Polymers for enhancing lipid nanoparticle delivery of nucleic acids

Abstract

The present invention provides delivery-enhancing polymers capable of being encapsulated in nanoparticles to enhance release of payload from the nanoparticle wherein the delivery-enhancing polymer comprises a polyamine comprising tertiary amine. The delivery-enhancing polymer may comprise methylated polyethylenimine (mPEI), branched PEI (bPEI), PAMAM dendrimer and / or histidine polymer.

Description

[0001] Polymers for Enhancing Lipid Nanoparticle Delivery of Nucleic Acids

[0002] Incorporation by Reference of a Sequence Listing XML

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “Delivery Enhancing Polymers NNS 2PCT.xml” created on March 25, 2025 and having a size of 166 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0004] Technical Field of the Invention

[0005] The present invention is related to delivery-enhancing polymers capable of being encapsulated in nanoparticles to enhance release of payload from the nanoparticle

[0006] Background of the Invention

[0007] Lipid nanoparticles (LNPs) are a widely recognized system for packaging, safeguarding, and transporting nucleic acids into cell cytosol. Typically, LNPs consist of four lipids: an ionizable cationic lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG)ylated lipid, each serving specific functions [1,2], Ionizable cationic lipids, facilitate the encapsulation and release of nucleic acids. Phospholipids and cholesterol contribute to the structural stability of the LNPs. PEGylated lipids minimize aggregation of the LNPs, owing to the hydrophilic nature of PEG. In the bloodstream, the PEG lipids rapidly dissociate from the LNPs, allowing serum proteins such as apolipoprotein E (ApoE) to adsorb onto the particle surface. Due to the high hepatic blood flow and the small particle size (~80 nm) of LNPs, a significant dose is directed to the liver, wherein LNPs can effectively penetrate the liver sinusoidal fenestrae and reach the hepatocyte compartment. The ApoE-coated LNPs can then be internalized by the hepatocytes through the LDL-receptor mediated endocytosis. Within the endosome, the ionizable lipids are protonated due to the acidic pH and interact with the anionic cndosomal membrane, destabilizing the membrane and leading to nucleic acid escape into the cytoplasm [3]. However, the endosomal escape of standard LNPs is not efficient, and more than 98% of the LNPs are either degraded in the lysosome or recycled back to the extracellular space [4,5].

[0008] One commonly used approach to improve the endosomal release of nanoparticles is to coat or incorporate polyamines containing a mixture of primary, secondary, and tertiary amines (such as polyethyleneimine) into the formulation [6]. Once the nanoparticles are internalized by cells and enclosed inside the acidic environment of the endosome, the polyamines absorb protons in the environment, leading to an influx of protons and chloride ions into the endosome to maintain charge balance. This influx increases osmotic pressure, causing water to enter theendosome, resulting in its swelling and eventual rupture [7,8], As a result, the nanoparticles are released from the endosome into the cytoplasm, where they can perform their intended functions, such as drug delivery or gene therapy.

[0009] We hypothesized that by incorporating a polyamine pH-sensitive polymer into the LNP formulation, we could introduce the proton sponge effect described above to facilitate the endosomal release of LNP. This approach was expected to reduce endosomal recycling and degradation of LNP, thereby increasing the overall cellular uptake and release of LNP, ultimately enhancing gene delivery. Here, we synthesized different poly amines and incorporated them into a standard LNP formulation at a range of ratios. These polyamine-modified LNP formulations (p-LNPs) were compared for their size, polydispersity index, zeta potential, and nucleic acid encapsulation efficiency. Their effects on pH buffering, cellular uptake, endosomal release, RNA stability, and gene delivery were evaluated in vitro. Finally, in vivo efficacy and safety of the optimal formulations were compared with standard LNPs in mice.

[0010] Summary of the Invention

[0011] The present invention provides a delivery-enhancing polymer, wherein the deliveryenhancing polymer comprises a polyamine that can be used to enhance the endosomal release of a nanoparticle comprising the delivery-enhancing polymer of the present invention.

[0012] The present invention also provides a nanoparticle comprising any embodiment of the delivery-enhancing polymer of the present invention, a first lipid comprising an ionizable lipid. In an embodiment, the nanoparticle further comprises a second lipid comprising neutral lipid, anionic lipid, PEGylated lipid, or a combination thereof. In an embodiment, the nanoparticle of the present invention further comprises a payload.

[0013] The present invention also provides a method of preparation of the nanoparticle of the present invention.

[0014] The present invention also provides a method for enhancing delivery of a payload to a subject comprising the step of administering a therapeutically effective amount of any embodiment of the nanoparticle of the present invention to the subject.

[0015] The present invention further provides a method of treatment of disease suffered by a subject comprising the step of administration to the subject of a therapeutically effective amountof any embodiment of the nanoparticle of the present invention.

[0016] Brief Description of the Drawings

[0017] FIG. 1 illustrates the synthetic scheme of delivery-enhancing polymer comprising the PAMAM dendrimers of the present invention.

[0018] FIG. 2 illustrates the synthetic scheme of delivery-enhancing polymer comprising the bPEI of the present invention.

[0019] FIG. 3 illustrates the 'H NMR (400 MHz) spectra of NS 101 and NS 102 in CDC13.

[0020] FIG. 4 illustrates the LC-MS of NS 102.

[0021] FIG. 5 illustrates theNMR (400 MHz) spectra of NS103 and NS104 in CDCI3.

[0022] FIG. 6 illustrates an embodiment of the method of preparation of the p-LNP of the present invention and the formulation coding logics of the p-LNP of the present invention.

[0023] FIG. 7 illustrates the proton NMR result of NS201.

[0024] FIG. 8 illustrates the proton NMR result of NS202.

[0025] FIG. 9 illustrates the proton NMR result of NS203.

[0026] FIG. 10 illustrates the physicochemical properties of LNPs and p-LNPs containing NS201, NS202 or NS203 and loaded with mRNA. Data= mean ± SD (n=3).

[0027] FIG. 11 illustrates the physicochemical properties of LNPs and p-LNPs containing NS211 or NS212 and loaded with mRNA. Data= mean ± SD (n=3).

[0028] FIG. 12 illustrates the in vitro transfection efficiency of MC3 standard LNP and p-LNP formulations. (A) HEK 293 cells were incubated with different formulations at 1 pg / mL of eGFP mRNA. eGFP fluorescence intensity was measured at 12 hours post-treatment using flow cytometry. All data are presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001; ‘not significant’ was not shown in figures. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. (*: comparing with MC-000-101; #: comparing with adjacent columns). (B) eGFP expression in HEK 293 cells 12 hours after treatment with p-LNP formulations loaded with eGFP mRNA in the presence or absence of Bafilomycin Al. All data are presented as mean± SD (n=3). ####p< 0.0001. The data were analyzed using t-test.

[0029] FIG. 13 illustrates the in vitro cellular delivery of LNP and NS201 -based p-LNP formulations. (A) Subcellular localization of Dil (red) labelled LNP and p-LNP formulations.Nuclei were stained with Hoechst (blue) and endosomes and lysosomes were stained with Lysotracker Green. Histograms were plotted using the RGB plot profile in Zen software for regions as denoted by the red arrows in the yellow rectangles. Images in the panel (A) were quantitatively analyzed using ImageJ for fluorescence intensity of total intracellular Dil-particels per field (B), fluorescence intensity of intracellular Dil-particels that are not overlayed with LysoTracker per field (C), fluorescence intensity of LysoTracker per field (D), and endosomal escape percentage of Dil particles (E). All data are presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001,0.0001; ‘not significant’ was not shown in figures. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. (*: comparing with MC-000-101; # comparing with MC-201-106.)

[0030] FIG. 14 illustrates the in vitro cellular delivery of LNP and NS202-based p-LNP formulations. (A) Subcellular localization of Dil (red) labelled LNP and p-LNP formulations. Nuclei were stained with Hoechst (blue) and endosomes and lysosomes were stained with Lysotracker Green. Histograms were plotted using the RGB plot profile in Zen software for regions as denoted by the red arrows in the yellow rectangles. Images in the panel (A) were quantitatively analyzed using ImageJ for fluorescence intensity of total intracellular Dil-particels per field (B), fluorescence intensity of intracellular Dil-particels that are not overlayed with LysoTracker per field (C), fluorescence intensity of LysoTracker per field (D), and endosomal escape percentage of Dil particles (E). All data are presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001,0.0001; ‘not significant’ was not shown in figures. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. (*: comparing with MC-000-101;#: comparing with MC-202-106.)

[0031] FIG. 15 illustrates the pH buffering effect of (A) MC3 standard LNP formulation and p- LNP formulations containing NS polymers and (B) p-LNP formulations containing 4-15 mol% NS202.

