can also be quantitatively measured by Pearson Correlation Coefficient (PCC) using WCIF Image J software. To evaluate the extent of the proton sponge effect, we utilize bafilomycin A1, a proton sponge effect inhibitor, and calcein, a membrane-impermeable dye (Hu et al. (2007) Nano Lett.7:3056-3064; Dröse & Altendorf (1997) J. Exp. Biol.200:1-8).

[0088] Finally, we investigate the in vivo delivery efficacy of both intracellular protein (e.g., FLuc) and secreted protein (e.g., EPO) after intravenous (I.V.) injection of mRNA LNPs into female C57BL / 6 mice at 6-8 weeks old (Example 4, Steps 67A-C). We assess their biodistribution and tolerability including weight retention assessment, histological evaluation, hematology testing, complete blood count (CBC), and clinical chemistry testing of liver and kidney function. I.V. injection is one commonly used administration route to evaluate the efficacy of mRNA LNPs in vivo (Wang et al. (2023) Nat. Protoc.18:265-291; Zhang et al. (2023) Acta Pharm. Sin. B 13:4105- 4126; Yin et al. (2014) Nat. Rev. Genet.15:541-555). Here, the mRNA LNPs enter directly into the bloodstream, ensuring their systemic distribution to various tissues and organs in comparison to other routes such as intramuscular (I.M.) injection or subcutaneous (S.C.) injection. Moreover, I.V. administration is commonly used in clinics, enhancing the translational capacity of pre-clinical research findings to clinical trials. Example 2: Materials

[0089] Biological Materials. All animal studies were approved by the UNC Institutional Animal Care and Use Committee, were consistent with local, state, and federal regulations as applicable, and were supported within the UNC Lineberger ASC at the University of North Carolina at Chapel Hill. All animal experiments were conducted in adherence to institutional and governmental regulations.Attorney Docket No.5470.971.WO

[0090] C57BL / 6 mice (female 6-8 weeks old, weight 18-21 g) were purchased from Jackson Laboratory. The weight of each mouse was monitored every two days to ensure mice were healthy and could be used for animal experiments. The mice were euthanized using a method approved by the AVMA Guidelines for the Euthanasia of Animals (ISBN 978-1-882691-09-8). HepG2 cell line (ATCC, cat. no. HB-8065) was regularly checked for Mycoplasma to ensure they are not cross contaminated.

[0091] Reagents. The reagents and their sources include the following: AlamarBlue™ cell viability reagent (Invitrogen™, cat. no. DAL1100), ARCA Cy5-EGFP mRNA (5-moUTP) (ApexBio, cat. no. R1009), Atto-488 DOPE (Sigma-Aldrich, cat. no. 67335), ALT kit (Alfa Wasserman, cat.no. SA1052), ALP kit (Alfa Wasserman, cat.no. RX2002), AST kit (Alfa Wasserman, cat.no. SA1053), Bafilomycin A1(Thermo Fisher Scientific, cat. no. J61835.MCR), Bright-Glo™ Luciferase assay system (Promega, cat. no. E2620), Bluing Reagent (Epredia™, supplier no.7301), BUN kit (Alfa Wasserman, cat.no. SA2024), C14-PEG-2000 (1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000] (ammonium salt)) (Avanti Polar Lipid, cat. no.880150), Calcein (Invitrogen, cat. no. C481), CellMask™ Deep Red Actin Tracking Stain (Thermo Fisher Scientific, cat. no. A57245), Cholesterol (Sigma-Aldrich, cat. no. 57885), Citric acid (Sigma-Aldrich, cat. no. 77929), CleanCap EPO mRNA (5moU) (Trilink Biotechnologies, cat. no. L-7209; aliquoted and stored at -80°C to avoid freeze-thaw cycles), CleanCap FLuc mRNA (5moU) (Trilink Biotechnologies, cat. no. L-7202; aliquoted and stored at -80°C to avoid freeze-thaw cycles), Clarifier™ 2 (Epredia™, supplier no. 7402), Creatinine kit (Alfa Wasserman, cat.no. RX1012), Cytoseal™ Mountant 60 (Thermo Fisher Scientific, cat. no.8310-4), DMEM (Genesee, cat. no.25-500), DMG-PEG-2000 (1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000) (Avanti Polar Lipid, cat. no. 880151), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (Avanti Polar Lipid, cat. no.850725), DPBS 1x (Genesee, cat. no. 25-508), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipid, cat. no.850365), Eosin Y (yellowish), Signature Series™ (Epredia™, cat. no.17372-87-1), Fetal bovine serum (FBS) (Genesee, cat. no.25-514; aliquoted and stored at -80°C to avoid freeze- thaw cycles), Hematoxylin 7211, Signature Series™ (Epredia™, cat. no. 517-28-2), Hoechst 33342, trihydrochloride, trihydrate (Invitrogen™, cat. no. H1399), Human EPO ELISA Kit (Invitrogen™, cat. no. BMS2035-2), Hydrochloric acid (VWR, cat no.7647010), IDEXX Procyte DX Reagent Kit (IDEXX Laboratories, cat. no.99-26306-00), Lipofectamine™ 2000 transfectionAttorney Docket No.5470.971.WO reagent (Thermo Fisher Scientific, cat. no. 11668500), Lysotracker™ Deep Red (Invitrogen™, cat. no. L12492; aliquoted and stored at -80°C to avoid freeze-thaw cycles), Opti-MEM™ (Gibco, cat. no. 31985070), PBS tablets (Sigma-Aldrich, cat. no. P4417), Penicillin-streptomycin (Genesee, cat. no. 25-512), Phenazine methosulfate (PMS) (Sigma-Aldrich, cat. no. 299-11-6), Pure ethanol (Sigma-Aldrich, cat. no. 64175), Quant-it™ RiboGreen™ RNA Assay Kit (Invitrogen™, cat. no. R11490), RPMI 1640 (Genesee, cat. no.25-506), RNase AWAY (Thermo Fisher Scientific, cat. no. 7000), RNase-free Water (Sigma-Aldrich, cat. no. 7732185), SM-102 (Broadpharm, cat. no. BP-25499), Sodium citrate dihydrate (Sigma-Aldrich, cat. no. 6132043), Sodium hydroxide (Sigma-Aldrich, cat. no. 1310732), Triton™ X-100 (Invitrogen™, cat. no. HFH10), Trypsin-EDTA 0.5% 10x (Gibco, cat. no. 15400054), Trypsin-EDTA, 0.25% 1x (Genesee, cat. no. 25-510), VivoGlo™ Luciferin, In vivo Grade (Promega, cat. no. P1041), Vybrant™ DiD Cell-Labeling Solution (Invitrogen™, cat. no. V22887), XTT sodium salt (Thermo Fisher Scientific, cat. no. J61726. MD), Adenosine 5’-triphosphate (ATP) disodium salt hydrate (Sigma-Aldrich, cat. no. 34369078), Calcium chloride (Sigma-Aldrich, cat. no. 10043524), Cyclosporine (Sigma-Aldrich, cat. no. 59865133), DC-Cholesterol • HCl (3ß-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) (Avanti Polar Lipid, cat. no. 700001), Heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich, cat. no.9041081), HIV-1 TAT protein peptide (Santa Cruz Biotechnology, cat. no.19193691-1; dissolved in 10 mM pH 3 citrate buffer, aliquoted, and stored at -20°C to avoid freeze-thaw cycles), Octaarginine (BOC Sciences, cat. no. 148796865; dissolved in 10 mM pH 3 citrate buffer, aliquoted, and store at - 20°C to avoid freeze-thaw cycles), Polyethyleneimine (PEI) (Sigma-Aldrich, cat. no.25987068), Sodium phosphate dibasic (Sigma-Aldrich, cat. no. 7558794) and Tannic acid (Sigma-Aldrich, cat. no.1401554).

[0092] Buffers may be stored at 4°C for several months. It is recommended to use the 0.22 µm filter to filter the buffers before use to minimize contamination.

[0093] PBS 1x (10 mM pH 7.4) was prepared by dissolving one PBS tablet per 200 mL of RNase- free water.

[0094] PBS 0.1x (pH 7.4) was prepared in a 50 mL tube by diluting

[0095] PBS 1x with RNase-free water at 1:10 ratio.

[0096] Citrate buffer (100 mM pH 3) was prepared by dissolving 1.379 g of sodium citrate dihydrate and 8.705 g of citric acid in 400 mL of RNase-free water. The final pH was adjusted toAttorney Docket No.5470.971.WO 3 using citric acid or NaOH and the RNase-free water was added until the final volume was 500 mL.

[0097] Citrate buffer (10 mM pH 3) was prepared in a 50 mL tube by diluting 100 mM pH 3 citrate buffer with RNase-free water at 1:10 ratio.

[0098] Citrate buffer (10 mM pH 4) was prepared in a 50 mL tube by dissolving 49.64 mg of sodium citrate dihydrate and 63.63 mg of citric acid in 40 mL of RNase-free water. The final pH was adjusted to 4 using citric acid or NaOH and RNase-free water was added until the final volume is 50 mL.

[0099] Citrate buffer (10 mM pH 5) was prepared in a 50 mL tube by dissolving 85.50 mg of sodium citrate dihydrate and 40.21 mg of citric acid in 40 mL of RNase-free water. The final pH was adjusted to 5 using citric acid or NaOH and RNase-free water was added until the final volume is 50 mL.

[0100] Citrate buffer (10 mM pH 6) was prepared in a 50 mL tube by dissolving 121.35 mg of sodium citrate dihydrate and 16.79 mg of citric acid in 40 mL of RNase-free water. The final pH was adjusted to 6 using citric acid or NaOH and RNase-free water was added until the final volume is 50 mL.

[0101] Hoechst 33342 (10 mg / mL) was prepared in a 1.7 mL tube by weighing 2.0 mg of Hoechst 33342 and adding 200 µL of DPBS to make the final concentration of 10 mg / mL. The Hoechst 33342 solution was aliquoted and stored at -20°C for up to 6 months.

[0102] Bright-Glo™ reagent was prepared by transferring the contents of one bottle of Bright- Glo™ Buffer to one bottle of Bright-Glo™ Substrate. The substrate was thoroughly dissolved by mixing by inversion. The reconstituted reagent was stored at -80°C for up to a month.

[0103] Equipment. The equipment used in the protocol and their sources include the following: 24-well cell culture plates flat bottom wells (Genesee, cat. No.25-102), 96-well cell culture plates flat bottom wells (Genesee, cat. No.25-109), 15 mL conical centrifuge tubes (Genesee, cat. No. 28-103), 50 mL conical centrifuge tubes (Genesee, cat. no.28-108), 25 mL reservoirs for 8 channel pipettes (Genesee, cat. No.28-133), 5.0 mL serological pipets (Genesee, cat. no.12-102), 10 mL serological pipets (Genesee, cat. no.12-104), 25 mL serological pipets (Genesee, cat. no.12-106), Autostainer (e.g., Lecia, Leica ST5010 Autostainer XL), Biosafety cabinet (e.g., Thermo Fisher Scientific, 1300 Series A2), Centrifuge (e.g., Eppendorf Centrifuge 5702R), CO2incubator (e.g., Thermo Fisher Scientific, Heracell Vios 160i), Confocal laser scanning microscope (e.g., ZeissAttorney Docket No.5470.971.WO LSM 980), Cover glass (e.g., Lecia, no.3800140ACS), Cuvette PS GRD 4.5 ML (VWR, cat. no. 58017880), Cuvette PS S-MCRO (VWR, cat. no. 97000586), Dialysis cassettes, 20K MWCO (Thermo Fisher Scientific, cat. no. 66005; cat. no. 66003), Dynamic light scattering (DLS) instrument (e.g., NanoBrook 90 Plus Zeta), Electrophoretic light scattering (ELS) instrument (e.g., NanoBrook 90 Plus Zeta), Flow cytometer (e.g., Thermo Fisher Scientific, Attune NxT Flow Cytometer), Hematology analyzer (e.g., IDEXX ProCyte DxTM), Hemocytometer (e.g. Reichert, HS-1490), Inverted microscope (e.g., VWR Trinocular Inverted Microscope), IVIS image system (e.g., PerkinElmer Inc.), Liquid nitrogen tank (e.g., VWR), Microfluidic chip (e.g., fabricated in PDMS (polydimethylsiloxane) according to standard soft lithographic procedures; Chen et al. (2012) J. Am. Chem. Soc.134:6948–6951), Microplate reader (e.g., SpectraMax iD3), MiniCollect tube 0.8 mL CAT serum separation (Greiner Bio-one, cat. no. 450472), Needles 27G (e.g., BD, cat. no.305136) and 21G (e.g., BD, cat. No.305167); Olympus 0.6 mL microtubes (Genesee, cat. no.24-272), Olympus 1.7 mL microtubes (Genesee, cat. no.24-282), Olympus 2.0 mL microtubes (Genesee, cat. no.24-283), pH meter (e.g., Thermo Fisher Scientific), Specimen transport systems- STS prefilled 10% NBF (VWR, cat. No. 48218-792), Syringe 1 mL (Norm-Ject, cat. no. NJ916601702), Syringe filters 0.22 µm (Genesee, cat. no.25-207), Syringe pump (e.g., Harvard Apparatus, item no. 703333), TC treated flasks (Genesee, cat. no. 25-207), Tubes with K2E (K2EDTA) (BD Microtainer, cat. no. 25240), Vet Axcel chemistry analyzer (e.g., Alfa Wasserman), Vortex (e.g., VWR, cat. no.10153-688), µ-Slide 8-Well (ibidi, cat. no.80826).

