High throughput method for screening lipid nanoparticle efficacy and cytotoxicity

Abstract

The present disclosure provides compositions and methods for high throughput screening of LNP formulations in vitro to identify top candidates based on cytotoxicity and effective endosomal escape. LNPs are membrane-active agents, which can compromise the permeability barrier constituted by phospholipid plasma membranes leading to cytotoxicity. Following effective cellular uptake, efficacious LNPs must successfully escape the endosome, through LNP-endosomal membrane fusion to deliver their cargo to the cytosol. The methods described herein are based on synthetic cells that encapsulate a water-soluble fluorescent dye or whose membranes are labeled with a lipophilic fluorescent dye at self-quenching dye concentrations.

Description

HIGH THROUGHPUT METHOD FOR SCREENING LIPID NANOPARTICLE EFFICACY AND CYTOTOXICITYFIELD

[0001] The present disclosure relates to synthetic cell membranes and their use in screening the toxicity and efficacy of lipid nanoparticle formulations in vitro, in a high throughput format.BACKGROUND

[0002] Lipid nanoparticles (LNPs) have demonstrated great promise in recent years in the delivery of nucleic acid-based therapeutics. In 2018, the United States Food and Drug Administration (FDA) approved the first-ever RNA interference (RNAi) therapeutic, ONPATTRO® by Alnylam, for the treatment of hereditary transthyretin amyloidosis (El-Mayta et al. (2023) J Vis Exp. (191 ):10.3791 / 64810). This was an important step forward for lipid nanoparticles and RNA-based therapies. More recently, Moderna and Pfizer / BioNTech received FDA approvals for their first-in-class mRNA-LNP vaccines against SARS-CoV-2; COMIRNATY® (U.S. Food and Drug Administration, BLA Approval 125742 / 0, Aug 23, 2021) and SPIKEVAX® respectively (U.S. Food and Drug Administration, BLA Approval 125752 / 0, Jan 31 , 2022). While LNPs have demonstrated significant promise for nucleic acid delivery applications, their therapeutic potential is limited by inefficient delivery to target cells and tissues in vivo (Zhang et al. (2014) J Control Release. 174:7-14). Cellular uptake of LNPs begins with endocytosis followed by endosomal escape, LNP degradation, and cargo release into the cytosol (Zhang et al. (2014) J Control Release. 174:7-14). LNPs however face several delivery pharmacokinetic and biodistribution barriers, including nonspecific serum protein interactions, rapid clearance, off-target localization (Zhang et al. (2014) J Control Release. 174:7-14). Moreover, the ability of LNP encapsulated cargo to escape early endosomes is critical for efficacy, failure results in residence in late-stage endosomes or lysosomes, where mRNA degradation occurs (Zhang et al. (2014) J Control Release.174:7-14). It has been consistently shown that most LNPs are sequestered and degraded in lysosomes, significantly limiting their efficacy (Zheng et al. (2023) Proc Natl Acad Sci U S A. 120(27):e2301067120). Expanding the use of nucleic acid-based therapies for different indications hinges on the ability to continuously evolve the design of LNPs with high potency, better endosomal escape, cellular-specific targeting, and low toxicity (Zheng et al. (2023) Proc Natl Acad Sci U S A. 120(27):e2301067120) . Most lipid-based nucleic acid delivery platforms that are undergoing clinical studies or on the market consist of four components: an ionizable lipid, cholesterol, a PEGylated lipid, and a helper phospholipid (e.g., 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC)) (Zhu et al. (2022) Nat Common.13(1 ):4282). Recent studies have reported that both the choice of lipid components, and therelative proportions of the lipid ingredients in the formulation, greatly influence in vivo transfection efficiency and tissue-specific delivery (Zhu et al. (2022) Nat Common. 13(1 ):4282). Development and screening of LNP libraries with different compositions and properties will accelerate the identification of the most suitable delivery system for a given biological application (Tome et al. (2021) APL Bioeng. 5 (3): 031511 ). Yet, developing suitable screening platforms with high accuracy, coupled with a rapid method of analysis, is challenging (Tome et al. (2021) APL Bioeng. 5 (3): 031511). In many cases, the selection of lead compounds relies on an extensive characterization of the LNP library using cellular models for the in vitro screening followed by the selection of a limited number of formulations for in vivo testing(Akinc et al. (2008) Nature biotechnology. 26, 561-569, Jayaraman, M. et al. (2012) Angewandte Chemie. 51 , 8259-8533, Wang, M. et al (2012) ACS Synthetic Biology 1 (9): 403-407). Yet this paradigm is changing since recent studies have adopted to test the LNP formulations after their synthesis immediately in animal models (Tome et al. (2021) APL Bioeng. 5 (3): 031511). Considering the cost and labor associated with in vivo screening, there is need for a well-designed and implemented high throughput (HTS) in vitro assay for screening LNP libraries that still relies on testing of interaction with lipids and lipid membrane targets for the selection of the best performing formulations for further development and formulation into therapeutics.SUMMARY

[0003] The present disclosure provides first-pass high throughput screening methods for determining LNP cytotoxicity or efficacious biomolecular cargo endosomal release into the cytosol, thus allowing the prioritization of the most promising LNP formulations for further development or in vivo testing. The disclosure features two complementary fluorescencebased methods and compositions for measuring LNP-induced membrane permeabilization and LNP- membrane fusion, which are proxies for LNP toxicity and efficacy respectively.

[0004] In one embodiment, the disclosure provides a method of measuring membrane permeabilization of a synthetic cell by a lipid nanoparticle (LNP), the method comprising: a) preparing a mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells; b) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells comprise a water-soluble fluorescent dye; and wherein an increase in fluorescence correlates with membrane permeabilization.

[0005] In an embodiment, the synthetic cell is a large unilamellar vesicle (LUV).

[0006] In one embodiment, the LUV is approximately from 30-400 nm in diameter.

[0007] In an embodiment, the water-soluble fluorescent dye is present in the synthetic cell at a self-quenching concentration.

[0008] In an embodiment, the dye is included in the assay at or above its self-quenching concentration.

[0009] In an embodiment, the increase in fluorescence intensity results from a decrease in self-quenching of the water-soluble fluorescent dye.

[0010] In another embodiment, the water-soluble fluorescent dye is selected from the group consisting of sulforhodamine B, FITC, 5(6)-carboxyfluorescein, and calcein.

[0011] In an embodiment, the water-soluble fluorescent dye is 5(6)-carboxyfluorescein (CF).

[0012] In another embodiment, the fluorescence signal intensity is measured using fluorescence spectroscopy.

[0013] In an embodiment, the preparing in step (a) and / or the measuring in step (b) occurs in a multi-well plate.

[0014] In one embodiment, the LNP comprises a cargo selected from the group consisting of a peptide, a nucleic acid, a small molecule, and a protein or combinations of the same.

[0015] In some embodiments, the LNP comprises a ribonucleic acid, protein, or drug cargo.

[0016] In another embodiment, the ribonucleic acid is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer- substrate RNA (dsRNA), an antisense oligonucleotide (ASO), an RNA aptamer, a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (IncRNA), a guide RNA (gRNA).

[0017] In an embodiment, the nucleic acid is a messenger RNA (mRNA).

[0018] In an embodiment, the conditions in step (a) optionally include a range of incubation parameters selected from the group consisting of temperature range, salinity range, ionic strength range, and pH range.

[0019] In an embodiment, the conditions in step (a) include a pH of approximately pH 7.4.

[0020] In yet another embodiment, the conditions in step (a) include a pH of approximately pH 5.5.

[0021] In another embodiment, the disclosure provides a method measuring fusion of a LNP and a synthetic cell, the method comprising: a.) preparing a mixture of LNPs andsynthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b.) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells comprise a lipophilic fluorescent dye; and wherein an increase in fluorescence correlates with fusion.

[0022] In an embodiment, the synthetic cell is a large unilamellar vesicle (LLIV).

[0023] In one embodiment, one embodiment, the LLIV is approximately from 30-400 nm in diameter.

[0024] In one embodiment, the lipophilic fluorescent dye is present in the synthetic cell at a self-quenching concentration.

[0025] In an embodiment, the dye is included in the assay at or above its self-quenching concentration.

[0026] In an embodiment, the increase in fluorescence results from a decrease in selfquenching of the lipophilic fluorescent dye.

[0027] In some embodiments, the lipophilic fluorescent dye is selected from the group consisting of Octadecyl Rhodamine B Chloride (R18), Diphenylhexatriene (DPH), N- (lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), and rac-2,3- dioleoylglycerol ester of rhodamine B (DORh-B).

[0028] In an embodiment, the lipophilic fluorescent dye is R18.

[0029] In another embodiment, the fluorescence signal intensity is measured using fluorescence spectroscopy.

[0030] In an embodiment, the preparing in step (a) and / or the measuring in step (b) occurs in a multi-well plate.

[0031] In some embodiments, the LNP comprises a cargo selected from the group consisting of a peptide, a nucleic acid, a small molecule, and a protein or combinations of the same.

[0032] In an embodiment, the LNP comprises a ribonucleic acid, protein, or drug cargo.

[0033] In yet another embodiment, the ribonucleic acid is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer- substrate RNA (dsRNA), an antisense oligonucleotide (ASO), an RNA aptamer, a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (IncRNA), a guide RNA (gRNA).

[0034] In one embodiment, the nucleic acid is a messenger RNA (mRNA).

[0035] In an embodiment, the conditions in step (a) optionally include a range of incubation parameters selected from the group consisting of temperature range, salinity range, ionic strength range, and pH range.

[0036] In one embodiment, the conditions in step (a) include a pH of approximately pH7.4.

[0037] In another embodiment, the conditions in step (a) include a pH of approximately pH5.5.

[0038] In one embodiment, the disclosure also provides a method of concurrently measuring permeabilization of synthetic cell membranes by an LNP and fusion of synthetic cell membranes with LNPs, the method comprising: a.) preparing a first mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b.) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells in the first mixture comprise a water-soluble fluorescent dye; wherein the synthetic cells in the second mixture comprise a lipophilic fluorescent dye; wherein an increase in fluorescence measured in the first mixture correlates with membrane permeabilization; and wherein an increase in fluorescence measured in the second mixture correlates with membrane fusion.

[0039] In another embodiment, the disclosure provides a method of concurrently measuring (i) membrane permeabilization and (ii) fusion of a synthetic cell by a lipid nanoparticle (LNP), the method comprising: a.) preparing a first mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b.) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells in the first mixture are LUVs and comprise 5(6)-carboxyfluorescein; wherein the synthetic cells in the second mixture are LUVs and comprise R18; wherein an increase in fluorescence measured in the first mixture correlates with membrane permeabilization; wherein an increase in fluorescence measured in the second mixture correlates with fusion.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a simplified, schematic representation of outcomes of the interaction between LNPs encapsulating cargo and target cells; A) Cargo loaded LNPs cross the plasma membrane and are internalized into the cell through endocytic pathways where they become entrapped in endosomes, for successful cytosolic cargo delivery, the cargo must undergo endosomal escape before endosomes fuse with lysosomes as the resulting acidification could lead to cargo degradation, B) LNPs rupture the plasma membrane preventing cellular internalization and cargo delivery and cause cytotoxicity.