[0032] FIG. 16 illustrates the HEK 293 cellular uptake of Dil labelled MC3 standard LNP and p- LNP formulations under normal conditions at 37°C in complete medium (A), at 4°C (B), in the absence of FBS (C), in the presence of phagocytosis inhibitor Amiloride (EIPA) (D), in the presence of caveolin-mediated endocytosis inhibitor Genistein (GEN) (E) and in the presence of clathrin-mediated endocytosis inhibitor Chlorpromazine (CPM) (F). All data are presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001 ; ‘not significant’ was notshown in figures. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. (*: comparing with MC-000-101;#: comparing with MC-202-106.)

[0033] FIG. 17 illustrates the whole body bioluminescence images of mice after IV delivery of NS201-based p-LNP formulations (A), NS202-based p-LNP formulations (B) and MC3 standard LNP formulation (C) loaded with luciferase mRNA. Total flux of bioluminescence in the liver after IV delivery of different LNP and p-LNP formulations (D)&(E). All data are presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001,0.0001; ‘not significant’ was not shown in figures. The data were analyzed using t test and one-way ANOVA followed by Dunnett’s multiple comparison test. (*: comparing with MC-000-101; # comparing with MC- 201-106 or MC-202-106.)

[0034] FIG. 18 illustrates the physicochemical properties of LNPs and p-LNPs containing NS201, NS202 or NS203 and loaded with mRNA. Data= mean ± SD (n=3).

[0035] FIG. 19 illustrates the whole body biolumincsccncc images of mice and total flux of bioluminescence in the liver after IV delivery of SM-102 standard LNP formulation and NS202 series p-LNP formulations (A). Whole body bioluminescence images of mice and total flux of bioluminescence in the liver after IV delivery of ALC-0315 standard LNP formulation and NS202 series p-LNP formulations (B). All data are presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001,0.0001; ‘not significant’ was not shown in figures. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test, (comparing with standard formulations SM-000-101 or ALC-000-101)

[0036] FIG. 20 illustrates the in vitro cellular delivery of SM-102 or ALC-0315 LNP and NS202-based p-LNP formulations. (A) Subcellular localization of Dil (red) labelled SM-102 LNP and p-LNP formulations. Nuclei were stained with Hoechst (blue) and endosomes and lysosomes were stained with Lysotracker Green. Histograms were plotted using the RGB plot profile in Zen software for regions as denoted by the red arrows in the yellow rectangles. Images in the panel (A) were quantitatively analyzed using ImageJ for fluorescence intensity of total intracellular Dil-particels per field (B), fluorescence intensity of intracellular Dil-particels that are not overlayed with LysoTracker per field (C), fluorescence intensity of LysoTracker per field (D), and endosomal escape percentage of Dil particles (E). (F) Subcellular localization of Dil (red) labelled ALC-0315 LNP and p-LNP formulations. Nuclei were stained with Hoechst (blue) and endosomes and lysosomes were stained with Lysotracker Green. Histograms were plotted using the RGB plot profile in Zen software for regions as denoted by the red arrows in the yellowrectangles. Images in the panel (F) were quantitatively analyzed using ImageJ for fluorescence intensity of total intracellular Dil-particels per field (G), fluorescence intensity of intracellular Dil-particels that are not overlayed with LysoTracker per field (H), fluorescence intensity of LysoTracker per field (I), and endosomal escape percentage of Dil particles (J). All data are presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001,0.0001; ‘not significant’ was not shown in figures. The data were analyzed using t test.

[0037] FIG. 21 illustrates the cytotoxicity and immunototxicity of LNP and P-LNP formulations. Cell viability of HEK 293 cells treated with empty standard LNP formulation and optimal p-LNP formulation with different total lipid concentrations at 24 h (A)&(B). Levels of IFN-y, IL 1 -|3, IL6 and TNF-a cytokines in the serum collected from untreated mice and mice received IV injection of empty MC3 standard LNP formulation and the optimal p-LNP formulation MC-202-006 (C- F); levels of IFN-y, IL1-P, IL6 and TNF-a cytokines in the serum collected from untreated mice and mice received IV injection of empty SM-102 standard LNP formulation and the optimal p- LNP formulation SM-202-004 (G-J). ). All data arc presented as mean± SD (n=3). *p<0.05, **p< 0.01, ***p< 0.001,0.0001; ‘not significant’ was not shown in figures. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test.

[0038] FIG. 22 illustrates the physicochemical properties of the LNPs and the -LNPs loaded with dsDNA (calf thymus). Data = mean ± SD (n=3).

[0039] FIG. 23 illustrates the physicochemical properties of the LNPs and the -LNPs loaded with eGFP mRNA. Data = mean ± SD (n=3).

[0040] FIG. 24 illustrates in vitro transfection efficiency and cytotoxicity of LNP and -LNP formulations. A. HEK 293 cells were incubated with different formulations at 1 pg / mL of eGFP mRNA. eGFP fluorescence intensity was measured at 4 h post-treatment using flow cytometry. B. Viability of HEK 293 cells treated with different formulations at 24 h. Data are presented as mean ± S.D (n = 3) and analyzed by one-way ANOVA with Dunnet’s post-test. ***P < 0.001.

[0041] FIG. 25 illustrates the physicochemical properties of empty LNPs and -LNPs, Data = mean ± SD (n=3).

[0042] FIG. 26 illustrates the pH buffering effect of LNP and NS 102-based p-LNP formulations measured by the acid titration assay.

[0043] FIG. 27 illustrates the physicochemical properties of LNPs and p-LNPs loaded with fluorescein-siRNA. Data = mean ± SD (n=3).

[0044] FIG. 28 illustrates the in vitro and in vivo delivery of siRNA formulated in LNP and p- LNP formulations. A. Cellular uptake of fluorescein- siRNA formulated in LNP and p-LNP formulations by flow cytometry. B. Subcellular localization of fluorescein- siRNA delivered by LNP and p-LNP formulations. Nuclei were stained with DAPI (blue) and endosomes and lysosomes were stained with Lysotracker Red. Histograms were plotted using the RGB plot profile in ImageJ for a particular region as denoted by the yellow line. Images in the panel B were quantitatively analyzed using ImageJ for total intracellular fluorescence intensity of fluorescein-siRNA (C), fluorescence intensity of escaped fluorescein-siRNA in the cytoplasm (D), endosomal escape % of fluorescein-siRNA (E) and total fluorescence intensity of lysotracker in the cells (F). G. Plasma levels of FVII 24 h after treatment with siRNA formulated in MC-000-201 and MC- 102-204.

[0045] FIG. 29 illustrates the physicochemical properties of the LNPs and p-LNPs encapsulated with fluorcsccin-mRNA. Data = mean ± SD (n=3).

[0046] FIG. 30 illustrates the cellular uptake and localization of fluorescein-mRNA delivered by LNPs and p-LNPs. A. HEK 293 cellular uptake and mean fluorescence intensity of fluorescein- mRNA delivered by LNPs and p-LNPs 4 h post treatment. B. Subcellular localization of fluorescein-mRNA (green) delivered by LNPs and p-LNPs. Nuclei were stained with DAPI (blue), and lysosomes were stained with lysotracker (red). Histograms were plotted using the RGB plot profile in ImageJ for a particular region as denoted by the yellow line. Images in panel B were quantitatively analyzed using ImageJ for total intracellular fluorescence intensity of fluorescein-mRNA. Images in panel B were quantitatively analyzed using ImageJ for total intracellular fluorescence intensity of fluorescein-mRNA (C), fluorescence intensity of escaped fluorescein-mRNA in the cytoplasm (D), endosomal escape % of fluorescein-mRNA (E) and total fluorescence intensity of lysotracker in the cells (F).

[0047] FIG. 31 illustrates the physicochemical properties of the LNPs and p-LNPs encapsulated with eGFP-mRNA. Data = mean ± SD (n=3).

[0048] FIG. 32 illustrates the in vitro eGFP expression in HEK 293 cells 4 h after treatment with different LNP and p-LNP formulations loaded with eGFP mRNA (A) in the presence or absence of Bafilomycin AL Data are presented as mean ± SD, n = 3 for all experiments and analyzed by one-way ANOVA with Dunnet’s post-test. ***p < 0.001, and **P < 0.01.

[0049] FIG. 33 illustrates the eGFP mRNA content in LNP and p-LNP formulations at different time points after incubation with FBS (A) and mouse scrum (B). Data arc presented as mean ±SD, n = 3 for all experiments and analyzed by two-way ANOVA with Dunnet’s post-test. (*: comparing MC-000-101 with MC-102-104).

[0050] FIG. 34 illustrates the physicochemical properties of the LNPs and p-LNPs encapsulated with luciferase-mRNA. Data = mean ± SD (n=3).

[0051] FIG. 35 illustrates the cryo-EM images of different LNP and p-LNP formulations prepared with various ionizable lipids.