[0104] HepG2 Cell Splitting and Dilution. The HepG2 cell line was selected as a representative cell line for in vitro evaluation. The details of HepG2 cell splitting and counting are summarized below. 1. Spray 70% ethanol on a paper towel and gloves, then wipe down the bench of the biosafety cabinet. 2. Culture cells in cell culture media (containing DMEM medium +10% FBS+1% Penicillin- streptomycin) in a T-75 flask at 37°C in a 5% CO2incubator for 2 days until they reach around 80-90% confluence. 3. Transfer the T-75 flask from the CO2incubator to the biosafety cabinet. 4. Remove the media from the T-75 flask using a 10 mL serological pipet.Attorney Docket No.5470.971.WO 5. Add 5 mL DPBS to wash the T-75 flask twice using a 5 mL serological pipet. Make sure the DPBS is not added directly to the bottom wall of the T-75 flask to avoid washing away the cells. Instead, gently pipet the DPBS down the side of the well. 6. Add 3 mL Trypsin (0.25%)-EDTA (0.02%) into the T-75 flask using a 5 mL serological pipet. Transfer the T-75 flask back to the 5% CO2incubator and incubate at 37°C for 5 minutes. Gently agitate the T-75 flask to ensure that the trypsin comes into full contact with the cells adhered to the bottom of the flask. Make sure the incubation time is not too long and the concentration of the Trypsin-EDTA is not too high as this might induce cell death. 7. Take the T-75 flask out from the CO2incubator and check the cells under the microscope to ensure all the cells are detached from the bottom of the flask. 8. Spray 70% ethanol on a paper towel, wipe down the T-75 flask, and put it back in the biosafety cabinet. 9. Add 7 mL cell culture media using a 10 mL serological pipet and gently mix up and down. 10. Take 7.5 mL of the cell suspension out from the T-75 flask and add it into a 15 mL tube. 11. Centrifuge the 15 mL tube at 300 g, 4°C, 5 minutes. At the same time, add 15-18 mL of the cell culture media using a 25 mL serological pipet into the T-75 flask and transfer it back to 37°C in a 5% CO2incubator. 12. After centrifuging, aspirate the supernatant using a 2 mL aspirating pipet via vacuum aspiration and discard, and resuspend the pellet with 5 mL cell culture media. Make sure the pump is not too strong to avoid discarding the cell pellet. 13. Aspirate 20 µL of the cell suspension and add it into a hemocytometer and cover it with a microscope cover glass. 14. Count the cell number and then dilute the cell suspension with cell culture media (DMEM medium with 10% FBS and 1% Penicillin-streptomycin) to make the final concentration 1×104cells or 2×104cells per 100 µL. 15. Pour the diluted cell solution into a 25 mL reagent reservoir. Example 3: Methods for preparing complete lipid mix solutions for the organic phase of mRNA LNP formulations 1. In a 1.7 mL tube, weigh 10 mg of SM-102 on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 10 mg / mL.Attorney Docket No.5470.971.WO 2. In a 1.7 mL tube, weigh 10 mg of DOPE on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 10 mg / mL. 3. In a 1.7 mL tube, weigh 10 mg of Cholesterol on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 10 mg / mL. 4. In a 1.7 mL tube, weigh 10 mg of C14-PEG-2000 on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 10 mg / mL. a. The lipids may be sonicated in a bath sonicator and vortexed to ensure that they are fully dissolved in ethanol before use. The reconstituted lipid components are stable for 1 week at 4°C. However, it is recommended to freshly prepare these lipid components. 5. In a 1.7 mL tube, weigh 2 mg of tannic acid (TA) on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 2 mg / mL. 6. In a 1.7 mL tube, weigh 2 mg of polyethyleneimine (PEI) on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 2 mg / mL. 7. In a 1.7 mL tube, weigh 2 mg of DC-Cholesterol • HCl (DC-Chol) on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 2 mg / mL. 8. In a 1.7 mL tube, weigh 2 mg of cyclosporine on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 2 mg / mL. 9. In a 1.7 mL tube, weigh 3 mg of calcium chloride on a balance. Add 1 mL of pure ethanol to dissolve to make a final concentration of 3 mg / mL. 10. To prepare a complete lipid mix solution for the organic phase of LNPs, in a new 1.7 mL tube, add 430 µL of SM-102 from Step 1; add 90 µL of DOPE from Step 2; add 200 µL of Cholesterol from Step 3; add 50 µL of C14-PEG-2000 from Step 4; add 230 µL of pure ethanol. Vortex the solution to mix well. 11. To prepare a complete lipid mix solution for the organic phase of TA LNPs, in a new 1.7 mL tube, add 430 µL of SM-102 from Step 1; add 90 µL of DOPE from Step 2; add 200 µL of Cholesterol from Step 3; add 50 µL of C14-PEG-2000 from Step 4; add 230 µL of TA from Step 5. Vortex the solution to mix well. 12. To prepare a complete lipid mix solution for the organic phase of PEI LNPs, in a new 1.7 mL tube, add 430 µL of SM-102 from Step 1; add 90 µL of DOPE from Step 2; add 200 µLAttorney Docket No.5470.971.WO of Cholesterol from Step 3; add 50 µL of C14-PEG-2000 from Step 4; add 230 µL of PEI from Step 6. Vortex the solution to mix well. 13. To prepare a complete lipid mix solution for the organic phase of DC-Chol LNPs, in a new 1.7 mL tube, add 430 µL of SM-102 from Step 1; add 90 µL of DOPE from Step 2; add 200 µL of Cholesterol from Step 3; add 50 µL of C14-PEG-2000 from Step 4; add 230 µL of DC- Chol from Step 7. Vortex the solution to mix well. 14. To prepare a complete lipid mix solution of the organic phase of cyclosporine LNPs, in a new 1.7 mL tube, add 430 µL of SM-102 from Step 1; add 90 µL of DOPE from Step 2; add 200 µL of Cholesterol from Step 3; add 50 µL of C14-PEG-2000 from Step 4; add 230 µL of cyclosporine from Step 8. Vortex the solution to mix well. 15. To prepare a complete lipid mix solution of the organic phase of Ca3(PO4)2LNPs, in a new 1.7 mL tube, add 430 µL of SM-102 from Step 1; add 90 µL of DOPE from Step 2; add 200 µL of Cholesterol from Step 3; add 50 µL of C14-PEG-2000 from Step 4; add 230 µL of calcium chloride from Step 9. Vortex the solution to mix well. Example 4: Protocol for Preparing LNPs