[0041] Figure 2 provides schematic representations of a fluorescence-based assay measuring LNP interactions with synthetic cell membranes. Figure 2A is a schematic drawing illustrating the dye leakage assay of the present disclosure, LNP interaction results in LUV membrane permeabilization and leakage of the aqueous CF dye whose fluorescence increases from the dye dequenching. In this assay, the extent of dye leakage (increase in fluorescence) is a proxy for LNP toxicity. Figure 2B is a mock graph exemplifying three possible traces of fluorescence intensity depending on the release rate of CF from LUVs; fast, slow or minimal, after mixing with LNPs. At t=0, LNPs are added to dye-encapsulating synthetic cells, the x axis shows the speed while the y axis shows the extent of LNP-induced dye leakage from membrane permeabilization or instabilities. Figure 2C is a schematic drawing illustrating the membrane lipid-mixing assay of the present disclosure, the assay is based on increased R18 fluorescence due to dye dilution (dequenching) upon fusion of LUVs with fluorescently labeled membranes with non-labeled LNPs, the extent of membrane fusion (fluorescence increase) is a proxy for LNP efficacy. Figure 2D is a mock graph exemplifying three possible R18 fluorescence profiles after mixing R18 labeled LUVs with unlabeled LNPs depending on whether the membranes fuse fast and completely, interact and mix slowly, or they do not fuse at all. At t=0, LNPs are added to synthetic fluorophore- labeled cell membranes, the x axis shows the speed while the y axis shows the extent of LNP-induced synthetic cell membrane fusion.

[0042] Figure 3 is a schematic illustrating an embodiment of a workflow of the fluorescence -based assays of the disclosure. A multi well plate of the user’s preferred format can be loaded with the fluorescent synthetic cells of the present disclosure. An automated fluorescence well plate reader can measure the time-dependent interactions between the LNP drug carrier or vaccine candidates and said synthetic cells. Curves unique to each LNP under a range of conditions (temperature, pH etc.) can be analyzed for their kinetic data and compared to identify LNP formulations with optimal efficacy vs toxicity profiles.

[0043] Figure 4 shows characteristics of the disclosed LLIVs and LNPs by Dynamic light scattering (DLS). Figure 4A is a schematic drawing illustrating one embodiment of the LUVs (synthetic cells) of the present disclosure. Figure 4B shows chemical structures of POPC and POPG lipids. Figure 4C is an image showing size distribution of dye loaded LUVs by Transmission Electron Microscopy (TEM), the scale bar = 500 nm. Figure 4D is a graph of DLS measurements demonstrating that both POPC or POPG liposomes can be extruded in such a way that their size distributions are indistinguishable. Figure 4E is a bar graph showing size change of LUVs by DLS before and after complete solubilization with Triton detergent. Figure 4F is schematic drawing of a cargo loaded LNP. Figure 4G is a bar graph showing the z-average diameter of LNP formulated with 35 mol% of 200oi10, 304oi10, 306o10, or 306oi10 lipidoids in either pH 7.4 or pH 5.5 1X PBS buffer as measured by DLS. Figure 4H is a representative TEM image of LNPs showing size distribution and uniformity. The LNPs in this image are made with 35 mol% 200oi10 lipidoid in 1X PBS pH 7.4 buffer and imaged using uranyl acetate negative-staining transmission electron microscopy. Scale bar = 200 nm.

[0044] Figure 5 provides graphs showing differences in the rate and extent of CF dye leakage and R18 dilution depending on the LUV composition and LNP formulation and concentration. Figure 5A is a graph of CF leakage from POPC LUVs at pH 7.4 after the addition of different LNP formulations (LNPs A-D) at the same concentration. Figure 5B and Figure 5C are graphs of R18 dilution of POPC LUVs by different LNP formulations (LNPs A- D) at the same concentration, at pH 7.4 and pH 5.5 respectively. Figure 5D provides graphs of CF leakage from POPC and POPG LUVs after the addition of different concentrations of LNP made with 35 mol% 200oi10 lipidoid at pH 7.4 and 35°C. Figure 5E provides graphs of R18 dilution from POPC and POPG LUVs after the addition of different concentrations of the same LNP made with 35 mol% 200oi10 lipidoid at 35 C at pH 7.4 (top panel) or pH 5.5 (bottom panel).

[0045] Figure 6 provides Arrhenius plots for calculating the activation energy for membrane permeabilization at pH 7.4 (Figure 6A) or membrane fusion at pH 5.5 (Figure 6B) or pH 7.4 (Figure 6C). Curves are fit to a linear regression by relative LNP concentration (0.01-0.5X). Collapse of these curves to a single line demonstrates high confidence that the fluorescence dequenching has linear dependence on LNP concentration and Arrhenius dependence on temperature. Prominent examples of curve collapse can be seen in Figure 6A for POPC liposomes and LNP with 306o10 lipidoid (3rdrow, 2ndcolumn), in Figure 6B for POPC and POPG liposomes mixed with LNP with 200oi10 lipidoid (3rdrow), and in Figure 6C for POPC liposomes mixed with LNP with 306oi10, 306o10, and 200oi10 lipidoids (3rd-5throw, 2ndcolumn).

[0046] Figure 7 provides a comparison of predicted values for each mRNA-carrying LNP and LUV pair. The toxicity score on the vertical axis is derived from results of the neutral pH CF-LUV membrane leakage assay, with lower values implying faster membrane permeation and higher toxicity. The efficacy score on the horizontal axis is derived from results of the endosomal pH R18-LUV membrane fusion assay, with lower values implying faster membrane fusion and higher delivery efficiency. Error bars describe a 95% confidence interval around the predicted value.DETAILED DESCRIPTION

[0047] The present disclosure addresses the aforementioned needs in the art and provides, in various embodiments, a high throughput dual dye-dequenching screening method where increases in the fluorescence signal results from LNP-mediated cell membrane permeabilization or LNP-membrane fusion, which are read outs for LNP cytotoxicity and efficacy respectively. The disclosed methods further enable the acquisition of large quantities of predictive structure-activity data of different LNP formulations. As used herein, the term “dequenching,” refers to an increase in the fluorescence intensity of fluorophores due to their dilution. Using the methods and workflows described herein, it will be appreciated that the present disclosure also contemplates methods for measuring vaccine efficacy and toxicity profiles in vitro and in a high-throughput manner. Likewise, methods for determining LNP components, and component percentages, capable of permeabilizing a cell membrane (e.g., causing cellular toxicity) / fusing with a cell membrane (enabling endosomal escape, which thus determines vaccine efficiency) are also provided herein.

[0048] Several methods are available for screening LNPs. However, the proper characterization of nanoparticles requires an assessment of numerous properties, including size, morphology, encapsulation efficiency, aggregation, uniformity, and presence of contamination. LNP particle size for example is one of the most important physical characteristics that affects biodistribution, cellular uptake, efficacy, toxicity, aggregation, and stability of LNP formulations. Light scattering methods, such as dynamic light scattering (DLS) and Nanoparticle Tracking Analysis (NTA), are typically used to characterize some physical properties of LNPs. DLS and NTA are label-free techniques that provide information about the size of LNPs, including the mean particle size, shape, diffusion coefficient, and the polydispersity index (PDI) of LNP formulations. DLS measures intensity changes of scattered light which can be converted to a relative particle size distribution, whereas NTA calculates the diffusivity of particles from optical videos which can be converted to particle size and concentration. Both techniques can be used to monitor the stability of LNPs over time. By measuring the changes in size distribution, they can indicatepotential aggregation, particle growth, or degradation of LNPs. Stability studies using either instrument can assess the effects of storage conditions, temperature, pH, and other factors on the LNP formulation. Though DLS and NTA are popular and useful screening tools, they do not provide any information on dynamics of LNP interaction, such as membrane permeability or fusion (toxicity or efficacy). Complementary information using different approaches is therefore necessary to completely characterize any LNP sample to support process development and optimization.

[0049] As there are still a number of drawbacks associated with current state-of-the-art techniques used for investigating LNP-mediated fusion and membrane permeabilization processes, there is need for improved and alternative assays overcoming these limitations.

[0050] As described herein, the present disclosure presents a method of measuring membrane permeabilization of a synthetic cell by a lipid nanoparticle (LNP). Synthetic cells are supramolecular chemical systems designed to mimic the behavior, function, and structure of living cells (Guindani et al. (2022) Angew Chem Int Ed Engl. 61 (16): e202110855).

[0051] Liposomes remain the most widely employed type of synthetic cells because of their close structural similarity to cell membranes. Like cells, liposomes allow functional macromolecules (e.g., proteins, electron carriers) to be incorporated into their selfassembled membranes (Guindani et al. (2022) Angew Chem Int Ed Engl.61 (16):e202110855). The hydrophobic layer of these cell-like compartments may contain functional biomolecules, such as pore-forming proteins, or be synthetically tuned to be endowed with the desired permeability properties (Guindani et al. (2022) Angew Chem Int Ed Engl. 61 (16):e202110855). A common feature of liposomes is that they can be formed with a wide range of sizes (10 nm-100 pm), depending on how they are prepared (Guindani et al. (2022) Angew Chem Int Ed Engl. 61 (16):e202110855; Walde et al. (2010) Chembiochem. 11 (7):848-65.). For example, small unilamellar vesicles (SUVs) are 10-100 nm in diameter, large unilamellar vesicles (LUVs) are 100-1000 nm in diameter, and giant unilamellar vesicles (GUVs) are 1000nm or larger in diameter. As a result of their wide size range, polymers and liposomes can be used to create synthetic nanocompartments with sizes comparable to that of cellular organelles, as well as microcompartments with sizes comparable to whole biological cells (Guindani et al. (2022) Angew Chem Int Ed Engl.61 (16):e202110855). The chemical tunability of liposomes also allows liposomes to be synthesized with a broad range of compositions including cholesterol, transmembrane proteins, and different lipid chemistries, that can represent different cell types.