[0052] FIG. 36 illustrates the in vivo luciferase mRNA delivery by different LNP and p-LNP formulations via IV and IM injection. (A-C) Total flux of bioluminescence in the liver at different time points after IV delivery of different LNP and -LNP formulations loaded with luciferase mRNA. Corresponding bioluminescence images of mice 6 h after IV delivery of different LNP and -LNP formulations arc also shown. (D-F) Total flux of bioluminescence in the injection site at different time points after IM delivery of different LNP and p-LNP formulations loaded with luciferase mRNA. Corresponding biolumincsccncc images of mice 6 h after IM delivery of different LNP and -LNP formulations are also shown. Data are presented as mean ± SD, n = 3 for all experiments, standard LNPs and p-LNPs were analyzed by t-tests. (*: comparing LNP and p-LNP).

[0053] FIG. 37 illustrates the levels of serum ALT (A), serum AST (B) and total bilirubin (C) measured 24 h post injection. Data are presented as mean ± SD, n = 3 for all experiments and analyzed by one-way ANOVA with Dunnet’s post-test. ns= not statistically significant.

[0054] FIG. 38 illustrates the remaining eGFP mRNA % in -LNP formulations with month-long storage at —80 °C. The eGFP mRNA-loaded p-LNPs were synthesized and diluted in PBS containing 12.5% w / v sucrose to an mRNA concentration of 10 p.g / mL and stored at -80 °C for 4 weeks. At each time point (week 1, 2, 3 and 4), mRNA content in the p-LNP samples was determined by gel electrophoresis as described in the main article.

[0055] FIG. 39 illustrates the eGFP mRNA transfection in HEK293 cells 12 hours after incubation with freshly prepared or -80°C-stored MC-102-104 (4 weeks).

[0056] FIG. 40 illustrates the physicochemical properties of the LNPs and the -LNPs loaded with dsDNA (calf thymus) or plasmid DNA (pDNA). Data = mean ± SD (n=3).

[0057] FIG. 41 illustrates the developing and screening polyhistidine-lipid nanoparticle (pLNP) formulations. A. The formulation coding logics (mRNA = luciferase mRNA). B. Structure of linear polyhistidine with capping at both N and C termini. C. Total flux of bioluminescence in the liver 5 h after I.V. delivery of different LNP and pLNP formulations loaded with luciferasemRNA (n=3). X-axis represents different polyhistidines (different numbers of repeat units of histidine) used to prepare pLNP formulations. Y-axis represents different lipid / polyhistidine compositions. D. Represented images of whole-body bioluminescence images of mice 5 h after IV delivery of different pLNPs. E. pH buffering effect of different pLNP formulations measured by the acid titration assay.

[0058] FIG. 42 illustrates the size, PDI, Zeta potential and encapsulation efficiency (EE %) of LNP and pLNP formulations. NA = data not available.

[0059] FIG. 43 illustrates the size, PDI, Zeta potential and encapsulation efficiency (EE %) of pLNP formulations.

[0060] FIG. 44 illustrates the total flux of bioluminescence in the liver 5 h after I.V. delivery of different pLNP formulations loaded with luciferase mRNA (n=3). X-axis represents different polyhistidines used to prepare pLNP formulations. Y-axis represents different lipid / polyhistidine compositions.

[0061] FIG. 45 illustrates the pLNP formulations prepared with different branched Hio. A. structures of branched Hio. B. Total flux of bioluminescence in the liver 5 h after I.V. delivery of different LNP and pLNP formulations loaded with luciferase mRNA (n=3). X-axis represents different polyhistidines (different numbers of repeat units of histidine) used to prepare pLNP formulations. Y-axis represents different lipid / polyhistidine compositions. C. Represented images of whole-body bioluminescence images of mice 5 h after I.V. delivery of different pLNPs. D. pH buffering effect of different pLNP formulations measured by the acid titration assay.

[0062] FIG. 46 illustrates the size, PDI, Zeta potential and encapsulation efficiency (EE %) of pLNP formulations.

[0063] FIG. 47 illustrates the pLNP formulations prepared with different branched Hx. A. structures of branched Hs. B. Total flux of bioluminescence in the liver 5 h after I.V. delivery of different LNP and pLNP formulations loaded with luciferase mRNA (n=3). X-axis represents different polyhistidines (different numbers of repeat units of histidine) used to prepare pLNP formulations. Y-axis represents different lipid / polyhistidine compositions.

[0064] FIG. 48 illustrates the size, PDI, Zeta potential and encapsulation efficiency (EE %) of pLNP formulations.

[0065] FIG. 49 illustrates the in vivo distribution and intracellular uptake of MC-402-103 and MC-000- 103. Biodistribution (A) of DiR labeled particles and the quantitative results (B) in main organs in mice 5 h after I.V. injection (Data=mcan ± SD, n=3). C. Intracellular uptake ofDil-label particles (yellow) loaded with FITC-mRNA (green) in the liver of mice 5 h after I.V. injection. Nuclei were stained with DAPI (blue). Scale bar = 20 pm. Quantitative results of intracellular Dil-label particles (D) and FITC-mRNA (E). P < 0.01 (**), n.s. no significant difference.

[0066] FIG. 50 illustrates the intracellular interaction between MC-402-103 or MC-000-103 with the endosomal network within cells in the liver of mice. A. Livers were collected from mice 5 h after receiving an I.V. dose of Dil-labeled particles. Liver sections were imaged using confocal microscopy. Yellow squares indicate the high-magnification view on the right. White arrows indicate association of endosomal markers with Dil particles. Nuclei were stained with DAPI (blue), MC-402-103 and MC-000-103 were labeled with Dil (green), and endosomal markers were stained with Alexa 647-antibodies (red). Scale bar = 5 pm. B. Quantification of % vesicles associated with particles. C. Quantification of % particles associated with vesicles markers. P < 0.05 (*), P < 0.01 (**), and P < 0.0001 (****).

[0067] FIG. 51 illustrates dynamic interaction of particles with endosomes. A and B. Live cells videos MC-000-101 and MC-402-103 uptake process by using conforcal microscopy. Nuclei were stained with Hochest (blue), MC-402-103 and MC-000-103 were stained with Dil (red) and endosomal markers (green) scale bar = 5 pm. White dash line circled cell edges, white arrows indicates cell membrane with particles, white stars indicates the particles outside of cells. Yellow arrows display particles outside of cells and yellow stars indicates aggregates of particles. C. Analyzing videos from 30 to 45 min in MC-000-101 and MC-402-103 by using Fiji to show the velocity of endosomes and particles.

[0068] FIG. 52 illustrates the dynamic interaction between particles and endosomes. HEK293 cells were seeded in a confocal dish and were imagined immediately by CLSM after particles were added to the culture medium. Dynamic analyses of endosomes (LysoTracker) and Dil- labeled particles at 30-35, 35-40, and 40-45 min after cells were incubated with MC-000-101 (A) and MC-402-103 (B) using Matlab and Eiji. Top panel shows interactions between endosomes (green) and particles (red). Particles panel shows the trails of particles (yellow dash line indicates cell edges). Right panel shows the trails of endosomes, scale bar = 20 pm. C. Total area of endosome cluster over 30 to 45 min.

[0069] FIG. 53 illustrates the delivery of mRNA encoded with adenosine deaminase base editor (ABE) for genome editing in LumA mice. A. Gene editing mechanism of ABE. Bioluminescence images (B) of and the quantitative results (B) in the LumA mic after I.V. injection of particlesloaded with ABE mRNA and sgRNA (data = mean ± SD, n=3). D. Quantification of base editing in the liver after different treatments.

[0070] FIG. 54 illustrates the size, PDI, Zeta potential and encapsulation efficiency (EE %) of pLNP and LNP formulations.

[0071] FIG. 55 illustrates the safety evaluation of pLNPs in vitro and in vivo. A. Cell viability of pLNPs. B and C. TNF-a and IL-6 levels in serum 3 h after mice receive pLNPs at dose 0.3 mg / kg.

[0072] FIG. 56 illustrates the liver and kidney function 24 h after mice receive 0.3 mg / kg pLNPs via I.V.

[0073] FIG. 57 illustrates the machine learning (ML) facilitates optimization of polyhistidine (Hio) conformation. A. Work flow of the ML model. MLP: multilayer perceptron; GNN: Graph Neural Network. First, pooled data from Figure 1C, Figure 2B, Suppl. Figure 1 and Suppl. Fig 2B. Second, created graph representation where animo acids arc nodes, and not atoms. Third, built GNN to predict bioluminescence from peptide structures. Fourth, using the trained GNN to predict NS304 to NS340, NS401 to NS404, and NS601 - NS603 to validate the model. B. New peptide structures were predicted by ML, which indicated branched structures were more effective compared to linear. C. Structures of NS506, NS533 and NS537 suggested by the ML method that would improve the transfection efficiency. NS534 was predicted to be inferior to NS402. D. Total flux of bioluminescence in the liver 5 h after I.V. delivery of different pLNP formulations loaded with luciferase mRNA (n=3). X-axis represents different branched of Hio being used in each series of formulations. Y-axis represents different Hio molar ratio in each formulation.