[0105] Part 1: Formulation of mRNA LNPs (Timing 0.5 days). We describe here a general method to formulate mRNA LNPs using microfluidic chips. We select a representative mRNA LNP formulation, which is composed of an organic phase containing ionizable lipid (SM-102), phospholipid (DOPE), cholesterol, and PEG-lipid (C14-PEG-2000) with the molar ratio of 48:10:40:2, and an aqueous phase containing mRNA. The chemical structures and details of the formulations are summarized in Tables 2-3. 1. Clean the workspace, gloves, and racks with RNase AWAY to prevent any RNAse contamination in the samples. Use RNase-free tubes and tips in this protocol. 2. Transfer 156 µL of mixed completed lipid to a 1.7 mL tube (prepared in Example 3, step 10). 3. Organic phase: Add 46 µL of ethanol to the tube above. Vortex to mix the solution. 4. Aqueous phase: Take another 1.7 mL tube, add 230 µL of RNase-free water, 40 µL of mRNA (1 mg / mL), and 30 µL of citrate buffer (100 mM pH 3). Vortex to mix the solution. The mRNA concentration may be confirmed with Ribogreen™ assay kit.Attorney Docket No.5470.971.WO 5. Aspirate each phase (organic and aqueous) into a separate 1 mL syringe with a 27-gauge needle, respectively. 6. Set up the syringe pump with a 300 µL / min flow rate for the ethanol phase and a 900 µL / min flow rate for the aqueous phase. Connect it to the microfluidic chip with tubing. 7. Use another 1.7 mL tube to collect the mRNA LNPs on the end of the microfluidic chip and start the syringe pump. 8. Aspirate the mRNA LNPs with a 1 mL syringe with a 21-gauge needle and inject into a dialysis cassette, 20K MWCO. 9. Dialyze the mRNA LNPs against 1x PBS for at least 2 hours at 4°C. 10. After the dialysis, transfer the mRNA LNPs into a 1.7 mL tube, and store it at 4°C. The mRNA LNPs should be stable at 4°C for several days. However, it is recommended to use the mRNA LNPs as soon as possible. Do not freeze the mRNA LNPs prior use. Table 2. mRNA LNP formulation details for mixing (note: The Na2HPO4, ATP, OctaR, TAT were dissolved in 10 mM pH 3 citrate buffer). Total aqueous phase for each formulation was 300 µL. 100 mM 2 0.2 0.2 1 mg / mL pH 3 RNase- e er )b0 0 0 0 ol.Table 3. Molar composition ratios for the formulation of mRNA LNPs. Or Aqueous PhaseN fganic Phase (Molar Ratio)t ti L )TPAttorney Docket No.5470.971.WO ATP LNPs 48 10 40 2 X X X X X X X X 2 TA LNPs 47 9 40 2 2 X X X X X X X X PEI LNPs 46 9 38 1 X 6 X X X X X X X11. After formulation of the mRNA LNPs, it is necessary to characterize before use. To determine the size and size distribution (characterized by polydispersity index (PDI)) of the mRNA LNPs, perform the steps in option A. To determine the zeta potential of the mRNA LNPs, perform steps in option B. To determine the encapsulation efficiency of the mRNA in LNPs, perform steps in option C. To determine the stability of the mRNA LNPs, perform steps in option D. To use the mRNA LNPs for further experiments, the anticipated size of mRNA LNPs is < 200 nm; the anticipated PDI of mRNA LNPs is < 0.3; the anticipated zeta potential is between –5 mV and 5 mV; the anticipated encapsulation efficiency is >50%; the LNPs are anticipated to be stable at 4°C in 1x PBS (pH 7.4) for up to 2 weeks. However, we recommend using the mRNA LNPs as soon as possible after the formulation to mitigate any degradation of the mRNA LNPs. (A) Measurement of the size and PDI of mRNA LNPs by DLS (Timing 10 minutes) (i) Add 1 mL of 1x PBS into a 1.7 mL tube. (ii) Add 10 µL of mRNA LNPs into the above tube. Vortex to mix well. (iii) Transfer the solution in the above tube to a disposable Cuvette PS S-MCRO. (iv) Read the sample on a DLS (e.g., NanoBrook 90 Plus Zeta) for 60 seconds. Ensure that the material is set to phospholipid (refractive index 1.450, absorption 0.001), and the dispersant is set to 1x PBS (viscosity 1.02 cp, refractive index 1.335). (B). Measurement of the zeta potential of mRNA LNPs by ELS (Timing 10 minutes) (i) Add 1.8 mL of 0.1x PBS into a 2.0 mL tube. (ii) Add 20 µL of mRNA LNPs into the above tube. Vortex to mix well. (iii) Transfer the solution in the above tube to a disposable Cuvette PS GRD 4.5 ML.Attorney Docket No.5470.971.WO (iv) Insert the ELS probe into the cuvette and connect it to the ELS. Read the sample on an ELS instrument (e.g., NanoBrook 90 Plus Zeta) for 60 seconds. Ensure that the material is set to phospholipid (refractive index: 1.450, absorption: 0.001), and the dispersant is set to 0.1x PBS (viscosity: 0.8882 cp, dielectric constant: 79.00). (C). Measurement of the encapsulation efficiency of mRNA in LNPs by Quant-iT Ribogreen™ Assay (Timing 1 hour) (i) Prepare TE solution: In a 15 mL tube, add 500 µL of 20x TE buffer (from Quant-iT Ribogreen™ RNA Assay Kit) to 4500 µL water to make 2x TE buffer. Vortex to mix well. Do not store the diluted TE buffer. Prepare it freshly. (ii) Prepare Triton™ solution: In a 1.7 mL tube, add 1470 µL of 2x TE buffer to 30 µL of Triton™ X-100 to make 2% v / v Triton™ detergent. Vortex to mix well. Note that to make the Triton™ solution for PEI LNPs, 10 mg heparin was added into 1 mL of 2% v / v Triton™ detergent. (iii) Prepare standard RNA stock solution: In a 1.7 mL tube, add 245 µL of 1x PBS to 5 µL of standard RNA (from Quant-iT Ribogreen™ RNA Assay Kit) to make diluted standard RNA stock. Vortex to mix well. Do not store the diluted RNA stock solution for the standard curve. Prepare it freshly. (iv) Prepare diluted sample solution: In a 1.7 mL tube, add 4 µL of the mRNA LNPs from Step 10 to 396 µL of 1x PBS. Vortex to mix well. (v) In a 96-well plate (transparent with a flat bottom), prepare a standard curve by adding 50 µL 1x PBS to 14 wells (wells B1-H1 and B2-H2). (vi) Add 100 µL of diluted stock RNA (from Step 11C (iii)) to wells A1 and A2, respectively. (vii) Aspirate 50 µL of diluted stock RNA in well A1 and add it to well B1. Take 50 µL of this mixture from well B1, add it to well C1, and repeat down to well G1 to make a serial dilution. Repeat this same serial dilution in wells B2-G2. (viii) Add 50 µL of diluted LNPs (from Step 11C (iv)) to 6 wells of the 96-well plate (wells A4-C4 and A6-C6). (ix) Add 50 µL of 2x TE buffer (from Step 11C (i)) to wells A1-H1 and to three of the wells of the mRNA LNPs (wells A4-C4). Add 50 µL of the 2% v / v Triton™Attorney Docket No.5470.971.WO in TE buffer (Step 11C (ii)) to wells A2-H2 and to the other three wells of the mRNA LNPs. (wells A6-C6). After adding 2% v / v Triton™ in TE buffer into each well, there might be some bubbles in the wells; use needles to prick the bubbles. (x) Incubate the plate at room temperature (22°C) for at least 20 minutes with 200 rpm shaking speed. (xi) In a 15 mL tube, add 2895 µL of 2x TE buffer to 15 µL of Quant-iT Ribogreen™ reagent (from Quant-iT Ribogreen™ RNA Assay Kit). The Quant-iT Ribogreen™ reagent needs to be protected from light. Use the diluted Quant-iT Ribogreen™ reagent within 30 minutes. (xii) Pour the above solution into a 25 mL reservoir. Add 100 µL of the solution into each standard and sample well using a multichannel pipette for a final volume of 200 µL per well. (xiii) Incubate the plate at room temperature for 3 minutes and read the fluorescence at 480 nm (excitation) and 520 nm (emission). The incubation step is optional. It is recommended to read the results within 15 minutes. The tested samples and standard curve should be on the same plate. (xiv) Use a linear regression model on your standards in PBS (wells A1-H1) and standards in Triton™ buffer (wells A2-H2). See Table 4 for an example data set calculation. (xv) Use your linear regression line to interpolate the total and unencapsulated mRNA concentration. Unencapsulated mRNA Concentration ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ = 1 −Total mRNA Concentration Table 4. Example of step-by-step guide to quantifying mRNA encapsulation within mRNA LNPs (Data are shown for triplicates). Standard Curve of Standard Curve of Diluted mRNA )Attorney Docket No.5470.971.WO 0.25 1659096 1838701 0.125 1097696 1224094 s ) l(D). Measurement of the stability of mRNA LNPs (Timing 5 days) (i) In separate 1.7 mL tubes, add 20 µL of mRNA LNPs into 80 µL of pH 4 citrate buffer (10 mM), pH 5 citrate buffer (10 mM), pH 6 citrate buffer (10 mM), pH 7.4 PBS (10 mM) buffer, DMEM + 10% FBS and RPMI + 10% FBS media, respectively. Vortex to mix well. (ii) Incubate the samples at either 4°C or 37°C for 5 days. This measurement may be adjusted to evaluate the stability of mRNA LNPs across different conditions, time periods, or temperatures depending on the application of the formulated mRNA LNPs. (iii) Measure the size of each sample by repeating Steps 11A (i)-(iv), using 10 µL of samples from Step 11D (ii) dissolved 1 mL of 1x PBS. (iv) Compare the size of the mRNA LNP sample with its original size measured on Day 0 (determined in Step 11A) to elucidate the stability of the mRNA LNPs. An alternative method to monitor the stability of mRNA LNPs is to measure the in vitro protein expression over a specified period. Cells may be treated with mRNAAttorney Docket No.5470.971.WO LNPs, samples stored at 4°C at the designated time points (days 0, 7, 14, and 21), and Steps 12-25 repeated to assess protein expression at each time point.

[0107] Part 3: In vitro Efficacy Evaluation of mRNA LNPs. Depending on the applications, different cell lines or cell types may be selected to evaluate the in vitro delivery efficacy of the mRNA LNPs. Culture the cells and count the cell numbers before usage. We selected the HepG2 cell line as one representative for the in vitro evaluation. The details of HepG2 cell splitting and counting is described in Example 2.

[0108] Quantitatively assess cell viability and in vitro FLuc expression of mRNA LNPs (Timing 2-3 days) 12. Add 100 µL of HepG2 cells at a concentration of 1×104cells per 100 µL (Example 2) to a 96-well plate (transparent with flat bottom). Incubate at 37°C in a 5% CO2incubator overnight to allow cellular adhesion to the substrate. 13. Dilute FLuc mRNA LNPs with HepG2 cell culture media (DMEM medium with 10% FBS and 1% Penicillin-streptomycin) to a final concentration of 500 ng / mL. Make sure the final diluted FLuc mRNA concentration is the total mRNA concentration, not the encapsulated mRNA concentration. 14. Positive controls include naked mRNA and Lipofectamine™ 2000. Prepare naked FLuc mRNA at a concentration of 500 ng / mL by diluting 1 mg / mL naked FLuc mRNA with HepG2 cell culture media. Prepare Lipofectamine™ 2000 control: In a 0.6 mL tube, add 5 µL Lipofectamine™ 2000 in 25 µL of Opti-MEM™ medium. In another 0.6 mL tube Add 5 µL of 1 mg / mL naked FLuc mRNA in 25 µL of Opti-MEM™ medium. Mix the above solution and incubate at room temperature for 5 minutes. Dilute the solution with HepG2 cell culture media to the final concentration of 500 ng / mL. The concentration of FLuc mRNA may be adjusted depending on the application. 15. Negative cell viability control: Dilute Triton™ X-100 in HepG2 cell culture media to get 1% v / v Triton™, as a negative viability control to ensure the assay was accurately functioning. It is optional to add PBS, cells without media, or other kill reagents as negative control for cell viability assay. 16. Aspirate the HepG2 cell culture media from the 96-well plate in Step 12 using a 2 mL aspirating pipet via vacuum aspiration and discard.Attorney Docket No.5470.971.WO 17. Add 100 µL 500 ng / mL of FLuc mRNA LNPs from Step 13 to three of the wells (A4-C4), 100 µL of fresh HepG2 cell culture media to eight of the wells (A2-H2) (as 100% cell viability control), 100 µL 500 ng / mL of naked FLuc mRNA to three of the wells (A6-C6), and 100 µL of 1% v / v Triton™ to three of the wells (F12-H12). Incubate at 37°C in a 5% CO2incubator for 24 hours. Wells without cells or samples will be used as the plate background. The incubation time may be adjusted depending on the application. All the samples and controls should be run on the same plate to ensure that results will be comparable. 18. To run the AlamarBlue™ cell viability assay, in a 15 mL tube, add 1 mL of AlamarBlue™Cell viability reagent into 9 mL of cell culture media (1:10). AlamarBlue™cell viability assay relies on resazurin-based reagents to gauge cell metabolic activity. Other cell viability assays may also be applied according to specific applications. 19. Aspirate the supernatant from the 96-well plate in Step 17 using a 2 mL aspirating pipet via vacuum aspiration and discard. 20. Pour the diluted AlamarBlue™solution from Step 18 into a 25 mL reagent Reservoir. 21. Add 100 µL of the AlamarBlue™solution into each well of the plate from Step 19 using a multichannel pipette. 22. Transfer the 96-well plate to a 5% CO2incubator and incubate at 37°C for 2-4 hours. 23. Read the absorbance at 570 nm and background at 600 nm in a microplate reader. 24. Calculate the cell viability. See Table 5 for an example data set calculation for AlamarBlue™cell viability assay. 25. In another 96-well plate, repeat Steps 12-17. Add 100 µL of Bright-Glo™ Luciferase assay buffer into each treated well. Incubate for 3 minutes and read the luminescence value on a microplate reader. After adding Bright-Glo™ Luciferase Assay buffer into each well, there might be some bubbles in the wells; use needles to prick the bubbles. The incubation step is optional. It is recommended to read the results within 15 minutes.Attorney Docket No.5470.971.WO Table 5. Example of step-by-step guide to calculate cell viability using AlamarBlue™cell viability assay (Data are shown for triplicates). Cells Treated 100 µL of 500 100 µL of 1% with Fresh ng / mL mRNA v / v Triton™ in 2) ™ a ™Attorney Docket No.5470.971.WO 0.260625 / 0.246 0.018625 0%=105.94 / 0.246 ×1051×100%=7.571138

[0109] Quantitatively assess cell uptake by fluorescence-labeled molecules in LNP formulation (Timing 2-3 days). To evaluate the mechanism of the mRNA LNPs in cells, a fluorescent tag is included on the LNPs. In this protocol, we include Atto-488 DOPE molecules in the mRNA LNP formulation (see Step 27). 26. Count the HepG2 cell number (from Example 2) and then dilute the cell suspension with HepG2 cell culture media to a final concentration of 2×104cells per 100 µL. Add 400 µL of cells per well in a 24-well plate (transparent with flat bottom). The final cell density should be 8×104cells per well. Incubate at 37°C in a 5% CO2incubator overnight to allow cellular adhesion to the substrate. 27. Prepare Atto-488 labeled FLuc mRNA LNPs as described in Steps 1-10, using Atto-488 DOPE molecules. Dilute Atto-488 labeled FLuc mRNA LNPs with the cell culture media to a final concentration of 500 ng / mL. Make sure the final diluted Atto-488 labeled FLuc mRNA LNP concentration is the total mRNA concentration, not the encapsulated mRNA concentration. 28. Aspirate the supernatant from the 24-well plate using a 2 mL aspirating pipet via vacuum aspiration and discard. 29. Add 400 µL 500 ng / mL of Atto-488 labeled FLuc mRNA LNPs per well to the cells. Add 400 µL of fresh cell culture media into other cells (as unstained cell control). Incubate at 37°C in a 5% CO2incubator for 24 hours. The incubation time is adjustable depending on the specific application. 30. Wash the cells with DPBS three times by adding 400 µL of DPBS to each well for each wash. Incubate for 10 seconds, then aspirate the supernatant using a 2 mL aspirating pipette with vacuum aspiration and discard the liquid. Do not add the DPBS directly to the bottom of the 24-well plate to avoid washing away the cells. Instead, gently pipet the DPBS down the side of the well.Attorney Docket No.5470.971.WO 31. Dissociate cells with 150 µL of Trypsin-EDTA 2x (1:5 dilution of Trypsin-EDTA 10x) and incubate at a 5% CO2incubator for 5 minutes. Check the 24-well plate under the microscope and make sure all the cells are detached from the bottom of the plate. 32. Once all the cells are detached from the plates, add 200 µL of DPBS into each well and transfer the cells from each well into separate 1.7 mL tubes. 33. Wash the cells with DPBS twice by adding 400 µL of DPBS to each well for each wash. Incubate for 10 seconds, then aspirate the supernatant using a 2 mL aspirating pipette with vacuum aspiration and discard the liquid and resuspend the cell pellets in 200 µL of DPBS. 34. Analyze the samples on a flow cytometer, such as the Attune NxT Flow Cytometer. Set up FSC voltage at 100 and SSC voltage at 280 to ensure clear observation of single-cell clusters. Then select ‘BL1’ channel to measure the samples. See FIGS.3A-3B for an example data of gating strategy. 35. Assess the uptake of Atto-488 labeled FLuc mRNA LNPs using the percentage of cells with stronger fluorescence intensity than the untreated cell control gate or characterized by the geometric fluorescence mean intensity (GMFI) using FlowJo software.