[0052] Liposomes can be used to characterize LNP-induced membrane permeabilization and the subsequent content leakage. There are various options to characterize induced membrane permeabilization. Most commonly, a fluorescent dye or a dye-quencher pair is entrapped in large unilamellar vesicles (LUVs), a perturbant molecule (such as a peptide) is added, and the rate and extent of release of the entrapped probe is measured (Guha et al. (2019) Chem Rev. 2019; 119(9):6040-6085). If and how much of the vesicle content is released or retained is then assessed either by fluorescence intensity modulated by the extent of quenching or via the fluorescence lifetime modulated by concentration-dependent self-quenching (Patel, H.; Tscheka, C.; Heerklotz, H.(2009) Soft Matter. 5:2849). Described herein is a method for measuring membrane permeabilization using a self-quenching assay with fluorescein or its derivatives (Marsden HR, Tomatsu I, Kros A. (2011) Chem Soc Rev. 40(3):1572-1585). The fluorescence of fluorescein or its derivatives is almost completely self-quenched at concentrations higher than 30 mM in HEPES Buffer. Concentrated solutions of these water-soluble dyes are encapsulated in liposomes and the external solution is washed of residual dye. Interaction with LNPs leads to mixing of the encapsulated and unencapsulated aqueous environments. The dye diffuses to extinguish the concentration gradient which results in overall dilution and increase in fluorescence (Weinstein JN, Blumenthal R, Klausner RD. (1986) Methods Enzymol. 128:657-68).

[0053] In some embodiments, provided herein are methods and compositions for measuring membrane permeabilization of a synthetic cell by a lipid nanoparticle (LNP).

[0054] As used herein, the term “membrane permeabilization,” refers to structural destabilization of the liposomal bilayer and increased permeability following interaction between LNPs and target liposomes. The increased permeability coupled with simultaneous partial disruption of LNPs themselves results in leakage / exchange of liposomal content into the LNP’s aqueous phase and external buffer solution. This interaction mimics LNP-induced disruption of plasma membrane integrity at physiological pH of ~7.4 contributing to LNP cytotoxicity and decreased efficacy. Membrane permeabilization has been demonstrated in lipid vesicles by dye leakage evaluation (Weinstein JN, Blumenthal R, Klausner RD. (1986) Methods Enzymol. 128:657-68; Ladokhin AS, White SH. (2001) Biochim Biophys Acta. 1514(2):253-60; Moghadam (2012) Langmuir. 28(47):16318-26).

[0055] Endosomal escape of LNPs relies on endosomal-LNP membrane fusion followed by translocation of nucleic acid cargo into the cytosol (Patel et al. (2020) Nat Common. 11 (1 ):983). Although the mechanism for LNP endosomal escape has not yet been fully understood, it has been suggested that protonation of the ionizable lipid within the LNPs at low endosomal pH promotes electrostatic interactions with the anionic endosomal membrane, which triggers the release of nucleic acid to the cytosol (Patel et al. (2020) NatCommun. 11 (1 ):983). Model systems for studying the organization, dynamics, and properties of the lipid bilayer component of biological membranes e.g., membrane fusion, are predominantly based on liposomes (Subczynski WK, Raguz M, Widomska J. (2022) Membranes (Basel) 26;12(7):657). Liposomes can be subdivided into small unilamellar vesicles (SUVs, < 100 nm), large unilamellar vesicles (LUVs, 100-500 nm) and giant unilamellar vesicles (GLIVs, >1000 nm). The present disclosure further provides a liposome- LNP fusion assay at the endosomal pH of ~5.5 which mimics LNP-endosomal membrane fusion during endosomal escape of nucleic acid nanoformulations.

[0056] The term “nanoformulation” as used herein means a formulation containing nanoparticles as an active ingredient (U.S. Patent Application Publication No. 20090104269). The term “nanoparticle,” as used herein, refers to a particle having a diameter, such as an average diameter, from about 10 nm up to but not including about 1 micron, preferably from 100 nm to about 1 micron (International Patent Publication No. WO 2021 / 184010). The particles can have any shape. Nanoparticles having a spherical shape are generally referred to as "nanospheres" (International Patent Publication No. WO 2021 / 184010). The term "lipid nanoparticles" or “LNP”, as used herein, refers to lipid-based particles in the submicron range (International Patent Publication No. WO 2021 / 184010). Lipid nanoparticles can have structural characteristics of liposomes and / or have alternative non-bilayer types of structures (International Patent Publication No. WO 2021 / 184010).Lipid nanoparticles may comprise one or more lipid species (International Patent Publication No. WO 2021 / 184010).

[0057] In a membrane fusion or lipid mixing assay, two sets of membranes are mixed - one membrane that is fluorescently-labelled and one membrane that is not fluorescently- labelled - under controlled experimental conditions (Gnopo YMD, Putnam D. Methods. 177:74-79). Exemplary fluorescent probes that are used are either self-quenching (R18) or are a pair of fluorescent acceptor and donor probes that undergo a process known as FRET whereby the acceptor probe quenches the fluorescence of the donor probe when they are in close proximity (< 10 nm) (Gnopo YMD, Putnam D. Methods. 177:74-79). The fluorescence of self-quenching probes like R18 is greatly reduced when single molecules are close enough to interact. Labelled membranes contain a high surface density of these probes, which allows quenching to occur (Gnopo YMD, Putnam D. Methods. 177:74-79). Other probes are contemplated and described herein.

[0058] Regardless of the probes or dyes used, lipid mixing or fusion between labelled and unlabeled membranes causes dilution of the probes, reducing their surface density (Gnopo YMD, Putnam D. Methods. 177:74-79). This reduction in surface density increases the distance between self-quenching probes or FRET pairs, which leads to an increase influorescence signal of the donor, and a decrease in fluorescence signal of the acceptor, for a total decrease in FRET efficiency. Kinetic experiments monitor de-quenching with time (Gnopo YMD, Putnam D. Methods. 177:74-79). FRET can only be used to measure changes in distance between ~2-8 nm due to the distance range over which FRET can occur. Additionally, to compare the fusogenic effect of various experimental conditions, a quantity known as the fusion efficiency is calculated from the raw fluorescence data by relative proportionality to the fluorescence signal when all probes are diluted (infinite dilution) by membrane disruption through the addition of a detergent (Gnopo YMD, Putnam D. Methods. 177:74-79). The detergent of choice is usually Triton X-100. Both the R18 lipid mixing assay and the FRET assay are based on these principles and have allowed many insights into the roles played by various proteins involved in viral entry (e.g.: influenza HA domain) and the transport of neurotransmitters (e.g.: SNAREs) (Gnopo YMD, Putnam D. Methods. 177:74-79).Synthetic Cells

[0059] As used herein, “synthetic cells”, or “artificial cells”, or “protocells” mean chemical or biochemical systems based on micro-compartments that enclose a set of reacting molecules, mimicking cell structure and behavior (Stano P. (2018) Life (Basel, Switzerland) 9(1 ):3). Synthetic cells encompass vesicles, membrane-free compartments and colloidosomes. Vesicle models include liposomes, polymersomes, and hybrid lipopolymersomes.

[0060] In one embodiment, the synthetic cell is a liposome. The term “liposome” as used herein refers to an artificially prepared vesicle composed of a lipid bilayer. A liposome may be classified as a unilamellar vesicle or a multivesicular vesicle (U.S. Patent No. 9457082).

[0061] In another embodiment, the synthetic cell is a large unilamellar vesicle (LUV).

[0062] “Unilamellar liposomes,” also referred to as “single lamellar vesicles,” are spherical vesicles that include one lipid bilayer membrane that defines a single closed aqueous compartment (U.S. Patent Application Publication No. 20140079773). The bilayer membrane includes two layers (or “leaflets”) of lipids; an inner layer and an outer layer (U.S. Patent Application Publication No. 20140079773). The outer leaflet of lipid molecules is oriented with the hydrophilic head portions toward the external aqueous environment and the hydrophobic tails pointed downward toward the interior of the liposome (U.S. Patent Application Publication No. 20140079773). The inner leaflet of lipids lies directly beneath the outer leaflet with the lipids heads oriented towards the aqueous interior of the liposome and the tails oriented toward the tails of the outer layer of lipid (U.S. Patent Application Publication No. 20140079773).

[0063] “Multilamellar liposomes” also referred to as “multilamellar vesicles” or “multiple lamellar vesicles,” include more than one lipid bilayer membrane, which membranes define more than one closed aqueous compartment (U.S. Patent Application Publication No. 20140079773). The membranes are typically concentrically arranged so that the different membranes are separated by aqueous compartments, much like an onion (U.S. Patent Application Publication No. 20140079773).

[0064] The term “lipid bilayer” as used herein refers to a membrane made of two layers of lipid molecules (U.S. Patent No. 9457082). The lipid bilayer may have a similar thickness as that of a naturally existing bilayer, such as a cell membrane, a nuclear membrane, and endocytic membranes (U.S. Patent No. 9457082). For example, the lipid bilayer may have a thickness of about 10 nm or less, for example, in a range of about 1 nm to about 9 nm, about 2 nm to about 8 nm, about 2 nm to about 6 nm, about 2 nm to about 4 nm, or about 2.5 nm to about 3.5 nm (U.S. Patent No. 9457082). The lipid bilayer is a barrier that keeps ions, proteins, and other molecules in an area, and / or prevents them from diffusing into other areas (U.S. Patent No. 9457082). The “lipid molecules” forming the lipid bilayer may be a molecule including a hydrophilic head and hydrophobic tails (U.S. Patent No. 9457082). The lipid molecule may have 14 to 50 carbon atoms (U.S. Patent No. 9457082). In some embodiments, the lipid molecules have more than 50 carbon atoms.

[0065] The lipid bilayer may comprise, functionalized cholesterol, synthetic ionizable lipid- like amphiphilic molecules, lipid conjugated to polyethylene glycol (PEG), cholesterol, or any combination thereof (U.S. Patent No. 9457082).

[0066] The liposomes that are used in the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol (U.S. Patent Application Publication No. 20140079773). The selection of lipids is generally guided by consideration of, e.g., liposome size and stability of the liposomes in the bloodstream (U.S. Patent Application Publication No. 20140079773).

[0067] Various types of lipids are used to produce liposomes. For example, amphipathic lipids that find use are zwitterionic, anionic, cationic, and neutrally charged lipids (U.S. Patent Application Publication No. 20140079773). Examples of zwitterionic amphipathic lipids are phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, etc (U.S. Patent Application Publication No. 20140079773). Examples of anionic amphipathic lipids are phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, etc (U.S. Patent Application Publication No. 20140079773). Examples of cationic amphipathic lipids are diacyl trimethylammonium propanes (DOTAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), dimethyldioctadecylammonium bromide (DDAB), etc (U.S. Patent Application Publication No. 20140079773). Examples of neutral lipids include diglycerides, such as diolein, dipalmitolein, and mixed caprylin-caprin; triglycerides, such as triolein, tripalmitolein, trilinolein, tricaprylin, and trilaurin; and combinations thereof (U.S. Patent Application Publication No. 20140079773). Additionally, cholesterol or plant sterols are used in some embodiments, e.g., to make multivesicular liposomes (U.S. Patent Application Publication No. 20140079773).

[0068] In an embodiment, the cationic lipids can be DMTAP, DPTAP, DOTAP, DC-Chol, MoChol or HisChol, or combinations thereof, and the anionic lipids can be CHEMS5 DGSucc, Cet-P, DMGSucc, DOGSucc, POGSucc, DPGSucc, DG Succ, DMPS, DPPS, DOPS, POPS5 DMPG, DPPG5 DOPG, POPG, DMPA, DPPA5 DOPA, POPA or combinations thereof (International Patent Publication No. WO 2007 / 064857).