[0074] FIG. 58 illustrates the size, PDI, Zeta potential and encapsulation efficiency (EE %) of pLNP.

[0075] FIG. 59 illustrates the machine learning validation of polyhistidine for enhanced mLuci transfection in vivo. A. Structures of newly designed branched Hio from ML model. B. Total flux of bioluminescence in the liver 5 h after I.V. delivery of different pLNP formulations loaded with luciferase mRNA (n=3).

[0076] FIG. 60 illustrates the size, PDI, Zeta potential and encapsulation efficiency (EE %) of pLNP

[0077] FIG. 61 visually illustrates the delivery efficacy of mRNA-eGFP to ganglion cell layer (GCL) by LNP comprising MC3 lipid and NS308 delivery enhancing polymer of the presentinvention as compared to the same LNP without the delivery enhancing polymer of the present invention administered via intravitreal injection.

[0078] Detailed Description of the Invention

[0079] The compositions of the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any of the additional or optional ingredients, components, or limitations described herein.

[0080] As used in the specification and claims, the singular form “a” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a” cell includes a plurality of cells, including mixtures thereof.

[0081] “About” in the context of amount values refers to an average deviation of maximum ±20%, preferably ±10% or more preferably ±5% based on the indicated value. For example, an amount of about 30 mol % anionic lipid refers to 30 mol % ±6 mol %, 30 mol % ±3 mol % or 30 mol % ±1.5 mol % anionic lipid with respect to the total lipid / amphiphile molarity.

[0082] “Lipid” refers to its conventional sense as a generic term encompassing fats, lipids, and alcohol-cthcr soluble constituents of protoplasm, which arc insoluble in water. Lipids arc composed of fats, fatty oils, essential oils, waxes, steroid, sterols, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids, and fatty acids. The term encompasses both naturally occurring and synthetic lipids

[0083] As used herein, the term "nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The terms "polynucleotide," "oligonucleotide," "oligo" or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term "nucleotide" refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer.Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of nucleic acids contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acids contemplated herein include any types of RNA (e.g., antisense RNA, mRNA, siRNA, miRNA, shRNA, guide RNA, dicer substrate RNA, dicer substrate siRNAs (dsiRNAs) (dsiRNA are cleaved by the RNase I class endoribonuclease dicer into 21-23 base duplexes having 2-base 3'-overhangs siRNA), and any type of DNA, genomicDNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "duplex" in the context of nucleic acids refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0084] As used herein, the term “effective amount” or “a therapeutically effective amount” of a drug, compound, pharmacologically active agent, a pharmaceutical composition or LNP comprises administering an amount thereof necessary to achieve a desired result. The exact amount required will vary from subject to subject, depending on the species, age, general condition of the subject, the severity of the disease, the particular’ active agent, its mode of administration, the desired outcome, and the like. In certain embodiments of the present invention, a “therapeutically effective amount” of a drug, compound, pharmacologically active agent, a pharmaceutical composition or LNP is that amount effective for inhibiting progression or reversing of any disease disclosed herein in a subject or a biological sample (e.g., in cells). In certain embodiments, disease progression is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In certain embodiments, the drug, compound, pharmacologically active agent, a pharmaceutical composition or LNP inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, at least about 90% or at least about 100%. In certain embodiments of the present invention, a “therapeutically effective amount” refers to an amount of a drug, compound, pharmacologically active agent, a pharmaceutical composition or LNP sufficient to cause reversal of disease. In certain embodiments, the disease is reversed by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100% or any numbers and number ranges falling within these values.

[0085] A “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

[0086] The “polydispersity index” (PDI) is used herein as a measure of the heterogeneity of sizes of molecules or particles in a mixture such as a mixture of LNPs calculated as the square of thestandard deviation of the size distribution divided by the square of the mean of the size distribution.

[0087] Ionizable lipids are positively charged at acidic pH whose positive charge facilitates packaging nucleic acids that are negatively charged into LNPs. The same ionizable lipids become neutral at physiological pH which reduces toxicity as well as minimizes non-specific binding with cells and removal of LNPs by the reticuloendothelial system. After cellular uptake, the ionizable lipids are protonated in the acidic endosome which facilitates membrane fusion / disruption, endosomal escape, and cargo release into the cytosol. Without being bound by theory, delivery enhancing polymers of the present invention are capable of working in concert and / or interacting with the ionizable lipids and other components of the LNP to enhance packaging as well as delivery of the nucleic acid payload due to the geometry, the charge and / or protonation of the delivery enhancing polymer of the present invention.

[0088] The present invention provides a delivery-enhancing polymer capable of enhancing endosomal release of payload of a nanoparticle comprising the payload and the deliveryenhancing polymer of the present invention, wherein the delivery-enhancing polymer comprises a polyamine. In an embodiment, the delivery-enhancing polymer of the present invention comprises a polyamine wherein the poly amine comprises primary amine, secondary amine, tertiary amine, or a combination thereof. In an embodiment, the delivery-enhancing polymer of the present invention comprises a polyamine where in the polyamine consists of tertiary amine. In an embodiment, the delivery-enhancing polymer of the present invention comprises a polyamine where in the polyamine consists of tertiary amine and does not comprise any primary amine and / or secondary amine. In an embodiment, the delivery-enhancing polymer of the present invention comprises a polyamine wherein the polyamine consists of tertiary amine and secondary amine. In an embodiment, the tertiary amine of the delivery-enhancing polymer of the present invention comprises tertiary alkyl amine, imidazole, tertiary aromatic amine, or a combination thereof.

[0089] In an embodiment, the delivery-enhancing polymer of the present invention comprises 1 to 50 tertiary amines such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 including any numbers or number ranges falling within these values. In an embodiment, at least about 10% to about 100% of the amine of the delivery-enhancing polymer of the present invention comprises tertiary amine. In an embodiment, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the amines of the deliveryenhancing polymer of the present invention are tertiary amine including any percentage or percentage ranges falling within these values. In an embodiment, less than about 80% of the amine of the delivery-enhancing polymer of the present invention comprises secondary amine. In an embodiment, less than about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% including any percentage falling in between these values of the amine of the delivery-enhancing polymer of the present invention comprises secondary amine. In an embodiment, less than about 60% of the amine of the delivery-enhancing polymer of the present invention comprises primary amine. In an embodiment, less than about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% including any percentage falling in between these values of the amine of the delivery-enhancing polymer of the present invention comprises primary amine. In an embodiment, the delivery-enhancing polymer of the present invention does not comprise a secondary amine. In an embodiment, the delivery-enhancing polymer of the present invention does not comprise a primary amine.

[0090] In an embodiment, the delivery-enhancing polymer of the present invention comprises polyethylenimine (PEI). In an embodiment, the PEI of the present invention comprises linear PEI (1PEI), branched PEI (bPEI), or a combination thereof. In an embodiment, the PEI of the present invention comprises methylated PEI (mPEI). In an embodiment, the mPEI of the present invention is synthesized by methylating all amine groups of polyethyleneimine (PEI) to tertiary amines.

[0091] In an embodiment, the delivery-enhancing polymer of the present invention comprises highly branched poly(beta- amino ester) (HPAE). In an embodiment, the HPAE comprises polyamidoamine (PAMAM) dendrimer. In an embodiment, the core molecule of the PAMAM dendrimer of the present invention comprises a formula of (NtDL^Nth) wherein L1comprises a linear chain comprising a chain length of about 1 to 10 atoms. In an embodiment, L1comprises carbon, hetero atom, or a combination thereof. In an embodiment, hetero atom comprises N, O, S, P, B, or X, wherein X comprises halogen. In an embodiment, L1consists of alkyl. In an embodiment, the core molecule of the PAMAM dendrimer of the present invention comprises mcthancdiaminc, cthylcncdiaminc, 1,3-diaminopropanc, 1 ,4-diaminobutanc, 1,5-diaminopentane, or 1,6-hexanediamine. In an embodiment, the PAM AM dendrimer of the present invention comprises full-generation PAMAM dendrimer, half-generation PAMAM dendrimer, or a combination thereof. In an embodiment, the PAMAM dendrimer of the present invention consists of half-generation PAMAM dendrimer. In an embodiment, the PAMAM dendrimer of the present invention is prepared using materials comprising the core molecule of the PAMAM dendrimer of the present invention, methyl acrylate, and one or more repeating units comprising a formula of (NH2)L2(NH2) wherein L2comprises a linear chain comprising a chain length of about 1 to 10 atoms. In an embodiment, L2comprises carbon, hetero atom, or a combination thereof. In an embodiment, hetero atom comprises N, O, S, P, B, or X, wherein X comprises halogen. In an embodiment, L2consists of alkyl. In an embodiment, the one or more repeating units of the present invention each comprises methanediamine, ethylenediamine, 1,3- diaminopropane, 1 ,4-diaminobutane, 1,5-diaminopentane, or 1,6-hexanediamine. In an embodiment, the PAMAM dendrimer of the present invention is prepared using at least two different repeating units of the present invention.