[0110] Quantitatively assess cell uptake and protein expression using fluorescence labeled mRNA in LNP formulation (Timing 2-3 days). In this protocol, we encapsulate Cy5-EGFP mRNA into the desired LNP formulation, which is composed of ionizable lipid (SM-102), phospholipid (DOPE), cholesterol, and PEG-lipid (C14-PEG-2000) and Cy5-EGFP mRNA. 36. In a 24-well plate, repeat Steps 26-34. Assess the Cy5-EGFP mRNA LNPs uptake and expression using the percentage of cells with stronger fluorescence intensity than the control untreated cell gate, separated by four quadrants using FlowJo software.

[0111] Qualitatively assess cell uptake and protein expression by confocal microscopy (Timing 2-3 days) 37. Count the HepG2 cell number (from Example 2) and then dilute the cell suspension with HepG2 cell culture media to make the final concentration 1×104cells per 100 µL. Add 300 µL of cells, so the final cell density is 3×104cells per well in a µ-slide 8-well coverslip slide. Incubate at 37°C in a 5% CO2incubator overnight to allow cellular adhesion to the substrate. 38. Use Cy5-EGFP mRNA LNPs prepared as per Steps 1-10. Dilute Cy5-EGFP mRNA LNPs with the cell culture media with the final concentration of 500 ng / mLAttorney Docket No.5470.971.WO 39. Aspirate the supernatant from the µ-slide 8-well coverslip slide using a 2 mL aspirating pipet via vacuum aspiration and discard. 40. Add 300 µL 500 ng / mL of Cy5-EGFP mRNA LNPs into each well. Incubate at 37°C in a 5% CO2incubator for 24 hours. The incubation time is adjustable depending on the application. 41. Wash the cells with DPBS twice by adding 300 µL of DPBS to each well for each wash and repeating Step 33. 42. In a 15 mL tube, dilute 10 mg / mL of Hoechst 33342 to 1 µg / mL with DPBS. Add 300 µL of 1 µg / mL Hoechst 33342 to each well and incubate for 10 minutes at 37°C in a 5% CO2incubator. A recommended incubation time is typically 10 to 20 minutes at 37°C to minimize potential adverse effects on cell viability. 43. Wash the cells three times by repeating Step 41. 44. Add 200 µL of DPBS into each well. 45. Perform live cell imaging by confocal microscopy by putting the µ-slide 8-well coverslip under the confocal laser scanning microscopy to visualize the level of mRNA LNPs (select ‘Cy5’ channel) and EGFP expression (select ‘EGFP’ channel). Process the images with WCIF Image J software to add scale bars and merge different channels (blue: nucleus; red: cy5- mRNA; green: EGFP protein).

[0112] Part 4: Mechanism Investigation.

[0113] Assessing the endosomal escape (Timing 2-3 days). To quantify the endosomal escape ability of mRNA LNPs in cells, a fluorescent tag is included on the LNPs. We use Atto-488 labeled FLuc mRNA LNPs. 46. Repeat Step 37. 47. Repeat Step 27. 48. Repeat Step 39. 49. Add 300 µL 500 ng / mL of Atto-488 labeled FLuc mRNA LNPs into each well. Incubate at 37°C in a 5% CO2incubator for 4 hours. The incubation time is adjustable depending on the application. 50. Repeat Step 41. 51. In a 15 mL tube, dilute LysotrackerTMDeep Red to the final concentration of 100 nM with cell culture media. Prewarm the cell culture media to 37ºCAttorney Docket No.5470.971.WO 52. Add 300 µL of 100 nM of diluted LysotrackerTMDeep Red to each well of the cells and incubate for 1 hour at 37°C in a 5% CO2incubator. The incubation time can be extended, but it is recommended not to exceed 2 hours. 53. Repeat Step 41. This washing step is optional. 54. In a 15 mL tube, dilute 10 mg / mL of Hoechst 33342 to 1 µg / mL with DPBS. Add 300 µL of 1 µg / mL Hoechst to each well and incubate for 10 minutes at 37°C in a 5% CO2incubator. It is recommended the incubation time not exceed 15 minutes. 55. Repeat Step 41. 56. Add 200 µL of DPBS into each well. 57. Perform live cell imaging by confocal laser scanning microscopy by putting the µ-slide 8- well coverslip under the confocal laser scanning microscopy to visualize the endosomes (select ‘Lysotracker Deep Red’ channel), nuclei (select ‘Hoechst 33342’ channel) and Atto-488 labeled FLuc mRNA LNPs (select ‘Atto-488’ channel). Five representative cell images (>20 cells) are used to calculate the Pearson Correlation Coefficient (PCC) value. The images can be further processed by WCIF Image J software to add scale bars and merge different channels (blue: nucleus; red: endosome; green: Atto-488 labeled FLuc mRNA LNPs). To obtain the PCC value, import confocal images into WCIF ImageJ software with ‘JACoP’ add-in. Navigate to ‘Plugins’ and select ‘JACoP’, then choose ‘Pearson’s Coefficient’. Select Image A (Green channel) and Image B (Red channel) and click ‘Analyze’. The software will then calculate the PCC value. The endosomal escape ability is quantified using PCC where a PCC value of 1 indicates no endosomal escape whereas a PCC value of 0 indicates complete endosomal escape.

[0114] Assessing proton sponge mechanisms using bafilomycin A1(Timing 1.5 days) 58. Repeat Steps 46-48, but in Step 47, prepare FLuc mRNA LNPs as described in Steps 1-10. Use FLuc mRNA LNPs that are not fluorescently labeled to avoid interference with the calcein signal. 59. Add 180 µL of fresh HepG2 cell culture media to three wells or fresh HepG2 cell culture media containing bafilomycin A1(111.1 nM) to another three wells. 60. In a 1.7 ml tube, weigh 1.5 mg of calcein and add 1 mL of DPBS to make the final concentration of calcein 1.5 mg / mL.Attorney Docket No.5470.971.WO 61. Add 20 μL of 1.5 mg / mL of calcein into each well to obtain a final concentration of 150 μg / mL for calcein and 100 nM for bafilomycin A1. 62. Add 100 µL of 1500 ng / mL of mRNA LNPs into each well (to obtain the final concentration of 500 ng / mL of mRNA LNPs). Add 100 µL of fresh media to control cells. Incubate at 37°C in a 5% CO2incubator for 4 hours. The incubation time should not exceed 4 hours to ensure the inhibition of endosomal escape can be attributed to the proton sponge effects of bafilomycin A1. 63. After the 4 hours incubation period, users can optionally stain the cytoskeleton with CellMask™ Deep Red Actin Tracking Stain (option A) or cell nuclei with Hoechst 33342 (option B) to visualize the cytoskeleton and cell nuclei, respectively. (A) Stain the cytoskeleton (i) In a 15 mL tube, dilute CellMask™ Deep Red Actin Tracking Stain (1 mM) to the final concentration of 1 µM with cell culture media. (ii) Add 300 µL of 1 µM of diluted CellMask™ Deep Red Actin Tracking Stain into each treated well and incubate for 15 minutes at 37°C in a 5% CO2incubator. (iii) Repeat Step 41. (B) Stain the cell nuclei. To stain the cell nuclei with Hoechst, repeat Steps 54 and 55. 64. Wash the cells with DPBS four times, repeating Step 41. 65. Add 200 µL of DPBS into each well. 66. Perform live cell imaging by confocal microscopy by placing the µ-slide 8-well coverslip under the confocal laser scanning microscopy to visualize the calcein signal (select ‘calcein’ channel), nuclei (select ‘Hoechst 33342’ channel) and cytoskeleton (select ‘CellMask Deep Red’ channel). The images can be further processed by WCIF Image J software to add scale bars or merge different channels (blue: nucleus; red: cytoskeleton; green: calcein).

[0115] Part 5: In vivo Evaluation of mRNA LNPs 67. To evaluate the biodistribution and intracellular protein expression level, perform steps in option A. To evaluate the secreted protein expression level, perform steps in option B. To evaluate the toxicity of the mRNA LNPs, perform steps in option C. (A) Measurement of the biodistribution and intracellular protein expression level using FLuc mRNA LNPs (Timing 2-3 days).Attorney Docket No.5470.971.WO (i) Prepare FLuc mRNA LNPs as described in Steps 1-10. To evaluate the biodistribution of LNP formulation in Step 67A (viii), a 1% molar ratio of DiD labeling should be added into the organic phase to formulate FLuc mRNA DiD- LNPs. (ii) Intravenously inject FLuc mRNA LNPs into C57BL / 6 mice (Jackson Laboratory, 18-21 g) (at least n = 3 per group). The injection dose used is 0.65 mg / kg, but this dose may be adjusted for alternative applications. (iii) 24 hours after injection of FLuc mRNA LNPs, anesthetize the mice using 1.5-2.5% isoflurane. The time post-injection of FLuc mRNA LNPs in this protocol is set to 24 hours, but may be adjusted as needed for specific applications. (iv) Monitor the weight of the mice after the injection of FLuc mRNA LNPs to indicate their toxicity. Compare each animal's weight before and 24 hours after the injection. Generally, a weight loss of less than 10% of the animal’s pre-injection weight is considered acceptable and indicates good tolerance of the treatment. A loss of more than 10% of body weight indicates poor tolerance to the treatment. (v) Intraperitoneally inject 130 µL of D-luciferin (30 mg / mL in DPBS). (vi) After 15 minutes, collect 60 µL of blood from Step 67A (v) into K2EDTA tubes and 190 µL of blood from Step 67A (v) into a Minicollect tube for each mouse via cardiac puncture bleed at room temperature. (vii) Euthanize the mice by cervical dislocation. Use CO2inhalation along with the secondary means (cervical dislocation). Shoebox style mouse cages require 2.8 L / min CO2flow. (viii) Remove and image the organs (pancreas, spleen, liver, kidneys, ovaries, lung, and heart) with the IVIS image system. Use the following IVIS settings to measure the biodistribution of mRNA LNPs (excitation wavelength 646 nm, emission wavelength 670 nm). Stage temperature: 37°C, camera CCD temperature: -90°C, binning: medium = 4, exposure time: 120 seconds, FOV: 12.5. Store and fix the organs in 10% neutral buffered formalin containers after imaging for histology analysis in Step 67C (iv)-(viii). (ix) Quantify the luminescence using AuRA software.Attorney Docket No.5470.971.WO (B) Measurement of the secreted protein expression level using EPO mRNA LNPs (Timing 2-3 days). (i) Prepare EPO mRNA LNPs as described in Steps 1-10. (ii) Intravenously inject EPO mRNA LNPs into C57BL / 6 mice (Jackson Laboratory, 18-21 g) (at least n = 3 per group). The injection dose used is 0.65 mg / kg, but this dose may be adjusted for alternative applications. (iii) 24 hours after injection of EPO mRNA LNPs, collect 200 µL of blood from Step 67B (iii) into a Minicollect tube for each mouse via cardiac puncture bleed at room temperature. (iv) Repeat Step 67A (iv). (v) Centrifuge the Minicollect tube at 1300 g for 10 minutes at room temperature to collect the serum. (vi) Measure the concentration of EPO protein in the serum sample using a Human EPO ELISA kit according to manufacturer’s instructions. (C) Evaluation of the toxicity of mRNA LNPs (Timing 1-2 weeks). (i) To perform the hematology CBC testing and clinical chemistry testing of liver and kidney function, centrifuge the Minicollect tube from Step 67A (vi) at 1300 g for 10 minutes at room temperature to collect the serum. (ii) Using the Vet Axcel instrument, measure the serum levels of ALT, AST, ALP, BUN or Creatinine using the appropriate kits, respectively, following the manufacturer’s protocols. (iii) Using the IDEXX ProCyte Dx hematology analyzer, perform a CBC test on the blood samples collected in K2EDTA tubes in Step 67A (vi) using CBC kits, following the manufacturer's protocol. (iv) To perform histological tests of the organs collected in Step 67A (viii), begin by placing the tissue in a metal mold, add warm paraffin to cover the tissue, and insert a cassette for support. Add more paraffin if needed. Place the mold on a cooling plate until the tissue block solidifies. Once solidified, remove the block from the metal mold and trim any excess paraffin. (v) Section tissues on a microtome at 5 µm onto positively charged slides.Attorney Docket No.5470.971.WO (vi) Before performing H&E staining, air-dry the slides overnight, then bake them at 60°C for 30 minutes. (vii) Perform H&E stains using the autostainer XL. Stain the sections with Hematoxylin for 2 minutes and Eosin-Y for 1 minute. Use ClarifierTM2 and Bluing solutions to differentiate the reaction. (viii) After staining, dehydrate the slides in graded ethanol (95%), ending in xylene, and coverslip with Cytoseal™ Mountant 60. (ix) Perform histological analysis on each sample to assess cellular and tissue damage and to observe any inflammatory response, helping to determine the safety and potential adverse impacts of mRNA LNPs on different organs. Example 5: Formulation and Characterization of mRNA LNPs