[0069] In another embodiment, the liposomes also include neutral lipids. In a further embodiment, the neutral lipids include sterols and derivatives thereof (International Patent Publication No. WO 2007 / 064857). The neutral lipids may also include neutral phospholipids. In one embodiment, the phospholipids include phosphatidylcholines and phosphoethanolamines (International Patent Publication No. WO 2007 / 064857). In another embodiment, the phosphatidylcholines are POPC, OPPC, natural or hydrogenated soybean PC, natural or hydrogenated egg PC, DMPC, DPPC, or DOPC and derivatives thereof and the phosphatidylethanolamines are DOPE, DMPE, DPPE or derivatives and combinations thereof (International Patent Publication No. WO 2007 / 064857). In a further embodiment, the phosphatidylcholine is POPC, OPPC, soybean PC or egg PC and the phosphatidylethanolamines is DOPE (International Patent Publication No. WO 2007 / 064857).

[0070] In one embodiment, the CF-Liposomes of the present disclosure comprise 100 mol% POPC. In another embodiment, the CF-Liposomes described herein comprise 100 mol% POPG.

[0071] In a different embodiment, the R18-Liposomes described herein comprise 10 mol% R18 and 90 mol% of either POPC or POPG.

[0072] In some embodiments, the LNPs disclosed herein comprise a mol ratio of 35:16:46.5:2.5 of ionizable lipid, helper lipid, Cholesterol, and PEG-lipid. In one embodiment, the presently disclosed LNPs comprise a mol ratio of 0:56:42:2 ionizable lipid, helper lipid, cholesterol, and PEG lipid.

[0073] A variety of methods are available for preparing liposomes as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871 ; 4,501 ,728; and 4,837,028; the text Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1 , and Hope, et al., Chem. Phys. Lip. 40:89 (1986), all of which are incorporated herein by reference. One exemplary method produces multilamellar vesicles of heterogeneous sizes (U.S. Patent Application Publication No. 20140079773). In this method, the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film (U.S. Patent Application Publication No. 20140079773). Alternatively, the lipids may be dissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture that is in a more easily hydrated powder-like form (U.S. Patent Application Publication No. 20140079773). This film or powder is covered with an aqueous buffered solution and allowed to hydrate, typically over a 15-60 minute period with agitation (U.S. Patent Application Publication No. 20140079773). The size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate (U.S. Patent Application Publication No. 20140079773).

[0074] Many different types of organic solvents such as ethers, hydrocarbons, halogenated hydrocarbons, and / or Freons are used in some embodiments as the solvent in the lipid component (U.S. Patent Application Publication No. 20140079773). For example, diethyl ether, isopropyl ether, and other ethers; chloroform; tetrahydrofuran; halogenated ethers; esters, and combinations thereof find use in the present technology (U.S. Patent Application Publication No. 20140079773).

[0075] Several techniques are available for sizing liposomes to a desired size. One sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference.Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUV) less than about 0.05 microns in size (U.S. Patent Application Publication No. 20140079773). Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones (U.S. Patent Application Publication No. 20140079773). In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed (U.S. Patent Application Publication No. 20140079773). In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination (U.S. Patent Application Publication No. 20140079773).

[0076] Extrusion of liposomes through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution (U.S. Patent Application Publication No. 20140079773). Typically, the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved (U.S. Patent Application Publication No. 20140079773). The liposomes may be extruded through successively smaller-pore membranes to achieve a gradual reduction in liposome size (U.S. Patent Application Publication No. 20140079773). For use in embodiments of the present technologies, liposomes having a size of from about 0.05 microns to about 0.22 microns are preferred (U.S. Patent Application Publication No. 20140079773).

[0077] As used herein “phospholipid” refers to a compound lipid containing phosphate ester within a molecule, and is a main component of biological membranes, such as cell membranes, endoplasmic reticulum, mitochondria, and myelin sheath around nerve fibers (U.S. Patent No. 9457082). The phospholipid includes a hydrophilic head and two hydrophobic tails (U.S. Patent No. 9457082). When the phospholipids are exposed to water, they can arrange themselves into a two-layered sheet (a bilayer) with all of their tails pointing toward the center of the sheet (U.S. Patent No. 9457082). The center of this bilayer contains almost no water and also excludes molecules such as sugars or salts that dissolve in water but not in oil (U.S. Patent No. 9457082).

[0078] The liposome may be a stimulus-sensitive liposome (i.e., sensitive to one or more stimuli), and the stimulus-sensitive liposome may control release of materials that are encapsulated therein (U.S. Patent No. 9457082). As used herein, “sensitive” to stimuli refers to the ability of a liposome to release its contents in response to exposure to one or more stimuli or the like, or to disintegrate in response to one or more stimuli or the like (U.S. Patent No. 9457082). Examples of the stimulus-sensitive liposome include a temperaturesensitive liposome, a pH-sensitive liposome, a chemical-sensitive liposome, a radiationsensitive liposome, an ultrasound-sensitive liposome, or any combination thereof (U.S. Patent No. 9457082). The temperature-sensitive liposome, the pH-sensitive liposome, the chemical-sensitive liposome, the radiation-sensitive liposome, and the ultrasound-sensitive liposome may release materials that are contained therein at a certain temperature or temperature range, a certain pH or pH range, the presence of chemical substance, radiation conditions, and / or ultrasound conditions (U.S. Patent No. 9457082). The temperature may be, for example, in a range of about 10°C to about 60°C, about 20°C to about 40°C or about 20°C to about 60°C. The pH may be greater than, equal to, or less than about 5.5, which is the pH of saline solution (U.S. Patent No. 9457082).

[0079] The liposome may have, for example, a diameter in a range of about 50 nm to about 100 nm, about 50 nm to about 150 nm, about 50 nm to about 220 nm, about 100 nm to about 150 nm, or about 100 nm to about 220 nm.Lipid Nanoparticles (LNPs)

[0080] As used herein, the term lipid nanoparticle “LNP” refers to a lipid-nucleic acid particle or a nucleic acid-lipid particle (e.g., a stable nucleic acid-lipid particle) (International Patent Publication No. WO 2020 / 219941 ). A LNP represents a particle made from lipids (e.g., a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle), and a nucleic acid, wherein the nucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self- amplifying RNA, gRNA, or a plasmid, including plasmids from which an interfering RNA or mRNA is transcribed) is encapsulated within the LNP (International Patent Publication No. WO 2020 / 219941 ). In one embodiment, the nucleic acid is at least 50% encapsulated in the LNP; in one embodiment, the nucleic acid is at least 75% encapsulated in the LNP; in one embodiment, the nucleic acid is at least 90% encapsulated in the LNP; and in one embodiment, the nucleic acid is completely encapsulated in the LNP (International Patent Publication No. WO 2020 / 219941). LNPs typically contain a helper lipid, cholesterol, a PEG- lipid, and an ionizable lipid. LNPS are extremely useful for systemic applications, as they can exhibit extended circulation lifetimes following intravenous (i.v.) injection, they can accumulate at distal sites (e.g., sites physically separated from the administration site), and they can mediate expression of the transfected gene or silencing of target gene expression at these distal sites (International Patent Publication No. WO 2020 / 219941).

[0081] As used herein, the terms “encapsulation,” “entrapped,” “loaded with” or grammatical equivalents, refer to the process of confining an individual cargo molecule e.g., an mRNA molecule within a nanoparticle or a liposome. In the context of pharmaceutical sciences, encapsulation entails the enclosure and protection of an active compound against degradation, uncontrolled diffusion and, ultimately, loss. The active ingredient may be associated with the lipid bilayer or present in the aqueous interior of the nanoparticle or liposome, or both.

[0082] The lipid nanoparticle formulations described herein may include cationic lipids. Cationic lipids are lipids that carry a net positive charge. The positive charge is used for association with negatively charged therapeutics such as antisense oligonucleotides and anti-sense miRNA via electrostatic interaction (U.S. Patent No. 10555910).

[0083] Suitable cationic lipids include, but are not limited to: 3p-(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol hydrochloride (DC-Chol); 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP); dimethyldioctadecylammonium bromide salt (DDAB); 1 ,2-dilauroyl-sn-glycero-3- ethylphosphocholine chloride (DL-EPC); N-(1-(2,3-dioleyloyx) propyl)-N-N-N-trimethyl ammonium chloride (DOTMA); N-(1-(2,3-dioleyloyx) propyl)-N-N-N-dimethyl ammonium chloride (DODMA); N,N-dioctadecyl-N,N-dimethylammonium chloride (DODAC); N-(1 -(2,3- dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA); 1 ,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE); dioctadecylamidoglycylspermine (DOGS); neutral lipids conjugated to cationic modifying groups; and combinations thereof (U.S. Patent No. 10555910). In addition, a number of cationic lipids in available preparations could be used, such as LIPOFECTIN® (from GIBCO / BRL), LIPOFECTAMINE® (from GIBCO / MRL), siPORT NEOFX® (from Applied Biosystems), TRANSFECTAM® (from Promega), and TRANSFECTIN® (from Bio-Rad Laboratories, Inc.) (U.S. Patent No. 10555910). The cationic lipids of the present disclosure may be present at a concentration of about 35 percent to 56 molar percent of the lipids in the formulation.

[0084] In another embodiment, lipid nanoparticle formulations presently disclosed may include lipidoids. As used herein, the term “lipidoid,” refers to lipid-like structures containing multiple secondary and tertiary amine functionalities, prepared from the conjugate addition of alkylamines to acrylates, and which confer highly efficient interaction with anionic nucleic acids, for example siRNA molecules (de Groot et al. (2018) Mol Ther Nucleic Acids. 11 : 159- 169; Akinc et al. (2008) Nat Biotechnol. 26(5) :561 -9; Whitehead et al. (2014) Nat Commun. 27;5:4277). Non-limiting examples of lipidoids include the 200oi10, 304oi10, 306o10, and 306oi10 lipidoids of the present disclosure.

[0085] In certain embodiments, the lipid nanoparticle formulations presently disclosed may also include anionic lipids. Anionic lipids are lipids that carry a net negative charge at physiological pH (U.S. Patent No. 10555910). These anionic lipids, when combined with cationic lipids, are useful to reduce the overall surface charge of lipid nanoparticles and introduce pH-dependent disruption of the lipid nanoparticle bilayer structure, facilitating nucleotide release by inducing nonlamellar phases at acidic pH or induce fusion with the cellular membrane (U.S. Patent No. 10555910).