[0092] In an embodiment, the delivery-enhancing polymer of the present invention comprises a histidine polymer wherein the histidine polymer comprises one or more histidine residues. In an embodiment, the histidine polymer of the present invention comprises a linear histidine polymer. In another embodiment, the histidine polymer of the present invention comprises a branched histidine polymer. In an embodiment, the histidine polymer of the present invention further comprises one or more non-histidine amino acid residue. In an embodiment, the one or more non-histidine amino acid residues comprise lysine, arginine, aspartic acid, glutamic acid, tyrosine, threonine (Thr), homothreonine (Hth), glutamine, asparagine, cysteine, serine (Ser), homoserine (Hse), proline, phenylalanine, tryptophan, methionine, isoleucine, glycine, leucine, valine, or alanine. In an embodiment, the one or more non-histidine amino acid residues consist of lysine. In an embodiment, the histidine polymer of the present invention does not comprise three or more consecutive lysine residues. In an embodiment, the three or more consecutive lysine residues comprise an amide linkage between the N-terminal amine of a first lysine residue and the carboxyl end of a second lysine residue, an amide linkage between the side chain amine of a first lysine residue and the carboxyl end of a second lysine residue, or a combination thereof. In an embodiment, the histidine polymer of the present invention comprises an amide linkage between the N-terminal amine of a lysine residue and the carboxyl end of a histidine residue, an amide linkage between the side chain amine of a lysine residue and the carboxyl end of ahistidine residue, or a combination thereof. In an embodiment, one or more of the N-termini of the histidine polymer is modified with acetylation, wherein the acetylation results in the capping of the one or more N-termini of the histidine polymer with a — COCH3 functional group. In an embodiment, one or more of the C-termini of the histidine polymer is modified with amidation, wherein the amidation results in the capping of the one or more C-termini of the histidine polymer with a — NH2 functional group. In an embodiment, the histidine polymer of the present invention does not carry a negative charge at a pH value of about 4. In an embodiment, the histidine polymer of the present invention is not charged at a pH value of about 7.4. In an embodiment, the histidine polymer of the present invention does not carry a negative charge at pH 4 nor become charged after pH neutralization to 7.4. In an embodiment, the histidine polymer comprises about 2 to about 50 histidine residues, such as about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 histidine residues. In an embodiment, the histidine polymer comprises about 6 to about 15 histidine residues, such as about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 histidine residues. In an embodiment, the histidine polymer comprises about 1 to about 15 lysine residues, such as about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 lysine residues. In an embodiment, the histidine polymer of the present invention comprises L-amino acid, D-amino acid, or a combination thereof.

[0093] In an embodiment, the delivery-enhancing polymer of the present invention comprises one or more of the structure or formula provided in Table 1 and Table 2.

[0094] Table 1. Structures of the bPEI or PAMAM dendrimer of the present invention

[0095] Table 2. Formula of the delivery enhancing polymer comprising histidine polymer of the present invention (“H” denotes a histidine residue; “K” denotes a lysine residue; “a” denotes — COCH3; “n” denotes — NH2; parenthesis denotes a branch sequence linked to a lysine side chain)0096] In an embodiment, the linear histidine polymer of the present invention comprises about6 to about 15 histidine residues, such as about 6 (SEQ ID NO. 1, x = 3), about 7 (SEQ ID NO. 1, x = 4), about 8 (SEQ ID NO. 1, x = 5), about 9 (SEQ ID NO. 1, x = 6), about 10 (SEQ ID NO. 1, x = 7), about 11 (SEQ ID NO. 1, x = 8), about 12 (SEQ ID NO. 1, x = 9), about 13 (SEQ ID NO. 1, x = 10), about 14 (SEQ ID NO. 1, x = 11), or about 15 (SEQ ID NO. 1, x = 12) histidine residues.

[0097] In an embodiment, the molecular weight of the delivery-enhancing polymer of the present invention is from about 500 g / mol to about 30000 g / mol, such as about 500 g / mol, about 1000 g / mol, about 1500 g / mol, about 2000 g / mol, about 2500 g / mol, about 3000 g / mol, about 3500 g / mol, about 4000 g / mol, about 4500 g / mol, about 5000 g / mol, about 5500 g / mol, about 6000 g / mol, about 6500 g / mol, about 7000 g / mol, about 7500 g / mol, about 8000 g / mol, about 8500 g / mol, about 9000 g / mol, about 9500 g / mol, about 10000 g / mol, about 15000 g / mol, about 20000 g / mol, about 25000 g / mol, or about 30000 g / mol including any molecular weight or molecular weight ranges falling within these values. In an embodiment, the molecular weight of the delivery-enhancing polymer of the present invention is from about 1000 g / mol to about 3000 g / mol, such as about 1000 g / mol, about 1100 g / mol, about 1200 g / mol, about 1300 g / mol, about 1400 g / mol, about 1500 g / mol, about 1600 g / mol, about 1700 g / mol, about 1800 g / mol, about 1900 g / mol about 2000 g / mol, about 2100 g / mol, about 2200 g / mol, about 2300 g / mol, about 2400 g / mol, about 2500 g / mol, about 2600 g / mol, about 2700 g / mol, about 2800 g / mol, about 2900 g / mol, or about 3000 g / mol including any weight or weight ranges falling within these values.

[0098] In an embodiment, the delivery-enhancing polymer of the present invention comprises a pH-sensitive polymer. In an embodiment, the delivery-enhancing polymer of the present invention comprises a titratable polymer. In an embodiment, the delivery-enhancing polymer of the present invention comprises a titratable amine. In an embodiment, the titratable amines comprise primary amines, secondary amines, tertiary amines or a combination thereof. In an embodiment, the titratable amine consists of tertiary amines. In an embodiment, a change in pH changes the surface area, overall size, and / or conformation of the delivery-enhancing polymer of the present invention. In an embodiment, a reduced pH increases the surface area, overall size, and / or conformation of the delivery-enhancing polymer of the present invention.

[0099] In an embodiment, the delivery-enhancing polymer of the present invention comprises protonatable functional group wherein the protonatable functional group is protonated at low pH environment and deprotonated at high pH environment. The protonatable functional group of the delivery-enhancing polymer of the present invention is protonated at low pH environments by absorbing protons (known as proton sponge effect) which can reduce acidification of its surrounding microenvironment. In an embodiment, the surrounding microenvironment comprises an endosome interior. In an embodiment, the delivery-enhancing polymer of the present invention absorbs protons in an endosome interior resulting in reduction of acidification withinthe endosome. In an embodiment, the delivery-enhancing polymer of the present invention absorbs protons in an endosome facilitating endosome swelling and rupture. In another embodiment, the delivery-enhancing polymer of the present invention is encapsulated within a nanoparticle wherein the delivery-enhancing polymer of the present invention absorbs protons in an endosome facilitating membrane fusion between the nanoparticle and the endosome. In yet another embodiment, the delivery-enhancing polymer of the present invention absorbs protons in an endosome resulting in endosomal membrane permeability. In an embodiment, the deliveryenhancing polymer of the present invention absorbs protons in an endosome inducing membrane destabilization of the endosome. In an embodiment, the delivery-enhancing polymer of the present invention facilitates charge-charge interactions with the endosomal membrane to induce local membrane destabilization and / or endosomal release. In an embodiment, the deliveryenhancing polymer of the present invention increases the endosomal release of a nanoparticle comprising the delivery-enhancing polymer of the present invention.

[0100] The delivery-enhancing polymer of the present invention can be either biodegradable or non-biodegradable. In an embodiment, the delivery-enhancing polymer of the present invention is non-biodegradable and can be normally excreted by the body of a subject treated with said polymer.

[0101] The present invention also provides a delivery-enhancing nanoparticle comprising any embodiment of the delivery-enhancing polymer of the present invention and a first lipid wherein the first lipid comprises an ionizable lipid. In an embodiment, the delivery-enhancing nanoparticle of the present invention further comprises a payload.

[0102] In an embodiment, the size of the delivery-enhancing nanoparticle of the present invention is from about 20 nm to about 400 nm such as about 20 nm, about 40 nm, about 60 nm, about 80 nm, about 100 nm, about 120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about 220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about 320 nm, about 340 nm, about 360 nm, about 380 nm, about 400 nm including any size and size ranges falling within these values.