[0116] Applications of the Protocol. LNPs are a powerful mRNA delivery platform within the pharmaceutical science field. Here, we illustrate the utility of our protocol by demonstrating the formulation and evaluation of nine distinct mRNA LNPs based on Moderna ionizable lipid (SM- 102), DOPE, cholesterol, and C14-PEG-2000, with optional excipients. Our protocol is modular and applicable to many types of new mRNA LNP formulations, encompassing various ionizable lipids (e.g., ALC-0315, DLin-MC3-DMA, cKK-E12 and OF-Deg-Lin; Fenton et al. (2017) Adv. Mater.29:1606994; Polack et al. (2020) N. Engl. J. Med.383:2603-2615; Riley et al. (2021) Sci. Adv.7:p. eaba1028; Fenton et al. (2016) Adv. Mater.28:2939-2943), phospholipids (e.g., DSPC; Vogel et al. (2021) Nature 592:283-289; Corbett et al. (2020) Nature 586:567-571), cholesterol and its derivatives, and PEG-lipids (e.g., DMG-PEG-2000 and ALC-0159; Baden et al. (2021) N. Engl. J. Med.384:403-416; Webb et al. (2022) Mol. Pharm.19:1047-1058; Polack et al. (2020) N. Engl. J. Med. 383:2603-2615). Furthermore, we envision that this protocol could be readily adapted to investigate other types of mRNA nanoparticle delivery platforms, including polymeric nanoparticles or liposomes (Fenton et al. (2018) Adv. Mater.30:p.1705328).

[0117] Comparison with Other Methods. This protocol offers a workflow for the formulation, characterization, and evaluation of mRNA LNPs. This protocol allows researchers to assess LNP formulations with various types of mRNA transcripts, highlighting the versatility of specific LNP formulations – a key consideration for delivering therapeutically relevant genes. Additionally, this workflow provides thorough characterization through both mechanistic and functional readouts,Attorney Docket No.5470.971.WO offering insights not only into the efficacy of a given mRNA LNP formulation but also into the underlying reasons for its performance.

[0118] To formulate the mRNA LNPs in this protocol we use microfluidic mixing, which leverages mixing of an aqueous phase and organic phase at a fixed rate. However, other techniques may also be utilized, such as: extrusion of lipid vesicles, where a suspension of lipids is driven through pores of a fixed size (Hunter & Frisken (1998) Biophys. J. 74:2996-3002); nanoprecipitation, where solvent and non-solvent phases are combined with constant stirring (Martínez Rivas et al. (2017) Int. J. Pharm. 532:66-81; Fessi et al. (1089) Int. J. Pharm. 55:R1- R4); and thin film hydration, where lipids dissolved in an organic phase are dried in a thin layer and rehydrated in an aqueous solution (Bangham et al. (1967) Chem. Phys. Lipids 1:225-246; Umbarkar et al. (2021) J. Drug Delivery Ther. 11:72-76). Compared with other approaches to prepare mRNA LNPs, microfluidic mixing provides rigorous reproducibility with uniform size distribution and scalable production, making it useful for manufacturing for future therapeutic applications (Shepherd et al. (2021) Nano Lett.21:5671-5680).

[0119] In this protocol, the size distribution of mRNA LNPs is characterized using Dynamic Light Scattering (DLS). However, DLS may not provide precise measurements of an individual LNP size or reveal their morphology and shape. To evaluate these characteristics of LNPs, one may use techniques such as Transmission Electron Microscopy (TEM), Cryogenic Electron Microscopy (Cryo-EM), Atomic Force Microscopy (AFM), and Nanoparticle Tracking Analysis (NTA)(Malburet et al. (2022) Anal. Chem. 94:4677-4685; Herrera et al. (2021) Biomater. Sci. 9:4289-4300; Thelen et al. (2024) ACS Nano 18:1464-1476; Eygeris et al. (2020) Nano Lett. 20:4543-4549; Cui et al. (2022) Nanoscale 14:1480-1491). Furthermore, techniques like Small- Angle X-ray Scattering (SAXS) may not only provide information on the average size and general shape of LNPs but also valuable insight into the internal arrangement of lipid layers within mRNA LNPs, including details on lipid bilayer thickness (Hammel et al. (2023) ACS Nano 17:11454- 11465; Schwamberger et al. (2015) Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 343:116-122; Bauer et al. (2019) Nat. Commun. 10:1122).

[0120] For in vitro evaluation of protein expression and cell viability of mRNA LNPs, our protocol uses FLuc mRNA LNPs. The expression of FLuc protein may be quantified using a Bright-Glo™ Luciferase assay (Promega). There are also other techniques to quantify the in vitroAttorney Docket No.5470.971.WO protein expression such as western blot analysis (Rungta et al. (2013) Mol. Ther. Nucleic Acids 2:e136; Hsu et al. (2022) Int. J. Pharm. 627:122256), mass spectrometry (Wang et al. (2022) Transl. Med. Commun.7:9), and ELISA (Rafique et al. (2019) Int. J. Nanomedicine 14:2829-2846; Yang et al. (2020) Bioact. Mater.5:1053-1061). Cell viability is measured using an AlamarBlue™ assay. This approach offers a straightforward way to quantify cell viability. The AlamarBlue™ assay measures cell viability through the incorporation of a stable oxidation-reduction (REDOX) indicator based on metabolic activity, requiring fewer steps (Tiwari et al. (2003) Eur. J. Vasc. Endovasc. Surg. 25:325-329; La Peyre et al. (2006) Dis. Aquat. Org. 71:59-74). However, the AlamarBlue™ assay is only compatible with certain types of cell culture media, like DMEM. For specific cell experiments such as neuron cultures, alternative viability assays may be required, such as the lactate dehydrogenase (LDH) assay (Han et al. (2011) Toxicology 287:99-104; Parot et al. (2024) J. Control. Release 367:385-401).

[0121] This protocol evaluates the in vivo functional delivery of LNPs using FLuc mRNA. There are also other ways to evaluate their in vivo efficacy, such as DNA barcoding (Xue et al. (2024) Nat. Commun. 15:1884; Guimaraes et al. (2019) J. Control. Release 316:404-417), mass spectrometry (Ci et al. (2023) Drug Metab. Dispos.51:813-823; Hyldbakk et al. (2024) J. Control. Release 366:611-620), radiolabeled lipids on the LNP (Chen et al. (2016) J. Control. Release 235:236-244), or fluorescent mRNA (Goutayer et al. (2010) Eur. J. Pharm. Biopharm. 75:137- 147).

[0122] Other Considerations. The microfluidic mixing method offers precise control to achieve uniform size distribution of mRNA LNPs and minimize batch-to-batch variability, however, it is worth noting that microfluidic chips typically come with a high cost, which requires a large initial investment. Additionally, clogging issues within the microfluidic channels, often caused by lipid aggregates, may require further investment in maintenance. These clogs may be avoided through the use of single-use chips, though they may come at a high cost. Proper cleaning of microfluidic channels may be used to prevent cross-contamination issues. Moreover, there also may be dead volume in syringes using microfluidic chips for LNP formulations, leading to material wastage. Furthermore, this method may not be suitable for producing very small volumes of mRNA LNPs for specific applications, such as intrathecal administration of mRNA LNPs. In cases where very small volumes of mRNA LNPs are needed, one can consider formulating a larger volume and aliquoting smaller amounts for administration.Attorney Docket No.5470.971.WO

[0123] Our approach to assess cell uptake and endosomal escape incorporates the use of fluorescently tagged lipid molecules in the LNP formulation. If such lipid components are not commercially available, additional lipid labeling experiments may be used, which can add additional experimental considerations. Also, additional fluorescent labeling might change the properties of LNPs. Moreover, our approach to investigate endosome escape involves using Atto- 488 labeled mRNA LNPs (green) and endosomes stained using Lysotracker™ Deep Red (red). If the fluorescently tagged lipid molecules in LNP formulations have similar excitation and emission wavelengths as Lysotracker™ Deep Red, alternative Lysotracker stains must be selected to ensure differentiation of their wavelengths from those used in the LNPs.

[0124] Our approach to evaluate protein expression and cell uptake by encapsulating Cy5-EGFP mRNA into LNP formulations uses fluorescently labeled mRNA and the translation of labeled proteins. Despite the programmable nature of mRNA LNPs that allows for easy substitution of one mRNA transcript for another, the efficacy of mRNA LNPs containing therapeutic mRNA might require further application-specific measurements such as RNA sequencing or quantitative polymerase chain reaction (qPCR) to assess mRNA delivery efficiency into cells, and ELISA to quantify the concentration of the therapeutic protein.

[0125] Further, our stability studies indicate that the effectiveness of some formulated mRNA LNPs may decrease to as low as ~0.5 % after 21 days when stored at 4°C over time. For researchers evaluating new nanoparticle formulations, it may be most effective to prepare their LNP formulation immediately prior to evaluating the stability. This may minimize any degradation of mRNA or the LNPs themselves.

[0126] Experimental Design. The base mRNA LNPs in this study may be prepared by microfluidic mixing an organic phase including 4 components: ionizable lipid (SM-102), phospholipid (DOPE), cholesterol, and PEG-lipid (C14-PEG-2000) at a molar ratio of 48:10:40:2, and an aqueous phase including mRNA. For this protocol, we chose DOPE LNP formulations with the optional addition of excipients as an example because previous work from has shown its efficacy in delivering mRNA payloads (Ma & Fenton (2023) Adv. Therap. 6:p.2200305; Ma & Fenton (2023) J. Am. Chem. Soc. 145:19800-19811; Ma & Fenton (2023) J. Am. Chem. Soc. 145:11375-11386; Ma et al. (2024) Nano Lett.24:6092-6101), but the workflow may be adapted to analyze many formulations, such as the clinically relevant Moderna formulation (FIGS.4A-4B and Table 6). Additional excipients may be added into the organic phase of the base mRNAAttorney Docket No.5470.971.WO formulation such as tannic acid (TA) (molar ratio of 47:9:40:2:2), polyethyleneimine (PEI) (molar ratio of 46:9:38:1:6), DC-Cholesterol • HCl (DC-Chol) (molar ratio of 45:9:38:1:7), cyclosporine (molar ratio of 47:9:40:1:3), or calcium chloride (CaCl2) (molar ratio of 33:6:27:1:33) leading to five components in the complete lipid mix solution. Moreover, additional excipients may also be added into the aqueous phase at 1: 1 wt% mixing with mRNA such as HIV-TAT 1 (TAT, 0.2 mg / mL), octaarginine (0.2 mg / mL), ATP (2 mg / mL) and Na2HPO4(2 mg / mL) diluted in citrate buffer (10 mM pH 3) (Table 3). Table 6. Size, PDI, zeta potential and mRNA encapsulation efficiency of Moderna LNPs. mRNA Encapsulation Size (nm) PDI Zeta Potential (mV) Efficiency (%)e s s ou e e a y e aga s a o a eas ou s n a 20K WMCO dialysis cassette with approximately 1.78 nm pore size to remove the ethanol and any small contaminants or buffer salts (Erickson (2009) Biol. Proced. Online 11:32-51). The N:P ratio (the ratio of protonatable nitrogen (N) from the ionizable lipid (SM-102) and anionic phosphate groups (P) from the mRNA) has important effects on cellular uptake and transfection efficiency (Li et al. (2022) Nat. Commun. 13:5561; Suzuki & Ishihara (2021) Drug Metab. Pharmacokinet. 41:100424; Gujrati et al. (2014) Mol. Pharm. 11:2734-2744; Douka (2023) J. Control. Release 361:455-469). In all the LNP formulations herein, the N:P ratio is 5.1 (Table 7). Clinically relevant LNP formulations Moderna, Pfizer, and Onpattro have N / P ratios of 6, 6, and 3, respectively (Schoenmaker et al. (2021) Int. J. Pharm.601:120586). The modular nature of this protocol allows researchers to make changes to prepare mRNA LNP formulations of interest beyond the standard base LNP formulation used in this study. Table 7. The N / P ratio of the LNPs. 1. Molecular weight of SM-102: 710.182 μg / μmol 12Attorney Docket No.5470.971.WO