[0086] Examples of suitable anionic lipids include, but are not limited to: fatty acids such as oleic, linoleic, and linolenic acids; cholesteryl hemisuccinate; 1 ,2-di-O-tetradecyl-sn- glycero-3-phospho-(1 '-rac-glycerol) (Diether PG); 1 ,2-dimyristoyl-sn-glycero-3-phospho-(1 '- rac-glycerol) (sodium salt); 1 ,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1 - hexadecanoyl,2-(9Z,12Z)-octadecadienoyl-sn-glycero-3-phosphate; 1 ,2-dioleoyl-sn-glycero- 3-(phospho-rac-(1 -glycerol)) (DOPG); dioleoylphosphatidic acid (DOPA); and 1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); anionic modifying groups conjugated to neutral lipids; and combinations thereof (U.S. Patent No. 10555910). The anionic lipids of the present disclosure are present at concentrations up to about 60.0 molar percent of the formulation.

[0087] In certain embodiments, charged lipid nanoparticles are advantageous for transfection, but off-target effects such as cytotoxicity and RES-mediated uptake may occur. Hydrophilic molecules such as polyethylene glycol (PEG) may be conjugated to a lipid anchor and included in the lipid nanoparticles described herein to discourage lipid nanoparticle aggregation or interaction with membranes (U.S. Patent No. 10555910). Hydrophilic polymers may be covalently bonded to lipid components or conjugated using crosslinking agents to functional groups such as amines (U.S. Patent No. 10555910).

[0088] Suitable conjugates of hydrophilic polymers include, but are not limited to: polyvinyl alcohol (PVA); polysorbate 80; 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- PEG2000 (DSPE-PEG2000); D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS); dimyristoylphosphatidylethanolamine-PEG2000 (DMPE-PEG2000); and dip almitoylphosphatidlyethanolamine-PEG2000 (DPPE-PEG2000) (U.S. Patent No. 10555910). The hydrophilic polymer may be present at concentrations ranging from about 0 to about 15.0 molar percent of the formulation, or from about 5.0 to about 10.0 molar percent of the formulation (U.S. Patent No. 10555910). The molecular weight of the PEG used is between about 100 and about 10,000 Da, or from about 100 to about 2,000 Da (U.S. Patent No.10555910). In one embodiment, the molecular weight of the PEG used is 2,555 Da.

[0089] The lipid nanoparticles described herein may further comprise neutral and / or amphipathic lipids as helper lipids (U.S. Patent No. 10555910). These lipids are used to stabilize the formulation, reduce elimination in vivo, or increase transfection efficiency (U.S. Patent No. 10555910).

[0090] Neutral lipids have zero net charge at physiological pH. One or a combination of several neutral lipids may be included in any lipid nanoparticle formulation disclosed herein (U.S. Patent No. 10555910).

[0091] Suitable neutral lipids include, but are not limited to: phosphatidylcholine (PC), phosphatidylethanolamine, ceramide, cerebrosides, sphingomyelin, cephalin, diacylglycerols, glycosylated diacylglycerols, prenols, lysosomal PLA2 substrates, N- acylglycines, and combinations thereof (U.S. Patent No. 10555910).

[0092] Other suitable lipids include, but are not limited to: phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, and lysophosphatidylethanolamine; sterols such as cholesterol, demosterol, sitosterol,zymosterol, diosgenin, lanostenol, stigmasterol, lathosterol, and dehydroepiandrosterone; and sphingolipids such as sphingosines, ceramides, sphingomyelin, gangliosides, glycosphingolipids, phosphosphingolipids, phytoshingosine; and combinations thereof (U.S. Patent No. 10555910).

[0093] The lipid nanoparticle formulations described herein may further comprise fusogenic lipids or fusogenic coatings to promote membrane fusion (U.S. Patent No. 10555910). Examples of suitable fusogenic lipids include, but are not limited to, glyceryl mono-oleate, oleic acid, palmitoleic acid, phosphatidic acid, phosphoinositol 4,5- bisphosphate (PIP2), and combinations thereof (U.S. Patent No. 10555910).

[0094] Anionic polymers may be incorporated into the lipid nanoparticle formulations presently disclosed as well (U.S. Patent No. 10555910). Suitable anionic polymers include, but are not limited to: poly(propylacrylic acid) (PPAA); poly(glutamic acid) (PGA); alginates; dextrans; xanthans; derivatized polymers; and combinations thereof (U.S. Patent No. 10555910).

[0095] In certain embodiments, the lipid nanoparticle formulation includes conjugates of polymers (U.S. Patent No. 10555910). The conjugates may be crosslinked to targeting agents, lipophilic moieties, peptides, proteins, or other molecules that increase the overall therapeutic efficacy (U.S. Patent No. 10555910).

[0096] Suitable crosslinking agents include, but are not limited to: N-succinimidyl 3-(2- pyridyldithio)-propionate (SPDP); dimethyl 3,3'-dithiobispropionimidate (DTBP); dicyclohexylcarbodiimide (DCC); diisopropyl carbodiimide (DIG); 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC); N-hydroxysulfosuccinimide (Sulfo-NHS); N'-N'- carbonyldiimidazole (CDI); N-ethyl-5-phenylisoxazolium-3'sulfonate (Woodward's reagent K); and combinations thereof (U.S. Patent No. 10555910).

[0097] The lipid nanoparticle formulations may further comprise peptides and / or proteins. Peptides and proteins, especially those derived from bacteria and viruses or used as antibiotic agents, may aid in membrane permeation (U.S. Patent No. 10555910). The peptides or proteins may be directly mixed with lipids, covalently attached, or conjugated to lipid moieties with crosslinking agents (U.S. Patent No. 10555910).

[0098] Suitable peptides and proteins include, but are not limited to: gramicidin A, B, C, D, and S; HA2; JTS-1 ; proteinase K (PrK); trichorovin-Xlla (TV-Xlla); rabies virus glycoprotein (RVG); interleukin-P; HIV-Tat; herpes simplex virus (HSV) VP22 protein; and combinations thereof (U.S. Patent No. 10555910). In certain embodiments, JTS-1 and / or gramicidin is used at about 0 to about 40 molar percent. In certain embodiments, PrK at a concentration of about 0 to about 30 molar percent is applied by direct mixing with oligonucleotide orconjugation to hexadecyl isothiocyanate for lipid nanoparticle surface coating of PrK (U.S. Patent No. 10555910).

[0099] The addition of targeting agents to the lipid nanoparticle provides increased efficacy over passive targeting approaches (U.S. Patent No. 10555910). Targeting involves incorporation of specific targeting moieties such as, but not limited to, ligands or antibodies against cell surface receptors, peptides, lipoproteins, glycoproteins, hormones, vitamins, antibodies, antibody fragments, prodrugs, and conjugates or combinations of these moieties (U.S. Patent No. 10555910).

[0100] In certain embodiments, maximization of targeting efficiency includes the surface coating of the lipid nanoparticle with the appropriate targeting moiety rather than encapsulation of the targeting agent (U.S. Patent No. 10555910). This method optimizes interaction with cell surface receptors (U.S. Patent No. 10555910).

[0101] It is to be understood that targeting agents may be either directly incorporated into the lipid nanoparticle during synthesis or added in a subsequent step (U.S. Patent No. 10555910). Functional groups on the targeting moiety as well as specifications of the therapeutic application (e.g., degradable linkage) dictate the appropriate means of incorporation into the lipid nanoparticle (U.S. Patent No. 10555910). Targeting moieties that do not have lipophilic regions cannot insert into the lipid bilayer of the lipid nanoparticle directly and require prior conjugation to lipids before insertion or must form an electrostatic complex with the lipid nanoparticles (U.S. Patent No. 10555910).

[0102] Also, under certain circumstances, a targeting ligand cannot directly bind to a lipophilic anchor. In these circumstances, a molecular bridge in the form of a crosslinking agent may be utilized to facilitate the interaction (U.S. Patent No. 10555910). In certain embodiments, it is advantageous to use a crosslinking agent if steric restrictions of the anchored targeting moiety prevent sufficient interaction with the intended physiological target (U.S. Patent No. 10555910). Additionally, if the targeting moiety is only functional under certain orientations (e.g., monoclonal antibody), linking to a lipid anchor via crosslinking agent is beneficial (U.S. Patent No. 10555910). Traditional methods of bioconjugation may be used to link targeting agents to lipid nanoparticles (U.S. Patent No. 10555910).Reducible or hydrolysable linkages may be applied to prevent accumulation of the formulation in vivo and subsequent cytotoxicity (U.S. Patent No. 10555910).

[0103] Various methods of lipid nanoparticle preparation are suitable to synthesize the lipid nanoparticles of the present disclosure (U.S. Patent No. 10555910). For example, ethanol dilution, freeze-thaw, thin film hydration, sonication, extrusion, high pressure homogenization, detergent dialysis, microfluidization, tangential flow diafiltration, sterilefiltration, and / or lyophilization may be utilized (U.S. Patent No. 10555910). Additionally, several methods may be employed to decrease the size of the lipid nanoparticles (U.S. Patent No. 10555910). For example, homogenization may be conducted on any devices suitable for lipid homogenization such as an Avestin Emulsiflex C5® device (U.S. Patent No. 10555910). Extrusion may be conducted on an Avanti Mini-extruder using a polycarbonate membrane of appropriate pore size (0.05 to 0.4 pm). Multiple particle size reduction cycles may be conducted to minimize size variation within the sample (U.S. Patent No. 10555910). The resultant lipid nanoparticles may then be passed through a size exclusion column containing a Sephadex® G-25 resin or processed by tangential flow diafiltration to purify the lipid nanoparticles.

[0104] Any embodiment of the lipid nanoparticles described herein may further include ethanol in the preparation process (U.S. Patent No. 10555910). The incorporation of about 30-50% ethanol in lipid nanoparticle formulations destabilizes the lipid bilayer and promotes electrostatic interactions among charged moieties such as cationic lipids with anionic oligonucleotides, such as ASO and siRNA (U.S. Patent No. 10555910). Lipid nanoparticles prepared in high ethanol solution are diluted before administration. Alternatively, ethanol may be removed by dialysis, or diafiltration, which also removes non-encapsulated NA (U.S. Patent No. 10555910).

[0105] In certain embodiment, it is desirable that the lipid nanoparticles be sterilized. This may be achieved by passing of the lipid nanoparticles through a 0.2 or 0.22 pm sterile filter with or without pre-filtration (U.S. Patent No. 10555910).

[0106] Physical characterization of the lipid nanoparticles can be carried through many methods (U.S. Patent No. 10555910). Dynamic light scattering (DLS), atomic force microscopy (AFM), or Nanoparticle Tracking Analysis (NTA) can be used to determine the average diameter and its standard deviation (U.S. Patent No. 10555910). In certain embodiments, it is especially desirable that the lipid nanoparticles have about a 200 nm diameter (U.S. Patent No. 10555910). Zeta potential measurement via zeta potentiometer is useful in determining the relative stability of particles (U.S. Patent No. 10555910). Both dynamic light scattering analysis and zeta potential analysis may be conducted with diluted samples in deionized water or appropriate buffer solution (U.S. Patent No. 10555910). Cryogenic transmission electron microscopy (Cryo-TEM), Transmission electron microscopes (TEM), and scanning electron microscopy (SEM) may be used to determine the detailed morphology of lipid nanoparticles (U.S. Patent No. 10555910).