[0103] In an embodiment, the polydispersity index (PDI) of the delivery-enhancing nanoparticle of the present invention is less than about 0.5 such as about 0.5, about 0.45, about 0.4, about 0.35, about 0.3, about 0.25, about 0.2, about 0.15, about 0.1 including any PDI or PDI ranges falling within these values.

[0104] In an embodiment, the zeta potential of the delivery-enhancing nanoparticle of the present invention is from about -30 mV to about 10 mV such as about -30 mV, about -25 mV, about -20 mV, about -15 mV, about -10 mV, about -5 mV, about 0 mV, about 5 mV, or about 10 mV including any zeta potential or zeta potential ranges falling in between these ranges.

[0105] In an embodiment, the mol% of any embodiment of the delivery-enhancing polymer in the delivery-enhancing nanoparticle of the present invention is between about 0.1 mol % to about 50 mol % such as about 0.1 mol %, about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, about 16 mol %, about 18 mol %, about 20 mol %, about 22 mol %, about 24 mol %, about 26 mol %, about 28 mol %, about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, or about 50 mol % including any mol % or mol % ranges falling within these values.

[0106] In an embodiment, the delivery-enhancing nanoparticlc of the present invention comprises lipid nanoparticle (LNP), polymer nanoparticle, lipid-polymer hybrid nanoparticle, liposome, exosome, virus, or virus-like particle.

[0107] In an embodiment, the payload comprises nucleic acids, polynucleotides, amino acids, peptides, polypeptides, recombinant protein, small molecular drug, or a combination thereof. In an embodiment, the polynucleotide comprises any polynucleotide known in the art including but not limited to double stranded DNA (dsDNA), plasmid DNA (pDNA), long non-coding RNA (IncRNA), antisense RNA, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), single-guide RNAs (sgRNAs), or combination thereof. In an embodiment, the payload comprises an anionic payload.

[0108] In an embodiment, the ionizable lipid of the present invention comprises DLin-MC3- DMA (MC3), 4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM- 102), 3,6-bis [4- [bis [(9Z, 12Z)-2-hydroxy-9, 12-octadecadien- 1 -yl] amino]butyl] -2,5- piperazinedione (OF-02), alkyne ionizable lipid: (di(dec-3-yn-l-yl) 9-((4-(dimethylamino) butanoyl)oxy) heptadecanedioate) (A6), alkylene ketone-derived lipid: 1H -Imidazole-2- carboxylic acid, l-[3-(2-ethyl-l-piperidinyl)propyl]-5,5-di-(8Z )-8-heptadecen-l-yl-2,5-dihydro-, ethyl ester (ACI) A18-Iso5-2DC18, or a combination thereof. In an embodiment, the ionizable lipid of the present invention comprises multi-tail ionizable lipids such as Nl ,N16-didodecyl- 4,7, 13-tris[3-(dodccylamino)-3-oxopropyl]-4,7, 10, 13-tctraazahcxadccancdiamidc (98N12-5),1 ,1 ' -[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l - piperazinyl]ethyl]imino]bis-2-dodecanol (C 12-200), 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9) or 3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (cKK- E12). In an embodiment, the ionizable lipid of the present invention comprises ionizable polymer-lipids. In an embodiment, the ionizable lipid of the present invention comprises biodegradable ionizable lipids 9-[4-(dimethylamino)-l-oxobutoxy]-heptadecanedioic acid, 1,17- di-(2Z)-2-nonen-l-yl ester (L-319), 9Z,12Z-octadecadienoic acid, l,r,l",l'"-[(3,6-dioxo-2,5- piperazinediyl)bis(4,l-butanediylnitrilodi-2,l -ethanediyl)] ester (OF-Deg-Lin), tetrakis(2- (octyldisulfaneyl)ethyl) 3,3',3",3"'-(((methylazanediyl)bis(propane-3,l- diyl))bis(azanetriyl))tetrapropionate (306-012B), tetrakis(8-methylnonyl) 3, 3', 3", 3"'- (((methylazanediyl)bis(propane-3,l -diyl))bis(azanetriyl))tetrapropionate (3060il0).

[0109] In an embodiment, the mol% of the ionizable lipid in the delivery-enhancing nanoparticlc of the present invention is from about 0 mol % to about 60 mol % such as about 0%, about 0.1 mol %, about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 50 mol %, about 55 mol %, or about 60 mol % including any mol % or mol % ranges falling within these values. In an embodiment, the mol% of the ionizable lipid in the deliveryenhancing nanoparticle of the present invention is from about 34 mol % to about 50 mol % such as about 34 mol %, about 36 mol %, about 38 mol %, about 40 mol %, about 42 mol %, about 44 mol %, about 46 mol %, about 48 mol %, or about 50 mol % including any mol % or mol % ranges falling within these values.

[0110] In an embodiment, the N / P ratio of the delivery-enhancing nanoparticle of the present invention is from about 2 to about 50 such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 including any ratios or ratio ranges falling within these values. In an embodiment, the N / P ratio refers to the molar ratio of the positively-chargeable amine from the ionizable lipid over the negatively charged nucleic acid phosphate from the payload comprising polynucleotide of the nanoparticle of the present invention. In another embodiment, the N / P ratio refers to the molar ratio of the positively-chargeable amine from the ionizable lipid and the delivery-enhancing polymer of the present invention over the negatively charged nucleic acid phosphate from the payload comprising polynucleotide of the delivery-enhancing nanoparticle of the presentinvention. In an embodiment, the N / P ratio of the delivery -enhancing nanoparticle of the present invention is from about 2 to about 8 such as 2, 3, 4, 5, 6, 7, 8, including any ratio or ration ranges falling within these values, wherein the N / P ratio refers to the molar ratio of the positively- chargeable amine from the ionizable lipid over the negatively charged nucleic acid phosphate from the payload comprising polynucleotide of the nanoparticle of the present invention.

[0111] In an embodiment, the ratio of the number of the delivery-enhancing polymer of the present invention over the number of ionizable lipid in the nanoparticle of the present invention is between about 0 to about 1.5 such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.12, about 0.14, about 0.16, about 0.18, about 0.2, about 0.22, about 0.24, about 0.26, about 0.28, about 0.3, about 0.32, about 0.34, about 0.36, about 0.38, about 0.4, about 0.42, about 0.44, about 0.46, about 0.48, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5 including any ratio or ratio ranges falling within these values.

[0112] In an embodiment, any embodiment of the delivery-enhancing nanoparticle of the present invention further comprises a second lipid comprising neutral lipid, anionic lipid, PEGylated lipid, or a combination thereof. In an embodiment, the second lipid of the present invention comprises natural lipid, synthetic lipid, or a combination thereof. In an embodiment, the second lipid of the present invention comprises phosphatidylserines (PSs), phosphatidylglycerols (PGs), phosphatidylinositols (Pls, not limited to a specific sugar), fatty acids, sterols containing a carboxylic acid group for example, cholesterol, phosphatidylethanolamines (PEs) such as 1,2- diacyl-sn-glycero-3-phosphoethanolamines including, but not limited to 1,2- dioleoylphosphoethanolamine (DOPE), 1,2-distearoylphosphoethanolamine (DSPE), or 1,2- dihexadecoylphosphoethanolamine (DHPE), phosphatidylcholines (PC) such as L-a- phosphatidylcholine, l,2-diacyl-glycero-3-phosphocholines including, but not limited to 1,2- di stearoylphosphocholine (DSPC), 1,2-dipalmitoylphosphocholine (DPPC), 1,2- dimyristoylphosphocholine (DMPC), l,2-dioeoyl-sn-glycero-3-phosphocholine (DOPC), egg PC, soybean PC, hydrogenated soybean phosphatidylcholine (HSPC), and sphingomyelins. The fatty acids linked to the glycerol backbone are not limited to a specific length or number of double bonds. Phospholipids may also have two different fatty acids. In an embodiment, PEGylated lipid comprises DMG-PEG, DSG-PEG, or a combination thereof.

[0113] In an embodiment, the second lipid of the present invention comprises lipid comprising phospholipid, cholesterol, PEGylated lipid or a combination thereof. In an embodiment, the second lipid of the present invention comprises lipid consisting of phospholipid, cholesterol, and PEGylated lipid. In an embodiment, the mol% of the phospholipid in the delivery-enhancing nanoparticle of the present invention is from about 2% to 20% such as about 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% including any mol% falling within these values. In an embodiment, the mol% of the cholesterol in the delivery-enhancing nanoparticle of the present invention is from about 20% to 80% such as about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% including any mol% falling within these values. In an embodiment, the mol% of the PEGylated lipid in the delivery-enhancing nanoparticle of the present invention is from about 0.1% to 16% such as about 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, or 16% including any mol% falling within these values. In an embodiment, the second lipid of the present invention comprises DSPC, cholesterol, and DMG- PEG. In an embodiment, the second lipid of the present invention consists of DSPC, cholesterol, and DMG-PEG. In an embodiment, the second lipid of the present invention consists of DSPC, cholesterol, and DMG-PEG at a mol% of 10%, 38.5%, and 1.5%, respectively. In an embodiment, the molar ratio among the first lipid, any embodiment of the delivery-enhancing polymer of the present invention, and the second lipid in the nanoparticle of the present invention is about (50-x):x:50 wherein x is between about 0.1 and about 30 such as about 0.1, about 0.5about 2.5, about 5, about 7.5%, about 10, about 12.5%, about 15, about 17.5%, about 20, about 22.5%, about 25, about 27.5% or about 30 including any values or value ranges falling within these values.