[0128] The key parameters used to characterize mRNA LNPs include size, PDI, zeta potentials, and mRNA encapsulation efficiency. The freshly prepared mRNA LNPs may be diluted in 1x PBS to quantify particle size and PDI using a DLS instrument. The freshly prepared mRNA LNPs may also be diluted in 0.1x PBS to quantify the zeta potential on an ELS instrument. The reason for using 0.1x PBS to measure the zeta potential of LNPs is to reduce the ionic strength of the medium, since high ionic strength might lead to the aggregation of nanoparticles (Skoglund et al. (2017) PLoS ONE 12:e0181735; Wang et al. (2017) J. Environ. Sci. (China) 51:248-255). Additionally, measuring LNPs in a slightly buffered condition helps maintain a stable pH, as fluctuations in pH can significantly impact zeta potential values (Berg et al. (2009) Nanotoxicology 3:276-283). Therefore, it is recommended to use diluted PBS (i.e., 0.1x PBS) to measure the zeta potential of LNPs (Yap et al. (2024) J. Colloid Interface Sci. 656:409-423; Escalona-Rayo et al. (2023) Biomed. Pharmacother.165:115065; Zhang et al. (2020) Pharmaceutics 12:p.1042).

[0129] The mRNA concentration and encapsulation efficiency of the formulated LNPs may be assessed using a Quant-iT Ribogreen™ assay, following the provided assay protocols (Steps 11C, Table 4). Our protocol specifies a 100-fold dilution of the mRNA LNPs for analyzing mRNA concentration and encapsulation efficiency. Researchers may choose to optimize this ratio according to their specific application requirements. It is necessary to make sure that the mRNA concentration measured by Quant-iT Ribogreen™ assay is within the range of the standard curve. After dialysis, LNPs are stored in PBS. PBS, being aqueous in nature, can facilitate ester bond cleavage via hydrolysis, potentially leading to the degradation of the mRNA, LNPs, or both over time. To assess the stability of mRNA LNPs, their size changes can be measured following incubation under various conditions. In this protocol, we incubate the mRNA LNPs in pH 4, pH 5, pH 6, and pH 7.4 buffers, DMEM + 10% FBS, and RPMI + 10% FBS media at 4°C, 37°C for 5 days. One can make changes in these pH values, media compositions, buffers, temperatures, and incubation times for their specific applications. Additionally, the stability of mRNA LNPs may be monitored by measuring the in vitro protein expression over the desired time (Day 0, 7, 14, and 21), indicating the shelf life of the mRNA LNPs. Example 6: In vitro evaluation methods of mRNA LNPsAttorney Docket No.5470.971.WO

[0130] Eight formulations of mRNA LNPs (see Table 2), prepared via microfluidic mixing, are employed as examples to assess FLuc expression, cell viability, and cell uptake in cells. The FLuc mRNA LNPs can be diluted to a concentration of 500 ng / mL for the treatment of cells. Control groups can include naked FLuc mRNA, fresh media, and Lipofectamine™ 2000 to evaluate FLuc expression. Additionally, a negative viability control is required to ensure accuracy in the cell viability assay. In this protocol, we use 1% v / v Triton™ as the negative viability control for the cell viability assay. We select the HepG2 cell line (a hepatocellular carcinoma cell line) as an example, given that the liver is the primary target organ for many mRNA LNP formulations. One can opt to select an alternative negative viability reagent and other cell lines according to their specific applications. It is important to avoid selecting kill reagents that easily evaporate such as ethanol, as they may inadvertently cause cell death in other wells during the incubation process. The cells can then be treated with mRNA LNPs (100 µL 500 ng / mL of total mRNA per well, n = 3) for 24 hours, followed by the measurement of FLuc expression using Bright-Glo™ Luciferase assay system and viability using AlamarBlue™ assay (Example 4, Steps 12-25).

[0131] To evaluate cell uptake of mRNA LNPs, fluorescently-labeled molecules in the mRNA LNPs are used. In this protocol, we use Atto-488 labeled DOPE molecules in mRNA LNP formation (Example 4, Steps 26-35). To formulate Atto-488 labeled DOPE mRNA LNPs, we employ microfluidic mixing of an organic phase including SM-102, Atto-488 labeled DOPE, cholesterol, C14-PEG-2000 and optional excipients with an aqueous phase including mRNA. The Atto-488 labeled mRNA LNPs are then diluted to a concentration of 500 ng / mL. Cells are treated with Atto-488 labeled mRNA LNPs (400 µL 500 ng / mL of total mRNA per well, n = 3). Cells treated with fresh media are used as an unstained control. After 24 hours, the cells are washed and harvested with trypsin-EDTA (no phenol red) and quantified by flow cytometry. One can adjust the incubation time, cell type, and cell viability assay reagent based on specific application requirements. The cell uptake is quantified by the percentage of cells exhibiting higher fluorescence signals compared to media-treated control cells.

[0132] In this protocol, we also perform an alternative method for simultaneously quantifying both protein expression and cell uptake using Cy5-EGFP mRNA. In this approach the mRNA is fluorescently labeled, and the expressed protein is also fluorescent. The Cy5-EGFP mRNA LNPs are then diluted to a concentration of 500 ng / mL. Cells are treated with Cy5-EGFP mRNA LNPs (400 µL 500 ng / mL of total mRNA per well, n = 3). After 24 hours, the cells are washed andAttorney Docket No.5470.971.WO harvested with trypsin-EDTA (no phenol red). One may adjust the incubation time and cell type based on specific application requirements. The Cy5 signal indicates the presence of mRNA within cells, while the EGFP signal reflects the production of the target protein. Both fluorescent signals can be quantified using flow cytometry, with quadrant gates providing insights into the percentage of cells that: i. Have not taken up the mRNA (Cy5(-) / EGFP(-)) ii. Empty gate (i.e., a quadrant where one would expect no cells) (Cy5(-) / EGFP(+)) iii. Have taken up the mRNA but not translated it (Cy5(+) / EGFP(-)) iv. Have both taken up the mRNA and translated it into protein (Cy5(+) / EGFP(+)) This information is obtained in Step 36 (Example 4). Additionally, visualization and qualitative quantification may be conducted using confocal microscopy (Example 4, Steps 37-45). Example 7: Mechanism Investigation Methods

[0133] Endosomal escape is studied by incubating HepG2 cells with Atto-488 labeled mRNA LNPs and observing endosomal escape using confocal microscopy (Example 4, Steps 45-57). The Atto-488 labeled mRNA LNPs are diluted to a concentration of 500 ng / mL. Cells are treated with Atto-488 labeled mRNA LNPs (300 µL 500 ng / mL of total mRNA per well, n = 3). After 4 hours, the cells are stained with LysotrackerTMDeep Red, and Hoechst 33342. The live cell imaging is performed by a confocal laser scanning microscope. In the confocal images, cell nuclei are blue (stained by Hoechst 33342), Atto-488 mRNA LNPs are green, endosomes are red (stained by LysotrackerTMDeep Red), and mRNA LNPs trapped in endosomes are yellow (i.e., colocalization of the green and red signals). Moreover, the Pearson Correlation Coefficient (PCC, a coefficient where a value of 0 implies 100% endosomal escape and a value of 1 implies 0% endosomal escape) can also be quantified using WCIF Image J software to quantitatively indicate the endosome escape ability of mRNA LNPs (French et al. (2008) Nat. Protoc.3:619-628). Cells cannot be fixed for performing this protocol with live cell LysotrackerTMDeep Red dye. We recommend studying the endosomal escape of mRNA LNPs within 6 hours, as incubation periods shorter than 6 hours are generally sufficient for mRNA LNPs to escape from endosomes (Gilleron et al. (2013) Nat. Biotechnol. 31:638-646). The incubation time for endosomal escape studies can be adjusted according to specific application requirements.Attorney Docket No.5470.971.WO

[0134] To evaluate the proton sponge effect of nanoparticle escape from endosomes, we employ a calcein dye-based assay. Cells are treated with fresh media or media containing bafilomycin A1(inhibitor of ‘proton sponge effects’). Then the calcein is added into those cells, followed by the addition of the mRNA LNPs (the mRNA LNPs are diluted to a concentration of 500 ng / mL, 100 µL 500 ng / mL of total mRNA per well, n = 3). Non-fluorescently labeled mRNA is used to avoid interference with the calcein signal. Cells are treated with calcein, and cells treated with calcein + bafilomycin A1without adding any mRNA LNPs are used as control. After 4 hours, the live cell imaging is performed by a confocal laser scanning microscope (Example 4, Steps 58- 66). If the calcein in the mRNA LNP treated group spreads throughout the cells, it indicates that the endosomes have burst, allowing for mRNA LNP escape. If the calcein in the mRNA LNP and bafilomycin A1treated group exhibit only small, punctuated dots similar to the cell control groups, it suggests that the ‘proton sponge effect’ is one of the underlying mechanisms for their endosomal escape. One may adjust the incubation time and cell type according to specific application requirements. The benefits of using this calcein leakage assay include the ability to use 'label-free' LNPs, as fluorescent labeling might affect the physicochemical properties of LNPs, such as stability, size, and charge. Additionally, this technique has been validated for the evaluation of endosomal escape in particle systems (Wong et al. (2015) Soft Matter 11:2993-3002; Smith et al. (2019) Bioconjug. Chem.30:263-272; Chen et al. (2019) ACS Nano 13:11653-11664). However, we advise against incubation periods longer than 4 hours, as they may compromise the inhibitory efficacy of bafilomycin A1. Example 8: In vivo Evaluation of mRNA LNPs

[0135] To evaluate mRNA LNP efficacy and biodistribution in vivo, we use C57BL / 6 mice (female 6-8 weeks old, weight 18-21 g), intravenously injected with various FLuc mRNA LNPs formulations at a dose of 0.65 mg / kg (n = 3 each group) with a fluorescent molecule (DiD) incorporated into the LNPs to allow biodistribution to be assessed. Mice are injected with naked mRNA or PBS as a control. Mice should be monitored for any signs of sickness or distress, as this could hinder accurate data collection (Burkholder (2012) Curr. Protoc. Mouse Biol.2:145-165). After 24 hours, mice are intraperitoneally injected with D-luciferin, and the fluorescence and luminescence signals (indicative of intracellular protein) emitted by each dissected organ are quantified, revealing their biodistribution using an IVIS system. Tolerability studies are performedAttorney Docket No.5470.971.WO involving weight retention assessment, histological evaluation, hematology testing of CBC, and clinical chemistry testing of liver and kidney function, following the procedures in Step 67C (Example 4). The injection time may be adjusted according to specific application requirements. However, we recommend imaging the organs within 72 hours, as the luminescence signals tend to decrease over time. If the researchers are interested in the reactogenicity of the mRNA LNPs, they may also measure the cytokine and chemokine levels with ELISAs or similar assays (Korzun et al. (2024) ACS Nano 18:24842-24859; Parhiz et al. (2022) J. Control. Release 344:50-61). We have not tested other animal strains or sexes but expect that users could apply this protocol to other mouse strains that are relevant for their specific application as needed (Miller et al. (2017) FASEB J.31:29-34). Studies have shown that intramuscular injection of FLuc mRNA in both female and male BALB / c mice resulted in no significant differences in FLuc protein expression, but some differences in IgG values were observed between sexes (Binici et al. (2024) Vaccines (Basel) 12:p.282). While these studies involved a different mouse strain and used intramuscular rather than intravenous injection, we expect that our protocol may yield similar results across male, female, and mixed populations. Additionally, to measure the efficacy of mRNA LNP formulations to facilitate the expression of secreted proteins, we employ a murine model with erythropoietin (EPO) mRNA. Here, C57BL / 6 mice (female 6-8 weeks old, weight 18-21 g) are intravenously injected with various EPO mRNA LNPs formulations at a dose of 0.65 mg / kg (n = 3 each group). After 24 hours, a cardiac puncture bleed is performed to collect 200 µL of blood into a Minicollect tube for each mouse at room temperature. The concentration of EPO protein in the serum sample is measured using a Human EPO ELISA kit. One can adjust the injection time according to specific application requirements. However, we recommend collecting blood within 72 hours, as it has been suggested that most of the EPO protein expressed from EPO mRNA LNPs is quantified before this time point (Riley et al. (2021) Sci. Adv.7:p. eaba1028; Yang et al. (2020) Bioact. Mater. 5:1053-1061; Kevin et al. (2016) Front. Bioeng. Biotechnol. 4; Mrksich et al. (2024) J. Biomed. Mater. Res. A 112:1494-1505). The analysis time can be adjusted depending on the a specific application. Example 9: Experimental Results