[0107] The LNPs typically have a mean diameter of from about 100 nm to about 150 nm, from about 100 nm to about 200 nm, from about 100 nm to about 250 nm, from about 150nm to about 250 nm, and are substantially non-toxic. In addition, nucleic acids, when present in the lipid particles of the invention, are resistant in aqueous solution to degradation with a nuclease (International Patent Publication No. WO 2020 / 219941). Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031 , the disclosures of which are herein incorporated by reference in their entirety for all purposes (International Patent Publication No. WO 2020 / 219941 ).LNP Cargo

[0108] The lipid nanoparticles described herein can be used as platforms for therapeutic delivery of oligonucleotide (ON) therapeutics, such as siRNA, shRNA, miRNA, anti-miR, and antisense ODN. These therapeutics are useful to manage a wide variety of diseases such as various types of cancers, leukemias, viral infections, and other diseases.

[0109] Nucleic acid-based therapeutic agents are highly applicable to the lipid nanoparticle formulations of the present disclosure. Examples of such nucleic acid-based therapeutic agents include, but are not limited to: antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys and analogs thereof, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA), messenger ribonucleic acid (messenger RNA, mRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (asymmetrical interfering RNA), and siRNA with 1 , 2 or more mismatches between the sense and anti-sense strand to relevant cells and / or tissues, such as in a cell culture, subject or organism (International Patent Publication No. WO 2016 / 010840). Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified. In one embodiment the biologically active agent is mRNA (International Patent Publication No. WO 2016 / 010840).

[0110] To protect from serum nucleases and to stabilize the therapeutic agent, modifications to the substituent nucleic acids and / or phosphodiester linker can be made. Such modifications include, but are not limited to: backbone modifications (e.g., phosphothioate linkages); 2' modifications (e.g., 2'-O-methyl substituted bases); zwitterionic modifications (6'-aminohexy modified ODNs); the addition of a lipophilic moiety (e.g., fatty acids, cholesterol, or cholesterol derivatives); and combinations thereof (U.S. Patent No. 10555910). The modified sequences synergize with the lipid nanoparticle formulations disclosed herein (U.S. Patent No. 10555910). For example, addition of a 3'-cholesterol to anODN supplies stability to a lipid nanoparticle complex by adding lipophilic interaction in a system otherwise solely held together by electrostatic interaction (U.S. Patent No. 10555910). In addition, this lipophilic addition promotes cell permeation by localizing the ODN to the outer leaflet of the cell membrane (U.S. Patent No. 10555910). Applying a peptide such as gramicidin or JTS-1 further promotes cell permeation of the formulation due to its fusogenic properties (U.S. Patent No. 10555910). Alternatively, addition of an enzyme such as proteinase K could further aid the ODN in resisting degradation (U.S. Patent No. 10555910).Techniques for measuring membrane permeabilization

[0111] The method comprises preparing a mixture of LNPs and synthetic cells loaded with a water-soluble fluorescent dye under conditions that allow contact between the LNPs and the synthetic cells and determining membrane permeability by assaying for fluorescent dye release from the synthetic cells.

[0112] In some embodiments, the synthetic cells have mean diameters of about 50-100 nm, about 100-170 nm, about 150-170 nm, or about 150-220 nm.

[0113] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a synthetic cell with a nanoparticle means that the synthetic cell and a nanoparticle are made to share a physical connection. The contacting may involve varied amounts of nanoparticle compositions. Moreover, one synthetic cell may be contacted by more than one nanoparticle composition.Water-soluble Fluorescent Dyes

[0114] In some embodiments, the water-soluble fluorescent dye is selected from the group consisting of sulforhodamine B (SRB), FITC, calcein, 5(6)-carboxyfluorescein (CF) and other fluorescein and rhodamine derivatives.

[0115] In one embodiment, the water-soluble fluorescent dye is CF.

[0116] CF-LUVs have been described, for example, to demonstrate fluorescent marker uptake into cultured frog retinal cells (Weinstein et al. (1977) Science. 1977; 195(4277) :489- 492.), to study lipoprotein induced permeability (Weinstein JN, Blumenthal R, Klausner RD. (1986) Methods Enzymol.128:657-668) and, in determining cell permeability changes due to inorganic environmental nanoparticles (Moghadam et al. (2012) Langmuir. 28(47):16318- 26).

[0117] In some embodiments, the water-soluble fluorescent dyes are loaded into the synthetic cells at self-quenching concentrations. In one embodiment, the self-quenchingconcentration range of the water-soluble fluorescent dye is between 30-50 mM or between 35-100 mM.

[0118] In one embodiment, membrane permeability is evaluated by following dye leakage from synthetic cells with time.

[0119] In one embodiment, dye release reduces self-quenching (dequenching) and leads to an increase in fluorescence, the magnitude of which is proportional to the extent of leakage of the dye from the lipid vesicle.

[0120] In another embodiment, the reduced self-quenching can be monitored by the fluorescence intensity, which is normalized to the maximum fluorescence corresponding to the complete release of the dye after complete disruption of the synthetic cells using a detergent. In some embodiments, the synthetic cells are lysed using detergents selected from the group consisting of T riton X-100, sodium dodecyl sulphate (SDS) and sodium cholate (SC), Dodecylmaltoside (DM), octyl glucoside (OG), and octaethylenglycol monododecylether.

[0121] In one embodiment, the synthetic cells are lysed by the addition of Triton X-100. In yet another embodiment, the Triton X-100 is added to a final concentration of between 0-10 mM.

[0122] In some embodiments, fluorescence spectroscopy is used to monitor dye leakage.Techniques for measuring membrane fusion

[0123] The present disclosure further provides compositions and methods for measuring membrane fusion between a synthetic cell and an LNP.

[0124] As used herein, the term “membrane fusion,” or “lipid mixing” refers to the mechanism by which two apposed (docked) membrane bilayers coalesce in rapid, transient steps that enable the successive merging of the outer and inner leaflets allowing lipid intermixing and subsequent mixing of the two previously separate compartments (Dabral D, Coorssen JR. Int J Biochem Cell Biol. 85:1 -5). “Membrane fusion,” as used herein can also mean mixing of only outer leaflets of two docked bilayers.

[0125] Membrane fusion is a fundamental process in cell biophysics, involved in infection of eukaryotic host cells by enveloped viruses, membrane trafficking (endo- and exocytosis), endocrine hormone secretion, neuronal signaling, and fertilization (Risselada HJ, Bubnis G, Grubmuller H. (2014) Proc Natl Acad Sci U S A. 111 (30):11043-11048). Several assays have been developed to characterize in vitro these processes on model systems the most common of which relies on fluorescence dequenching to measure lipid mixing between small membranes such as liposomes (Frangois-Martin C, Pincet F. (2017) Sci Rep. 7:43860). Thelipid mixing assay monitors the transfer of lipids from the donor to the acceptor membrane (Frangois-Martin C, Pincet F. (2017) Sci Rep. 7:43860).

[0126] Endosomal-membrane fusion is required for LNP cargo release. Described herein is a lipid mixing assay for quantifying endosomal-membrane LNP fusions resulting from interactions between synthetic cells and LNPs.

[0127] The method comprises preparing a mixture of LNPs and synthetic cells with fluorescently labeled membranes under conditions that allow fusion between the LNPs and the labeled synthetic cells.

[0128] The term “fluorescent tagging,” or “labeling,” as used herein refers to the attachment of a reactive derivative of a fluorescent molecule known as a fluorophore to aid in the detection of a biomolecule e.g., membrane lipids.Lipophilic Fluorescent Dyes

[0129] In some embodiments, the membranes of the synthetic cells are labeled with a lipophilic fluorescent dye.

[0130] Examples of lipophilic fluorescent dyes include, but are not limited to, Octadecyl Rhodamine B Chloride (R18), Diphenylhexatriene (DPH), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s- indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (p-BODIPY- C12HPC), N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1 ,2- dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (BODIPY FL- DHPE), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (ammonium salt) (Fluorescein-DHPE), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1 ,3- benzoxadiazol-4-yl) (ammonium salt) (NBD-DOPE), N-(6- tetramethylrhodaminethiocarbamoyl)-1 ,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine (triethylammonium salt) (TMR-DHPE), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (LR-DOPE), 2- (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn- glycero-3-phosphocholine (P-BODIPY-C5-HPC), Texas Red 1 ,2-dihexadecanoyl-sn-glycero- 3-phosphoethanolamine (triethylammonium salt) (TR-DHPE), and rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B).

[0131] In one embodiment, the lipophilic fluorescent dye is R18.

[0132] The synthesis of the R18 dye was first described by Keller et al., where it was used to label cells and viruses to study viral membrane fusion by FRET (Keller PM, Person S, Snipes W. J Cell Sci. (1977) 28:167-177).

[0133] R18-LUVs have been described, for example, in fluorescence quenching assays in liposomes and small unilamellar vesicles (Arnhold, J, A Mops, M Krumbiegel, and K. Arnold. (1989) Studia Biophysica 133(2) :101 — 8), in liposome fusion induced by Ca2+ and PEG (Arnhold et al. (1994) Biochim Biophys Acta. 1191 (2):375-383), while R18 self-quenching properties have been characterized in phosphatidylcholine (PC) vesicles (MacDonald Rl. (1990) J Biol Chem. 265(23): 13533- 13539).

[0134] In an embodiment, the synthetic cells are labeled with self-quenching concentrations of the lipophilic fluorescent dye.

[0135] In some embodiments, the lipophilic fluorescent dye demonstrates self-quenching fluorescence when incorporated into lipid structures above 1 mol% dye.

[0136] In one embodiment, the lipid fusion assay is carried out at a temperature of about 20 °C, 25 °C, 30 °C, 35 °C, 37 °C, or 40 °C.

[0137] The terms "substantially", "approximately", "about", "relatively", or other such similar terms that may be used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing, from a reference or parameter. Such small fluctuations include a zero fluctuation from the reference or parameter as well. For example, fluctuations can refer to less than or equal to ±10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.

[0138] In another embodiment, the lipid fusion assay is carried out at a pH of about 5, 5.5, 6, 6.5, 7.0, or 7.5.

[0139] In some embodiments, the lipid fusion assay is recorded at pH 7.4 or pH 5.5.

[0140] In one embodiment, fusion of the unlabeled LNPs membranes, with the labeled membranes of the synthetic cells dilutes the probe, with a concomitant increase in fluorescence that allows for the measurement of lipid mixing.

[0141] In one embodiment, the extent of membrane fusion at each time point is calculated by normalizing the respective fluorescence values to maximal fluorescence dequenching seen upon detergent lysis of the synthetic cells.