[0114] In an embodiment, any embodiment of the delivery-enhancing nanoparticle of the present invention comprises delivery-enhancing polymer wherein the delivery-enhancing polymer comprises protonatable functional group and wherein the protonatable functional group is protonated at low pH environment and deprotonated at high pH environment. The protonatable functional group of any embodiment of the delivery-enhancing polymers of the present invention is protonated at low pH environments by absorbing protons (known as proton sponge effect) which can reduce acidification of its surrounding microenvironment. In an embodiment, the surrounding microenvironment comprises an endosome interior. In an embodiment, the nanoparticle of the present invention absorbs protons to reduce the acidification within an endosome. In an embodiment, the delivery-enhancing nanoparticlc of the present inventionabsorbs protons in an endosome which promotes endosome swelling and rupture. In another embodiment, the delivery-enhancing nanoparticle of the present invention absorbs protons in an endosome encapsulating said nanoparticle of the present invention facilitating increased membrane fusion between the nanoparticle and the endosome. In yet another embodiment, the delivery-enhancing nanoparticle of the present invention absorbs protons in an endosome resulting in increased endosomal membrane permeability. In an embodiment, the deliveryenhancing nanoparticle of the present invention absorbs protons in an endosome resulting in membrane destabilization of the endosome. In an embodiment, the delivery-enhancing nanoparticle of the present invention facilitates charge-charge interactions with the endosomal membrane to induce local membrane destabilization and / or endosomal release. In an embodiment, the delivery-enhancing nanoparticle of the present invention increases the endosomal release of an endosome. In an embodiment, the nanoparticle of the present invention facilitates the release of the nanoparticlc payload of the present invention from an endosome to the cytosol. In an embodiment, a protonated form of any embodiment of the delivery-enhancing polymer of the present invention aids in effective complexation with a polynucleotide payload of the present invention. In an embodiment, the nanoparticle of the present invention increases the transfection efficacy of the nanoparticle-encapsulated polynucleotide payload of the present invention.

[0115] In an embodiment, any embodiment of the delivery-enhancing polymer of the present invention is capable of boosting the delivery of payload encapsulated in the nanoparticle of the present invention to a subject. In an embodiment, delivery of the payload to a subject comprises release of the payload from an endosome into the cytosol of cells of a subject. In an embodiment, delivery of the payload to a subject comprises release of the payload in the cytosol of cells of a subject. In an embodiment, delivery of the payload to a subject comprises release of at least about 0.1% to about 100% the payload in the cytosol of cells of a subject such as about 0.01%, about 0.02%, about 0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.2%, about 0.4%, about 0.6%, about 0.8%, about 1%, about 2%, about 4%, about 6%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% including any percentage or percentage ranges falling within these values. In an embodiment, delivery of the payload to a subject comprises release of the payload in the cytosol of targeted cells of a subject. In an embodiment, the targeted cellscomprise cells afflicted by, affected by, is related to, is in proximity to or are at least partially responsible for causing a disease or condition suffered by the subject. In an embodiment, any embodiment of the delivery-enhancing polymer of the present invention boosts the delivery of the payload encapsulated by the nanoparticle of the present invention by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 75%, about 100%, about 125%, about 150%, about 175%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900% or about 1000% including any percentages or percentage ranges falling within these values as compared to the same nanoparticle without any deliveryenhancing polymer of the present invention.

[0116] In an embodiment, the delivery-enhancing polymer of the present invention does not affect the viability of cells treated with said polymer at a concentration up to about 5 to 1000 pg / mL such as up to about 5, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 pg / mL or any value or value ranges falling in between these values. In an embodiment, the delivery-enhancing polymer of the present invention does not affect the viability of cells treated with said polymer for at least about 12 to 96 hours such as about 12, 24, 36, 48, 60, 72, 84, 96 hours including any time duration or time duration ranges falling within these values. In an embodiment, the cells treated with the delivery-enhancing polymer of the present invention maintains cell viability of at least about 80% to about 100% such as about 80%, 85%, 90%, 95%, or 100% including any percentages or percentage ranges falling within these values. In an embodiment, the cells treated with the delivery-enhancing polymer of the present invention maintains cell viability of at least about 80% to about 100% such as about 80%, 85%, 90%, 95%, or 100% including any percentages or percentage ranges falling within these values. In an embodiment, the delivery-enhancing polymer of the present invention is delivered to the cells using the deliver-enhancing nanoparticle of the present invention.

[0117] In an embodiment, the delivery-enhancing polymer of the present invention does not affect the health condition of a subject treated with said polymer. In an embodiment, the subject comprises a mammal such as but not limited to a mouse, a rat, a monkey, or a human. In an embodiment, the delivery-enhancing polymer of the present invention does not affect the inflammatory responses of a subject treated with said polymer. In an embodiment, the inflammatory response of the subject is indicated by parameters such as but not limited to the serum level of cytokines comprising IFN-y, IL1-P, IL6, TNF-a, or a combination thereof. In an embodiment, the delivery-enhancing polymer of the present invention docs not induce liverand / or kidney toxicity of a subject treated with said polymer. In an embodiment, the liver and / or kidney toxicity of the subject is indicated by parameters such as but not limited to the levels of glucose, creatinine, blood urea nitrogen (BUN), BUN:creatinine ratio, phosphorus, sodium, potassium, sodiur potassium ratio, calcium, chloride, bicarbonate, anion gap, total cations, total anions, total protein, albumin, globulin, albumimglobulin ratio, AST, ALT, ALP, total bilirubin, creatinine kinase, osmolality, gamma-glutamyl transpeptidase, hemolysis index, icterus index, lipemia index, or a combination thereof. In an embodiment, the delivery-enhancing polymer of the present invention does not affect the inflammatory responses and / or induce liver and / or kidney toxicity in a subject comprising a mouse at a concentration up to about 0.01 to 20 mg / kg mouse body weight such as up to about 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 mg / kg or any values falling in between these ranges. In an embodiment, the delivery-enhancing polymer of the present invention is delivered to the subject using the nanoparticlc of the present invention.

[0118] In an embodiment, the delivery-enhancing nanoparticle of the present invention enhances payload stability in the body fluid of a subject when the nanoparticles are incubated with the body fluid for up to about 1 to 96 hours such as about 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, 96 hours including any time duration or time duration ranges falling within these values. In an embodiment, the stability of the payload within the delivery-enhancing nanoparticle of the present invention is at least about 10% to 500% higher compared to the same nanoparticle formulation except the addition of the delivery-enhancing polymer of the present invention such as about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% higher including any percentages or percentage ranges falling within these values. In an embodiment, the body fluid comprises serum, plasma, or a combination thereof. In an embodiment, the delivery-enhancing polymer increases the transfection efficiency of the payload encapsulated in the nanoparticle of the present invention by increasing the stability of the payload when the nanoparticle of the present invention is in contact with the body fluid of a subject.

[0119] In an embodiment, the delivery-enhancing polymer of the present invention does not affect the storage stability of the delivery-enhancing nanoparticle of the present invention. In an embodiment, the delivery-enhancing polymer of the present invention does not affect the storage stability of the delivery-enhancing nanoparticle of the present invention after storing at about - 80°C for up to about 1 to about 24 weeks including 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24weeks or any time duration falling within these values. In an embodiment, the deliveryenhancing polymer of the present invention does not affect the payload encapsulated in the nanoparticle of the present invention after storing at about -80°C for up to about 1 to about 24 weeks including 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 weeks or any time duration falling within these values. In an embodiment, the delivery -enhancing polymer does not affect the transfection efficiency of the payload encapsulated in the nanoparticle of the present invention. In an embodiment, the delivery-enhancing polymer does not affect the transfection efficiency of the payload encapsulated in the nanoparticle of the present invention after storing at about -80°C for up to about 1 to about 24 weeks including 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 weeks or any time duration falling within these values.