[0136] The results of size (FIG. 5A), PDI (FIG. 5B), zeta potential (FIG. 5C) and mRNA encapsulation efficiency (FIG. 5D) demonstrate the expected outcomes of formulating mRNAAttorney Docket No.5470.971.WO LNPs using microfluidic mixing, with sizes (<300 nm), PDIs (<0.3), zeta potentials (-5 mV to 5 mV) and mRNA encapsulation efficiencies (39% to 75%) being observed. Additionally, there was no observable difference in mRNA encapsulation efficiency at 22°C and 37°C. Furthermore, our stability assessment revealed that TAT LNPs, Octaarginine LNPs, and Cyclosporine LNPs exhibited increased size over time (FIGS. 5E-5F), indicating that they may not be suitable for long-term storage or use in prolonged applications. To further investigate the dominant interactions that govern the stability of each LNP formulation, urea (hydrogen bonding), Tween® 20 (hydrophobic interactions) and NaCl (electrostatic interactions) were used to monitor the LNP size change. For this analysis, 20 µL of FLuc mRNA LNP, TA LNP, PEI LNP, DC-Chol LNP, Ca3(PO4)2LNP, TAT LNP, Octaarginine LNP, cyclosporine LNP, and ATP LNP suspensions were dissolved in 80 µL of 100 mM urea, 100 mM Tween® 20, or 100 mM NaCl solution and incubated in an Eppendorf thermomixer at 37°C and 500 rpm for the desired time. The suspensions were further diluted with PBS buffer and changes in the LNPs size were monitored over 48 hours via DLS measurements. In collectively analyzing these data, several trends were observed: LNPs were stabilized predominantly via hydrophobic interactions; and after adding various excipients, the dominant interactions for stabilizing the LNPs could change, such as hydrogen bonding for stabilizing ATP LNPs, and electrostatic interactions for stabilizing TA LNPs (FIG.5G).

[0137] The stability of mRNA LNPs may also be monitored by measuring in vitro FLuc protein expression over a specified period (days 0, 7, 14, and 21). The level of protein expression from FLuc mRNA LNPs decreased to as low as ~0.5% over 21 days at 4°C compared to day 0, indicating that mRNA LNPs lose their effectiveness when stored at 4°C over time (FIGS.6A-6C). Of note, FLuc expression increased with increased dosage of mRNA from 50 ng to 200 ng per well (FIG. 6D). It was further observed that cells treated with ATP LNPs exhibited the highest FLuc expression values, whereas cells treated with TAT and Octaarginine LNPs exhibited the lowest FLuc expression values (FIG.6D).

[0138] In the in vitro evaluation of FLuc expression in HepG2 cells, the goal of our protocol is to assess the efficacy of LNPs in delivering mRNA payloads and initiating protein expression. The results showed that all mRNA LNPs are well-tolerated as expected (FIG. 7A), with the 1% v / v Triton™ exhibiting less than 10% viability indicating the AlamarBlueTMcell viability assay works. For protein expression, ATP LNPs exhibited the highest FLuc expression compared to other formulations (Ma & Fenton (2023) J. Am. Chem. Soc.145:19800-19811; Ma & Fenton (2023) J.Attorney Docket No.5470.971.WO Am. Chem. Soc.145:11375-11386), while TAT LNPs and Octaarginine LNPs demonstrated lower FLuc expression (similar to the naked mRNA control and cells treated with fresh media control group) (FIG. 7B). For assessing cell uptake, our approach is to utilize Atto-488 labeled DOPE molecules in LNP formation and quantify the percentage of cells exhibiting higher fluorescence signals compared to media-treated controls. TAT LNPs, Octaarginine LNPs, and TA LNPs showed lower cell uptake compared to the LNP group (FIG. 7C). We also demonstrate an alternative approach for in vitro evaluation of mRNA LNPs by encapsulating Cy5-EGFP mRNA into LNP formulations. Protein expression and cell uptake can be quantitatively assessed by flow cytometry (FIG.8, FIGS.9A-9B), yielding similar results to those obtained from FIGS.7B-7C. With this approach, protein expression and cell uptake can be assessed simultaneously. In particular, flow cytometry results could be classified into three different categories: i. both Cy5 and EGFP negative signals, indicating no LNP uptake by cells; ii. Cy5 positive signals, and EGFP negative signals, indicating LNP uptake by cells but no protein expression; and iii. Both Cy5 and EGFP positive signals, indicating LNP uptake by cells resulting in protein expression (FIG. 8). The incubation time to evaluate the cellular uptake and EGFP protein expression may be adjusted according to the specific application, with an example at the 6-hour time point and Moderna formulation data shown in FIGS.10A-10C. To qualitatively evaluate protein expression and cell uptake, confocal microscopy can be used to visualize Cy5-EGFP mRNA LNPs, providing a more illustrative and direct way to evaluate the efficacy of mRNA LNPs (FIG.9C). As shown in FIG.9D, ATP LNPs exhibited higher protein expression (indicated by more EGFP protein and more color), whereas TAT LNPs, Octaarginine LNPs, and TA LNPs showed lower protein expression (indicated by less EGFP protein and less color), consistent with the results in FIGS.9A-9B.

[0139] Our protocol evaluates endosomal escape using the Atto-488 labeled mRNA LNPs with the lysotracker assay and determine their colocalization. In these confocal images, cell nuclei are blue, Atto-488 labeled mRNA LNPs are green, and endosomes are red; yellow (i.e., colocalization of the green and red signals) indicates that the mRNA LNPs are trapped in endosomes (FIG.11). TAT LNPs, ATP LNPs, TA LNPs, Cyclosporine LNPs, and Ca3(PO4)2LNPs showed improved endosomal escape, indicated by lower PCC values (FIG. 12A) and representative confocal microscopy images (FIG. 12B). To further evaluate one of the underlying mechanisms of endosomal escape – the 'proton sponge effects’ – we employed a proton sponge effect inhibitor (bafilomycin A1) and a membrane-impermeable dye (calcein) (FIG. 12C). Upon addingAttorney Docket No.5470.971.WO bafilomycin A1and calcein, we observed that calcein spread throughout the cells only in the presence of Octaarginine LNPs (FIG. 12D). This indicates that the ‘proton sponge effect’ is not one of the main mechanisms for their endosomal escape. By contrast, the 'proton sponge effect’ appeared to be one of the main mechanisms for endosomal escape for all the other mRNA LNP groups. This protocol may be customized by adding optional staining, such as Hoechst for nuclei and CellMask™ Deep Red Actin Tracking Stain for the cytoskeleton (F-actin), based on the specific application needs (FIGS.13A-13B).

[0140] Our protocol evaluates in vivo efficacy by injecting FLuc or EPO mRNA LNPs via I.V. injection into C57BL / 6 mice. Characteristics of example LNP formulations delivering mRNA encoding for FLuc (intracellular protein) or EPO (secreted protein) are presented in Tables 8-9. Researchers performing these injections randomized the experimental group to which each mouse was assigned and were blinded to the identity of the mRNA LNP that each experimental group received. The injections were performed at the same time of day and location for all groups. I.V. administration of FLuc mRNA LNPs facilitated the quantification of fluorescence and luminescence signals (indicative of intracellular protein) emitted by each dissected organ, revealing their biodistribution and functional protein expression level. Furthermore, I.V. administration of human erythropoietin (EPO) assessed the efficacy of the LNP formulation in inducing the expression of secreted proteins in the blood serum, measured via ELISA. Our protocol also includes weight retention assessment, histological evaluation, hematology CBC testing, and clinical chemistry testing of liver and kidney function to evaluate the tolerability of LNP formulations (FIG. 14A). The results indicate several key findings that demonstrate the applicability of our protocol for evaluating mRNA LNPs in vivo. First, TAT LNPs and Octaarginine LNPs exhibited lower FLuc signals, which was similar to the naked mRNA and PBS control group (FIG.14A and FIG.14C). Second, TA LNPs, PEI LNPs, and ATP LNPs exhibited higher FLuc expression (FIG. 14A and FIG. 14C). Third, LNP formulations without any excipients mainly expressed FLuc protein in the liver (Ma & Fenton (2023) Adv. Therap. 6:p.2200305; Ma & Fenton (2023) J. Am. Chem. Soc.145:19800-19811; Ma & Fenton (2023) J. Am. Chem. Soc. 145:11375-11386). The biodistribution of each representative LNP formulation indicated predominant accumulation in the liver and some accumulation in the spleen, similar to the biodistribution observed with the Moderna control (FIG. 14B). Fourth, various mRNA LNP formulations could facilitate the expression of the protein in different organs. Fifth, the amount ofAttorney Docket No.5470.971.WO EPO expression secreted into the blood serum followed similar trends as FLuc expression (FIG. 14E). Lastly, all mRNA LNP formulations demonstrated good tolerability, as analyzed by weight retention (FIG. 15), histology (FIG. 14F), hematological test-complete blood count and blood paneling to test liver and kidney function data (FIG.14G), including alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transferase (AST), blood urea nitrogen (BUN), and creatinine (CREAT) tests. These biomarkers in the treatment groups showed similar results to those in the PBS control group. Healthy levels of these markers for C57BL / 6 female mice can vary and may be within the following ranges: ALP (122-200 U / L), ALT (22-32 U / L), AST (46-221 U / L), BUN (20-40 mg / dL) and CREAT (0.1-1.8 mg / dL)(Wquimby & Hluong (2007) In The Mouse in Biomedical Research 171-216 (Elsevier); Wei & Dong (2012) Am. J. Physiol. Renal Physiol.303:F1487-94). No differences were observed between the mRNA LNP treatment groups and the naked mRNA or PBS control group. Table 8. Size, zeta, encapsulation efficiency, PDI of in vivo intravenous injected LNPs-FLuc mRNA. Zeta potential FLuc mRNA Encapsulation Formulation Size (nm) PDI (mV) Efficiency (%)Table 9. Size, zeta, encapsulation efficiency, PDI of in vivo intravenous injected LNPs-EPO mRNA. Zeta potential EPO mRNA EncapsulationAttorney Docket No.5470.971.WO DC-Chol LNPs 150.8±3.2 0.18±0.09 5.83±5.86 75.8±2.8 Ca3(PO4)2 LNPs 180.8±7.9 0.21±0.05 5.06±2.12 73.0±2.8be potentially used as vaccine (FIGS.16A-16H). Given the generality of the prior I.V. results, LNPs, TAT LNPs and ATP LNPs for intramuscular (I.M.) injection in vivo were investigated (FIGS.16I, 16J). Characteristics of example LNP formulations delivering mRNA encoding for FLuc are presented in Table 10. Table 10. Size, zeta, encapsulation efficiency, PDI of in vivo intramuscular injected LNPs-FLuc mRNA. Zeta potential EPO mRNA Encapsulation Formulation Size (nm) PDI (mV) Efficiency (%)

[0142] In collectively analyzing these data, several results emerged. First, RIME could be used to screen the activity of LNPs on DC2.4 cells regarding cellular uptake and protein expression (FIGS.16A-16C). Second, FLuc expression of each LNP formulation was consistent with RIME results (FIG.16D). Third, cellular association of each FLuc mRNA LNP formulation was 100%, indicating the challenging nature of analyzing cell association (FIG.16E). Therefore, geometric mean fluorescence intensity (GMFI) results could indicate that there was significantly less TAT and Octaarginine LNP uptake by DC2.4 cells than LNPs without any excipients (FIG. 16F). Similarly, DC-Chol, Ca3(PO4)2, cyclosporine and ATP LNPs had significantly higher cellular uptake than LNPs without any excipients. Fourth, lower PCC values and representative confocal microscopy images of each LNP formulation compared to LNPs without any excipients indicate better endosomal escape (FIGS. 16G-16H). Fifth, LNP formulation with different excipients might potentially alter the innate biodistribution via IM injection (FIGS.16I-16J). And sixth, theAttorney Docket No.5470.971.WO luminescence value between different mice groups treated with same LNPs was high, possibly due to the small injection volume via the I.M. routes (FIGS.16I-16J).