[0142] In one embodiment, the maximal fluorescence was achieved by the addition of 0.2% (v / v) Triton X-100.

[0143] In some embodiments, the fluorescence intensity is measured using a spectrofluorometer using a multi-well plate.

[0144] In some embodiments, the LNP comprises a cargo selected from the group consisting of a peptide, a nucleic acid, a small molecule, and a protein or combinations of the same.

[0145] In a further embodiment, the LNP comprises a ribonucleic acid, protein, plasmid, or a small molecule drug cargo.

[0146] Examples of ribonucleic acid cargo include but are not limited to small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicersubstrate RNA (dsRNA), an antisense oligonucleotide (ASO), an RNA aptamer, a small hairpin RNA (shRNA), a messenger RNA (mRNA), trans-activating clustered regularly interspaced short palindromic repeat RNA (tracrRNA), clustered regularly interspaced short palindromic repeat RNA (crRNA), and a long non-coding RNA (IncRNA), a guide RNA (gRNA).

[0147] In one embodiment, the ribonucleic acid cargo is mRNA.

[0148] The present disclosure further contemplates a method for concurrently measuring membrane permeabilization and membrane fusion of a synthetic cell by a lipid nanoparticle (LNP).

[0149] The method comprises, in some embodiments, preparing a first mixture of LNPs and synthetic cells encapsulating a water-soluble fluorescent dye under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells with fluorescently labeled membranes under conditions that allow fusion between the LNPs and the synthetic cells.

[0150] In still other embodiments, the method further comprises measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells after normalization to maximum fluorescence values from full homogenization by Triton X-100 or other surfactants.

[0151] In one embodiment, the extent of membrane permeabilization at each time point is calculated by normalizing the respective fluorescence values of the mixture comprising the synthetic cells encapsulating a water-soluble fluorescent dye to the maximal fluorescence dequenching seen upon detergent lysis of said synthetic cells.

[0152] In one embodiment, the extent of membrane fusion at each time point is calculated by normalizing the respective fluorescence values of the mixture comprising the synthetic cells with fluorescently labeled membranes to maximal fluorescence dequenching seen upon detergent lysis of the synthetic cells.

[0153] In one embodiment, the fluorescently labeled membranes are labeled with R18 dye.EXAMPLES

[0154] The following examples are intended to illustrate, but not limit the scope of the present disclosure.

[0155] Example 1 - Materials and MethodsTable 1 : List of chemicals utilized in the described studies.

[0156] A 100 nm polycarbonate filter and Mini-Extruder Set was purchased from Avanti Polar Lipids.Preparation of large unilamellar vesicles (LUVs)

[0157] Pure lipids (POPC, POPG) were purchased dissolved in chloroform from Avanti Polar Lipids and stored at -80 C. Octadecyl Rhodamine B chloride (R18) was purchased from Sigma Aldrich, dissolved in chloroform, and stored at -80 C.

[0158] For fluorescein leakage assay, lipids in chloroform were added to a glass cuvette and dried under a N2stream, then dried under vacuum in the dark at room temperature for at least 12 hours. For R18 fusion assay, lipid and R18 in chloroform were added to a glass cuvette and mixed by swirling before being dried under N2and then dried under vacuum in the dark at room temperature for at least 12 hours.

[0159] Lipids for the R18 assay were rehydrated with 20 mM HEPES buffer at either pH 7.4 or pH 5.5 and vortexed to disperse. Lipids for the CF assay were rehydrated with ~36 mM CF in 20 mM HEPES buffer at pH 7.4. Hydrated lipid underwent 5 freeze-thaw cycles and was extruded through a 100 nm polycarbonate filter (purchased from Avanti Polar Lipids) 29 times to produce unilamellar liposomes of a consistent size. LUV were stored at 4C in the dark.

[0160] Immediately before running the assay, CF-LUV were filtered through a column packed with Sephadex® G-25 Medium media, washed through with 20 mM pH 7.4 HEPES buffer. After filtration, CF absorbance was measured by UV-VIS spectrophotometer, size was measured using DLS, size was also measured by Transmission Electron Microscopy (TEM).Synthesis of Lipid Nanoparticles (LNPs)

[0161] Lipophilic LNP components (lipidoid, helper lipid, PEG lipid, and cholesterol) were dissolved in ethanol and stored at -80 °C. Aliquots of mRNA were stored at -80 C and thawed slowly on ice before use. LNP components were mixed at room temperature and diluted with ethanol and 10 mM sodium citrate buffer pH 4. In parallel, mRNA was diluted with 10 mM sodium citrate buffer at pH 4 at room temperature. Lipid solution was quickly added to mRNA solution, pipetting to mix, to spontaneously form conjugates. LNPs were then diluted with 1 X PBS at pH 7.4 or pH 5.5. Ethanol was removed by dialysis using 3.5k MWCO filters (Thermo Scientific) into the same PBS buffer at room temperature. LNP were stored at 4C in the dark for up to one week. Size characterization was performed by DLS and TEM.Dynamic Light Scattering (DLS) for Particle Size and Zeta- Potential

[0162] Size and charge distributions were measured using a Malvern Zetasizer.Liposomes were diluted in 20 mM HEPES at pH 7.4 or pH 5.5 to approximately 0.1 mg / mLbefore measurement. LNP were diluted to a concentration of approximately 5 zg / mL mRNA in 1X PBS at pH 7.4 or pH 5.5 before measurement.Morphological assessment of Liposomes and LNPs by TEM

[0163] First, carbon film grids were cleaned via glow discharge using a Pelco Easiglow. Next, LLIV or LNP samples were loaded onto the carbon grid and then stained with 1% uranyl acetate. Finally, carbon grids were loaded into a Tecnai 12 Transmission Electron Microscope for image acquisition.Membrane permeabilization and membrane fusion assays

[0164] LUVs and LNPs were brought to room temperature and mixed in a 96 well plate, then immediately placed in a temperature-controlled fluorescent plate reader. Fluorescence measurements were taken immediately, then every 5 minutes for 160 measurements. The plate was taken out of the plate reader and the plate cover was removed. Triton X-100 was added to each well to reach a final concentration of 0.2% (v / v) and vigorously pipetted to rupture and mix all LUVs and LNPs. The plate was placed back into the plate reader and three rapid fluorescence measurements were taken.Data processing

[0165] Raw fluorescence data was taken from the fluorescence plate reader and normalized according to Eqs. 1 and 2 for CF and R18 respectively.Equation 1where: t = timeFCF(t) = time dependent CF fluorescenceFCF O= average of first three CF fluorescence timepoints, measured immediately after plate is placed in fluorescence plate readerFC max= maximum CF fluorescence over all timepoints, typically from Triton additionEquation 2where: R18(t) = time dependent R18 fluorescenceFRIB.O = average of first three R18 fluorescence timepoints, measured immediately after plate is placed in fluorescence plate readerpRis.max = maximum R18 fluorescence over all timepoints, which comes from Triton X-100 additionRelease Kinetics Model Fitting of carboxyfluorescein and R18 release from LLIVs

[0166] The CF and R18 release curves obtained using equation 1 and 2 were averaged across technical replicates and fitted using the Modified Hill Equation given in Eq. 3. Equation 3where:FNormaiized .t') = Time dependent % CF Leakage or % R18 Dilution from Eqs 1 and 2. f = Fitted value representing maximum asymptotic normalized fluorescence value T = Fitted value representing time constant of reaction dequenching t = time n = Fitted value representing cooperativity coefficientStatistical analysis

[0167] Data were recorded with three or more technical replicates. Error bars in Figure 5 represent 95% confidence intervals.

[0168] EXAMPLE 2: Development of a Synthetic Cell Dual Assay to Measure Toxicity and Efficacy of LNPs (LUV and LNP Characterization)

[0169] Lipid nanoparticles (LNP) containing mRNA have shown success as drug delivery tools and as vaccines against infectious diseases such as the COVID-19 mRNA vaccines. LNP synthesis is cost-effective and amenable to scaling, however, the LNP design space is high-dimensional and it is not well known a priori how a given LNP will perform in animals or cell systems as a function of its chemical formulation. The present disclosure describes two complementary fluorescence-based assays (Figures 2 and 3) to quantify the degree and rate of LNP induced permeabilization (a measurement of LNP “toxicity”) as well as membrane fusion with a liposomal artificial cell target (a measurement of LNP “efficacy”), which inform LNP design for successful drug delivery formulation. These techniques enable high throughput in vitro characterization of the efficacy of LNP interaction with cell membranes. Information gleaned from the complementary assays can be used to screen promising LNP formulations for further in vivo study. Many environmental parameters including target membrane composition, size, buffer pH, salt / osmolyte concentration, and temperature can be varied so that LNP vaccine or drug delivery formulations can be assessed for their potential toxicity and efficacy profiles without using cell or mouse models.The disclosed techniques are best used as a first-pass delivery screen to triage lead LNP formulations for nucleic acid-based therapeutics delivery at high throughput. This can help to assess and prioritize LNP formulations (of which there are -tens of thousands) with the best efficacy and toxicity profiles, and identify the conditions (pH, ionizability, temperature stability, etc) under which those formulations are likely to exhibit maximal efficacy with minimal toxicity. Currently, testing of LNP formulations requires low-throughput cell assays or animal testing which are expensive and unable to accommodate the tens of thousands of unique formulations achievable with LNP synthesis.

[0170] To demonstrate differences in interactions between POPC LUVs and four different LNP formulations, a CF leakage at pH 7.4 (physiological pH) and an R18 dilution assay at pH 7.4 and pH 5.5 (endosomal pH) were performed at 25°C using 0.1X LNP concentration (Figure 7) and performed as described herein (Figure 5). 1X LNP concentration represents a characteristic in vivo dosage concentration of 50 zg / mL of mRNA. Each LNP was formulated with the same molar ratios of ionizable lipid, helper lipid, cholesterol, and PEG and from here on will be distinguished by its lipidoid identity. In the CF leakage assay, LNP with 200oi10 and 304oi10 lipidoids induced an order of magnitude greater leakage than LNP with 306o10 or 306oi10 lipidoids, indicating an aggressive interaction with the liposomal membrane at neutral pH that could lead to rupture or potential toxic effects (Figure 5A). In the R18 assay at pH 7.4 (Figure 5B), comparable degrees of fluorophore dequenching were observed. In combination with the CF assay, this implies that LNP with 306o10 or 306oi 10 lipidoids are in fact interacting and fusing with the cell membrane but via a mechanism that does not release the liposomal contents and therefore may not induce a toxic effect. In the R18 assay at pH 5.5 (Figure 5C), the extent of dequenching was highest for LNP with 304oi10, then LNP with 200oi10, 306o10, and 306oi10 lipidoids, implying the highest efficiency of endosomal escape in that order as well. Taken together, LNP formulated with 306o10 and 306oi 10 lipidoids are promising candidates for mRNA delivery due to their strong interaction with the membrane and efficient dequenching at pH 5.5 whilst inducing insignificant amounts of liposomal leakage, implying that they may induce a lower cytotoxic response when circulating in the bloodstream. These data suggest that the methods described herein can be used to compare the efficacy and toxicity profiles of different LNPs in a purely in vitro and high throughput format.