[0143] Following our protocol allows for an efficient assessment of the in vivo efficacy of mRNA LNPs, facilitating the selection of a lead formulation for further therapeutic testing and application. Taken collectively, this protocol can aid in designing optimized mRNA LNP formulations through the evaluation of their performance both in vitro and in vivo, thus facilitating worldwide research on mRNA LNP development.

[0144] The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

Attorney Docket No.5470.971.WO WHAT IS CLAIMED: What is claimed is:

1. A method of evaluating a polymer nanoparticle, the method comprising: exposing a cell to the polymer nanoparticle, the polymer nanoparticle comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; and sorting the exposed cell by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein; and evaluating the polymer nanoparticle for its ability to deliver the mRNA to the cell and express the protein based on the presence or absence of the signals.

2. The method of claim 1, wherein the sorting comprises flow cytometry.

3. The method of claim 1, wherein the polymer nanoparticle is a lipid nanoparticle (LNP).

4. The method of claim 3, wherein the LNP comprises an ionizable lipid, a phospholipid, a sterol, and / or a PEG molecule, and optionally one or more excipients.

5. The method of claim 4, wherein the one or more excipients is a small molecule, a biologic, or an inorganic compound.

6. The method of claim 4, wherein the one or more excipients is a hydrophobic, hydrophilic, or amphiphilic molecule.

7. The method of any one of claims 1-6, wherein the detectable label is a fluorescent label or radiolabel.

8. The method of claim 7, wherein the fluorescent label is selected from a cyanine (e.g., Cy2m Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 Cy7), rhodamine, Fluorescein isothiocyanate (FITC), Phycoerythrin (PE), Allophycocyanin (APC), Peridinin-Chlorophyll-Protein (PerCP), Alexa Fluor®, or DyLight® dye.Attorney Docket No.5470.971.WO 9. The method of any one of claims 1-8, wherein the detectable protein is a protein that fluoresces when exposed to light.

10. The method of any one of claims 1-9, wherein the detectable protein is enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), Venus, monomeric Infrared Fluorescent Protein (mIFP), Long Stokes Shift monomeric Orange (LssmOrange), Tag Red Fluorescent Protein 657 (TagRFP657), monomeric Apple (mApple), monomeric Orange2 (mOrange2), Sapphire, monomeric Tag Blue Fluorescent Protein (mTagBFP2), tdTomato, monomeric Cherry (mCherry), monomeric Ruby (mRuby), monomeric Cerulean3 (mCerulean3).

11. The method of any one of claims 1-10, comprising evaluating a plurality of LNPs.

12. The method of claim 11, wherein each LNP has a different formulation.

13. The method of any one of claims 1-12, comprising exposing a plurality of cells to the LNP.

14. The method of claim 13, wherein the plurality of cells comprises more than one cell type.

15. The method of claim 13 or claim 14, further comprising sorting the plurality of cells by cell type.

16. The method of any one of claims 1-15, wherein the detectably labeled mRNA and the detectable protein emit signals at different wavelengths, preferably comprising an at least 10 nm to 20 nm difference in wavelength.

17. The method of any one of claims 13-16, wherein the plurality of cells is in the range of about 50 cells to about 1,000,000 cells.

18. The method of any one of claims 1-17, further comprising assaying viability of the cell.Attorney Docket No.5470.971.WO 19. A method of identifying a polymer nanoparticle with increased endosomal escape ability, the method comprising: exposing a plurality of cells to a candidate polymer nanoparticle, the candidate polymer nanoparticle comprising a detectably labeled mRNA, the mRNA encoding a detectable protein; sorting the exposed cells by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein; quantifying the cells comprising both the detectably labeled mRNA and the detectable protein; and assessing the quantification of the cells relative to cells exposed to a reference polymer nanoparticle, to thereby identify the polymer nanoparticle with increased endosomal escape ability.

20. The method of claim 19, herein the sorting comprises flow cytometry.

21. The method of claim 19, wherein the polymer nanoparticle is a lipid nanoparticle (LNP).

22. The method of claim 21, wherein the lipid nanoparticle comprises an ionizable lipid, a phospholipid, a sterol, and / or a PEG molecule, and optionally one or more excipients.

23. The method of claim 22, wherein the one or more excipients is a small molecule, a biologic, or an inorganic compound.

24. The method of claim 22, wherein the one or more excipients is a hydrophobic, hydrophilic, or amphiphilic molecule.

25. The method of any one of claims 19-24, wherein the detectable label is a fluorescent label or radiolabel.

26. The method of claim 25, wherein the fluorescent label is selected from a cyanine (e.g., Cy2m Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 Cy7), rhodamine, Fluorescein isothiocyanate (FITC),Attorney Docket No.5470.971.WO Phycoerythrin (PE), Allophycocyanin (APC), Peridinin-Chlorophyll-Protein (PerCP), Alexa Fluor®, or DyLight® dye.

27. The method of any one of claims 19-26, wherein the detectable protein is a protein that fluoresces when exposed to light.

28. The method of any one of claims 19-27, wherein the detectable protein is enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), Venus, monomeric Infrared Fluorescent Protein (mIFP), Long Stokes Shift monomeric Orange (LssmOrange), Tag Red Fluorescent Protein 657 (TagRFP657), monomeric Apple (mApple), monomeric Orange2 (mOrange2), Sapphire, monomeric Tag Blue Fluorescent Protein (mTagBFP2), tdTomato, monomeric Cherry (mCherry), monomeric Ruby (mRuby), monomeric Cerulean3 (mCerulean3).

29. The method of any one of claims 19-28, wherein the plurality of cells comprises more than one cell type.

30. The method of any one of claims 19-29, further comprising sorting the cells by cell type.

31. The method of any one of claims 19-30, wherein the detectably labeled mRNA and the detectable protein emit signals at different wavelengths, preferably comprising an at least 10 nm to 20 nm difference in wavelength.

32. The method of any one of claims 19-31, wherein the plurality of cells is in the range of about 50 cells to about 1,000,000 cells.

33. A method of comparing the ability of polymer nanoparticles to deliver an mRNA and express a protein in a cell, comprising: exposing a plurality of cells to the polymer nanoparticles, each polymer nanoparticles comprising a detectably labeled mRNA, the mRNA encoding a detectable protein;Attorney Docket No.5470.971.WO sorting the exposed cells by the presence or absence of a signal from the detectably labeled mRNA and the detectable protein of each cell; and comparing each polymer nanoparticles for its ability to deliver the mRNA to the cell and express the protein based on the present or absence of the signal.

34. The method of claim 33, wherein sorting the cells comprises flow cytometry.

35. The method of claim 33 or claim 34, wherein the polymer nanoparticle is a lipid nanoparticle (LNP).

36. The method of claim 35, wherein the method predicts the ability of the LNPs to deliver the mRNA to the cell and express the protein in vitro and / or in vivo.

37. The method of claim 35 or claim 36, wherein the ability of the LNPs comprises cellular association and / or endosomal escape.

38. The method of any one of claims 33-37, wherein the lipid nanoparticle comprises an ionizable lipid, a phospholipid, a sterol, and / or a PEG molecule, and optionally one or more excipients.

39. The method of claim 38, wherein the one or more excipients is a small molecule, a biologic, or an inorganic compound.

40. The method of claim 38, wherein the one or more excipients is a hydrophobic, hydrophilic, or amphiphilic molecule.

41. The method of any one of claims 33-40, wherein the detectable label is fluorescent label or radiolabel.

42. The method of claim 41, wherein the fluorescent label is selected from a cyanine (e.g., Cy2m Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 Cy7), rhodamine, Fluorescein isothiocyanate (FITC),Attorney Docket No.5470.971.WO Phycoerythrin (PE), Allophycocyanin (APC), Peridinin-Chlorophyll-Protein (PerCP), Alexa Fluor®, or DyLight® dye.

43. The method of any one of claims 33-42, wherein the detectable protein is a protein that fluoresces when exposed to light.

44. The method of any one of claims 33-43, wherein the detectable protein enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), Venus, monomeric Infrared Fluorescent Protein (mIFP), Long Stokes Shift monomeric Orange (LssmOrange), Tag Red Fluorescent Protein 657 (TagRFP657), monomeric Apple (mApple), monomeric Orange2 (mOrange2), Sapphire, monomeric Tag Blue Fluorescent Protein (mTagBFP2), tdTomato, monomeric Cherry (mCherry), monomeric Ruby (mRuby), monomeric Cerulean3 (mCerulean3).

45. The method of any one of claims 33-44, wherein the plurality of cells comprises more than one cell type.

46. The method of any one of claims 33-45, wherein the detectably labeled mRNA and the detectable protein emit signals at different wavelengths, preferably comprising an at least 10 nm to 20 nm difference in wavelength.

47. The method of any one of claims 33-46, wherein the plurality of cells is in the range of about 50 cells to about 1,000,000 cells.

48. The method of any one of claims 1-47, wherein the polymer nanoparticle or LNP further comprises an mRNA encoding a protein of interest.

49. The method of any one of claims 1-47, wherein the polymer nanoparticle or LNP further comprises a nucleic acid barcode.Attorney Docket No.5470.971.WO 50. A lipid nanoparticle (LNP) comprising an ionizable lipid, a phospholipid, a sterol, and / or a PEG lipid, and one or more excipients selected from octaarginine, cyclosporine, calcium phosphate, or TAT (48-57) peptide.

51. The LNP of claim 50, wherein the excipient comprises calcium phosphate in the range of at about 10 mol% to about 40 mol%.

52. The LNP of claim 50, wherein the excipient comprises octaarginine, cyclosporine, calcium phosphate, or TAT (48-57) peptide in the range of about 0.05 mol% to about 10 mol%.

53. The LNP of any one of claims 50-52, wherein the LNP comprises ionizable lipid in the range of about 15 mol% to about 95 mol%, phospholipid in the range of about 5 mol% to about 30 mol%, sterol in the range of about 20 mol% to about 60 mol%, and / or a PEG lipid in the range of about 1 mol% to about 5 mol%.

54. A lipid nanoparticle (LNP) comprising a detectably labeled mRNA, the mRNA encoding a detectable protein, and an mRNA encoding a protein of interest.

55. A lipid nanoparticle (LNP) comprising a detectably labeled mRNA, the mRNA encoding a detectable protein, and a nucleic acid barcode.

56. The LNP of claim 54 or claim 55, wherein the LNP comprises an ionizable lipid, a phospholipid, a sterol, and / or a PEG molecule, and optionally one or more excipients.

57. The LNP of claim 56, wherein the one or more excipients is a small molecule, a peptide, or an inorganic compound.

58. The LNP of any one of claims 54-57, wherein the detectable label is a fluorescent label.

59. The LNP of claim 58, wherein the fluorescent label is selected from a cyanine (e.g., Cy2m Cy3, Cy3B, Cy3.5, Cy5, Cy5.5 Cy7), rhodamine, Fluorescein isothiocyanate (FITC),Attorney Docket No.5470.971.WO Phycoerythrin (PE), Allophycocyanin (APC), Peridinin-Chlorophyll-Protein (PerCP), Alexa Fluor®, or DyLight® dye.

60. The LNP of any one of claims 54-59, wherein the detectable protein is a protein that fluoresces when exposed to light.

61. The LNP of any one of claims 54-60, wherein the detectable protein is enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), Venus, monomeric Infrared Fluorescent Protein (mIFP), Long Stokes Shift monomeric Orange (LssmOrange), Tag Red Fluorescent Protein 657 (TagRFP657), monomeric Apple (mApple), monomeric Orange2 (mOrange2), Sapphire, monomeric Tag Blue Fluorescent Protein (mTagBFP2), tdTomato, monomeric Cherry (mCherry), monomeric Ruby (mRuby), monomeric Cerulean3 (mCerulean3).

62. The LNP of any one of claims 54-61, wherein the detectably labeled mRNA and the detectable protein emit signals at different wavelengths, preferably comprising an at least 10 nm to 20 nm difference in wavelength.

Images