[0171] Moreover, since different cell types in the body can have different compositions, the techniques described herein were tested on synthetic cells that are made with different lipid compositions. The interaction of LNP formulated with 35 mol% 200oi10 lipidoid with 16:0-18:1 PC (POPC) and 16:0-18:1 PG (POPG) liposomes at 35°C was measured by CF leakage at pH 7.4 (Figure 5D), and R18 dilution at pH 7.4 (Figure 5E) and pH 5.5 (Figure5F). The differences in permeability, rate of fusion, and extent of fusion in each plot demonstrate the importance of liposomal target head group. CF dye leakage was faster from POPC vesicles than from POPG vesicles, as was the rate of fusion at neutral pH. In comparison, differences in rate and extent of fusion were generally indistinguishable at pH 5.5 when the lipid nanoparticle is ionized, implying a very high delivery efficiency to either artificial cell type regardless of membrane charge (Figure 5F). These data suggest that cell membrane composition, another variable the presently disclosed methods can test in a high- throughput manner, can affect the efficacy and toxicity profiles of LNP based drug carriers or vaccine formulations. This data suggests the methods of the present disclosure can be used to test biodistribution (e.g., LNPs that target the liver vs. lungs vs. spleen or other organs) in vitro, by changing synthetic cell composition.

[0172] EXAMPLE 3: Interaction of POPC synthetic cells with LNP encapsulating mRNA made with 200oi10, 304oi10, and 306o10 lipidoids.

[0173] Splaying of curves demonstrates permeability dependence on both LNP Concentration and temperature, where sharper curves that reach higher values represent a higher “toxicity” profile of the LNP. 1X LNP represents a common in vivo LNP dosage of 50 pg / mL mRNA when LNPs are used as vaccines. Increase in fluorescent signal due to dilution of R18 in POPC liposomes due to fusion with the same mRNA lipid nanoparticles. Assay performed at pH 7.4 (Figure 5B) and pH 5.5 (Figure 5C) with raw fluorescence data normalized to 100% R18 Dilution. Increase in fluorescence over time is indicative of fusion between liposomal cellular targets and lipid nanoparticles and shows dependence on LNP concentration and temperature. The asymptotic final R18 % Dilution increases somewhat proportionally to the LNP concentration implying this is an equilibrium state with full mixing of the R18 liposomes and LNP (Figure 5E). While the pH 7.4 assay results (Figure 5F) indicate a similar equilibrium % R18 dilution, the curves rise slower and temperature plays a more essential role. Sharper and larger increases in fluorescence for these curves represent LNPs with better endosomal escape capabilities and thus better “efficacy”.

[0174] In Alvarez-Benedicto et al. (Alvarez-Benedicto, et al. (2022) Biomater Sci. 10(2):549-559.), the authors describe a FRET assay for determining lipid fusion of endosome and plasma membrane mimicking liposomes and different formulations and concentrations of LNPs. Fusion with the endosomal membrane is key to mRNA delivery. In this work, authors generate mRNA encapsulating LNP using phosphatidylethanolamine (PE) or phosphatidylserine (PS) helper lipids and mix them with liposomes containing FRET donor and acceptor pair, NBD-PE and N-Rh-PE. Fusion of the LNP and liposome dilute the FRET fluorophores, dequenching the N-Rh-PE fluorescence. Similarly, the presently disclosed compositions and methods allow for measurement of lipid fusion based on adequenching assay using label-free LNP, custom liposomal compositions, and variation of environmental conditions such as pH. In contrast, however, the present disclosure relies on a single dye to measure fusion, rather than a lipophilic dye pair. Further, unlike the present disclosure, Alvarez-Benedicto et al do not generate liposomes of a controlled size or lamellarity, but instead use intermittent sonication to generate a most likely polydisperse and multilamellar suspension of vesicles. Further, the present disclosure uses complementary assays to measure indicators of both membrane fusion and membrane permeabilization, which yield two metrics of LNP potential: LNP delivery efficiency and LNP toxicity

[0175] In Martens et al. (Martens et al. (2014) Nano Today. 9(3):344 - 364), the authors describe assays for detecting and quantifying endosomal escape of nanomaterials. In this review article, several methods describe membrane rupture and burst with subsequent leakage of tracers and membrane fusion, dye dilution and cytosolic cargo transfer. While the review does describe the use of an entrapped self-quenching concentration of fluorescent dye whose recovery of fluorescence signal upon release can be attributed to pore-formation, bursting, or otherwise rupturing the liposomal enclosure, this technique has been primarily used for viral particles and cell-penetrating peptides but has not been used for systematic analysis of oligonucleotide carrying engineered lipid nanoparticles. While the review also describes the use of a lipophilic fluorescent dye at self-quenching concentration whose recovery of fluorescence signal upon dilution can be attributed to lipid mixing or fusion, this technique has not previously been used for systematic analysis and comparison of oligonucleotide carrying engineered lipid nanoparticles. In the present disclosure, application of the membrane permeability and the membrane fusion / mixing assays are performed in tandem specifically to assess potential of engineered lipid nanoparticles in a high throughput fashion.

[0176] EXAMPLE 4: Dye release kinetics

[0177] Data was fitted according to Eqn. 4 below:where:F30= normalized fluorescence at 30 minutesCLNP= normalized LNP concentration EA= Activation Energy R = gas constant T = Temperature (K)

[0178] Normalized fluorescence data (such as that from Figure 5) is transformed according to Equation 4. Plotting ln(F30) - ln(CLWP) versus 1 / T , yields an Arrhenius plot. Using a linear regression to fit the resulting data for a unique LNP and LUV pair including multiple concentrations (0.1 X - 0.5X for LNP with 200oi10, 306o10, and 306oi10 lipidoids or 0.01X - 0.1X for LNP with 304oi10 lipidoid) and multiple temperatures (typically, 20 C - 40 C) allows for calculation of an activation energy (EA) and reaction rate pre-factor (k) for both membrane leakage and membrane fusion. These kinetic parameters can be used to compare different LNP formulations. An optimal LNP formulation would demonstrate (1) low toxicity corresponding to a small prefactor and / or high activation energy for membrane leakage at pH 7.4, as well as (2) high efficiency delivery corresponding to a large prefactor and / or low activation energy for membrane fusion at pH 5.5.

[0179] The present invention has been described by way of illustration and with reference to specific examples and implementations. However, this application is intended to cover those changes and substitutions which are apparent and may be made by those skilled in the art without departing from the spirit and scope of the claims. All of the references cited herein are incorporated by reference.

Claims

What is claimed is:1 . A method of measuring membrane permeabilization of a synthetic cell by a lipid nanoparticle (LNP), the method comprising: a. preparing a mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells; b. measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells comprise a water-soluble fluorescent dye; and wherein an increase in fluorescence correlates with membrane permeabilization.

2. A method of measuring fusion of a LNP and a synthetic cell, the method comprising: a. preparing a mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b. measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells comprise a lipophilic fluorescent dye; and wherein an increase in fluorescence correlates with fusion.

3. The method of claims 1 or 2, wherein synthetic cell is a large unilamellar vesicle (LUV).

4. The method of claims 1 or 3, wherein the water-soluble fluorescent dye is present in the synthetic cell at a self-quenching concentration.

5. The method of claim 4, wherein the increase in fluorescence results from a decrease in self-quenching of the water-soluble fluorescent dye.

6. The method of claim 5, wherein the water-soluble fluorescent dye is selected from the group consisting of sulforhodamine B, FITC, 5(6)-carboxyfluorescein, and calcein.

7. The method of claim 6, wherein the water-soluble fluorescent dye is 5(6)- carboxyfluorescein.

8. The method of 2 or 3, wherein the lipophilic fluorescent dye is present in the synthetic cell at a self-quenching concentration.

9. The method of claim 8, wherein the increase in fluorescence results from a decrease in self-quenching of the lipophilic fluorescent dye.

10. The method of claim 9, wherein the lipophilic fluorescent dye is selected from the group consisting of Octadecyl Rhodamine B Chloride (R18), Diphenylhexatriene (DPH), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), and rac-2,3- dioleoylglycerol ester of rhodamine B (DORh-B).11 . The method of claim 10, wherein the lipophilic fluorescent dye is R18.

12. The method of claim 4 or 8, wherein the dye is included in the assay at or above its self-quenching concentration.

13. The method of claim 3, wherein the LLIV are approximately from 30-400 nm in diameter.

14. The method of any one of claims 1-13, wherein fluorescence signal intensity is measured using fluorescence spectroscopy.

15. The method of any one of claims 1-14, wherein the preparing in step (a) and / or the measuring in step (b) occurs in a multi-well plate.

16. The method of any one of claims 1-15, wherein the LNP comprises a cargo selected from the group consisting of a peptide, a nucleic acid, a small molecule, and a protein or combinations of the same.

17. The method of claim 16, wherein the LNP comprises a ribonucleic acid, protein, or drug cargo.

18. The method of claim 17, wherein the ribonucleic acid is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer- substrate RNA (dsRNA), an antisense oligonucleotide (ASO), an RNA aptamer, a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (IncRNA), a guide RNA (gRNA).

19. The method of claim 18, wherein the nucleic acid is a messenger RNA (mRNA).

20. The method of any one of claims 1-19, wherein the conditions in step (a) optionally include a range of incubation parameters selected from the group consisting of temperature range, salinity range, ionic strength range, and pH range.21 . The method of claim 1 , wherein the conditions in step (a) include a pH of approximately pH 7.4.

22. The method of claim 2, wherein the conditions in step (a) include a pH of approximately pH 5.5.

23. A method of concurrently measuring (i) membrane permeabilization and (ii) fusion of a synthetic cell by a lipid nanoparticle (LNP), the method comprising: a. preparing a first mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b. measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells in the first mixture comprise a water-soluble fluorescent dye; wherein the synthetic cells in the second mixture comprise a lipophilic fluorescent dye; wherein an increase in fluorescence measured in the first mixture correlates with membrane permeabilization; and wherein an increase in fluorescence measured in the second mixture correlates with fusion.

24. A method of concurrently measuring (i) membrane permeabilization and (ii) fusion of a synthetic cell by a lipid nanoparticle (LNP), the method comprising: a. preparing a first mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b. measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells;wherein the synthetic cells in the first mixture are LLIVs and comprise 5(6)- carboxyfluorescein; wherein the synthetic cells in the second mixture are LLIVs and comprise R18; wherein an increase in fluorescence measured in the first mixture correlates with membrane permeabilization; wherein an increase in fluorescence measured in the second mixture correlates with fusion.

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