[0120] The present invention also provides a method of preparation 1000 of the nanoparticle of the present invention. In an embodiment, the method 1000 of the present invention comprises step 1010 of dissolving in a solvent such as ethanol any embodiment of the first lipid of the present invention, and any embodiment of the delivery-enhancing polymer of the present invention. In an embodiment, any embodiment of the second lipid of the present invention such as DSPC, cholesterol and / or pegylated lipids such as PEG-DMG can also be dissolved in ethanol. In an embodiment, the first lipid comprising an ionizable lipid of the present invention, any embodiment of the polymer of the present invention, any embodiment of the second lipid of the present invention are added at the molar ratio of about (50-x):x:50 wherein x is between about 0 and about 30 such as about 0, about 2.5, about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, about 25, about 27.5 or about 30 including any values or value ranges falling within these values. Next in step 1020, the pay load comprising polynucleotide is prepared in the aqueous phase such as but not limited to 25 mM acetate buffer (pH 4.0). In an embodiment, the payload comprising polynucleotide is dissolved in the aqueous phase such as but not limited to an acetate buffer at a concentration from about 1-1000 pg / mL such as about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 pg / mL including any values or value ranges falling within these values. In step 1030, the organic and aqueous phases obtained from step 1010 and step 1020 are mixed. In an embodiment, the mixing step is performed using a microfluidic device. In an embodiment, the mixing using a microfluidic device is performed at a flow rate ratio of about 1 : 1 to about 1 :5 such as about 1 :1 , about 1 :2, about 1 :3, about 1 :4, or about 1 :5 respectively including any ratios or ratio ranges falling within these values. In an embodiment, the total flowrate is about 5 mL / min to about 30 mL / min such as about 5 mL / min, about 10 mL / min, about 15 mL / min, about 20 mL / min, about 25 mL / min or about 30 mL / min including any flow rate falling within these values. The mixing step comprises a pivotal step in the formation of nanoparticle, yielding a nanoparticle-nucleic acid mixture. Next in step 1040, the nanoparticle-nucleic acid mixture is dialyzed. In an embodiment, dialyzing step is performed using phosphate buffered saline at about pH 7.4 using a 2 to 30 kDa molecular weight cut-off (MWCO) dialysis tubing such as about 2 kDa, about 4 kDa, about 6 kDa, about 8 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa including any MWCO falling within these values. In step 1050, the nanoparticle-nucleic acid mixture is then filtered and concentrated. In an embodiment, the filtering and concentration step is performed using a filter of about 0.1 pm to about 0.3 pm such as about 0.1 pm, about 0.15 pm, about 0.2 pm, about 0.25 pm or about 0.3 pm including any values or value ranges falling within these values. In an embodiment, all steps are carried out at about room temperature and the N (from ionizable lipid) / P (from polynucleotide) ratio is maintained at about 1-10 such as about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 including any ratios or ratio ranges falling within these values.

[0121] The present invention also provides a method for enhancing delivery of a payload to a subject comprising the step of administering a therapeutically effective amount of any embodiment of the nanoparticle of the present invention to the subject. In an embodiment, enhancing delivery of a payload of the present invention comprises enhancing the buffering effect of nanoparticle in the endosomal or endolysosomal environment, leading to faster pH neutralizing, osmolarity increase, endosomal or endolysosomal swelling and disruption, and eventually nanoparticle release in the cytosol of the cell.

[0122] The present invention further provides a method of treatment of disease suffered by a subject comprising the step of administration to the subject of a therapeutically effective amount of any embodiment of the nanoparticle of the present invention. In an embodiment, the disease comprises any disease treatable by downregulation or upregulation of expression of one or more polynucleotide in the subject. In an embodiment, the polynucleotide comprises a protein-coding gene, a non-coding gene such as but not limited to transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (IncRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), small nucleolar (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), or a combination thereof. In an embodiment, the disease comprises any disease that may be treated by expression of the polynucleotide pay load of thenanoparticle comprising any embodiment of the polymer of the present invention in the subject. In an embodiment, the disease comprises any disease that may be prevented by expression of the polynucleotide payload of the nanoparticle comprising any embodiment of the polymer of the present invention in the subject. In an embodiment, the disease may comprise various types of cancer. In an embodiment, the disease may be an infectious disease such as various types of influenza as well as prevention thereof such as vaccine. In an embodiment, the disease comprises any type of diseases caused by genetic mutation or excessive or under expression of proteins.

[0123] In an embodiment, the administration step of the present invention may be performed in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intravitreal, or intrathecal or intraventricular administration. The route and site of administration may be chosen to enhance delivery or targeting of the disrupting agent comprising a site-specific targeting moiety to a particular location. For example, to target liver cells, intravenous injection may be used.

[0124] In an embodiment, the disease or condition suffered by the subject may be inhibited or reversed by delivery of any embodiment of the nanoparticle comprising the polymer of the present invention to the cells of the subject by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%.

[0125] It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology. [000142] It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detaileddescription explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology.

[0126] Examples

[0127] Materials and Experimental Methods

[0128] Materials

[0129] Methyl acrylate, 1 ,4-diaminobutane and ethylenediamine were purchased from MilliporeSigma (Oakville, ON, Canada). Ionizable lipid Dlin-MC3-DMA (4-(Dimethylamino)- butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester) (MC3) was purchased from Cayman Chemicals (Ann Arbor, MI). ALC-0315 ((4-Hydroxybutyl) azanediyl) bis(hexane-6,l-diyl) bis(2-hexyldecanoate) and SM-102 (Heptadecan-9-yl 8-((2- hydroxyethyl) (6-oxo-6-(undecyloxy)hexyl) amino) octanoate) were purchased from BroadPharm (San Diego, CA). l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) and Cholesterol were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Luciferase mRNA (Luc-mRNA) and enhanced green fluorescent protein (cGFP) mRNA was purchased from Ccdarlanc Laboratories (Burlington, ON, Canada). siRNA against Factor VII (siFVII) was purchased from Integrated DNA Technologies (Coralville, IA). VivoGlo™ Luciferin was purchased from Promega Corporation (Madison, WI). Branched polyethylenimine (bPEI) (average Mw 800 or 25,000) was purchased from MilliporeSigma (Oakville, ON, Canada). The linear Luciferase DNA template was supplied by Genescript (Piscataway, NJ). DNase I, LiCl, and the HiScribe T7 mRNA Kit with CleanCap Reagent AG were purchased from New England Biolabs, Inc (Ipswich, MA). Fluorescein- 12-UTP was provided by Enzo Life Sciences (Farmingdale, NY). XTT salt was purchased from MilliporeSigma (Oakville, ON, Canada), Dil, Hoechst and LysoTracker Green were purchased from Thermofisher (Waltham, MA USA). Mouse IFN- gamma Elisa kit was purchased from Thermofisher (Waltham, MA USA), Mouse ILl-beta Elisa kit was purchased from VWR (Edmonton, AB, Canada), Mouse IL6 and TNF-alpha Elisa kits were purchased from Cedarlane Laboratories (Burlington, ON, Canada). Chlorpromazine, Amiloride, Genistein and Bafilomycin Al were purchased from MedChemExpress (Monmouth Junction, NJ). Polyhistidines of different lengths and conformations were synthesized bySynpeptide (Shanghai, China), with N- and C-termini capped with acetylation and amidation, respectively.

[0130] Synthesis of PAMAM dendrimers

[0131] The synthetic scheme is shown in FIG. 1. Methyl acrylate (166 g, 1.93 mol) was dissolved in 100 mL of methanol and cooled to 0 °C under a nitrogen atmosphere. A separate solution of 1,4-diaminobutane (15.15 g, 0.172 mol) in 75 mL of methanol was added dropwise to the methyl acrylate solution over 1 hour. The reaction mixture was maintained at 0 °C for 3 hours, followed by overnight stirring at room temperature. The excess methyl acrylate and methanol were then removed using a rotary evaporator to obtain NS200, which was used without purification.

[0132] NS200 (71.55 g, 0.167 mol) was dissolved in 850 mL of methanol. Ethylenediamine (EDA) was dissolved in 110 mL of methanol and cooled to 0 °C. The NS200 solution was gradually added to the EDA solution over 1 hour under a nitrogen atmosphere. The reaction mixture was kept at 0 °C for 3 hours and then stirred for 4 days at room temperature. Residual methanol and excess EDA were removed under vacuum, followed by azeotropic distillation using a methanol / toluene (1:9) mixture to ensure complete EDA removal. The excess toluene was subsequently eliminated by azeotropic distillation with methanol, yielding NS211 as a colorless oil.

[0133] NS201 was synthesized by dissolving methyl acrylate (3 equivalents per surface amine group) in methanol at a volume approximately equal to that of the methyl acrylate and cooling the solution to 0 °C. Under a nitrogen atmosphere, a 10% w / w methanol solution of NS211 was added dropwise over 1 hour. The mixture was kept at 0 °C for 3 hours and stirred at room temperature for 2 days. The Kaiser test (1% ninhydrin in ethanol) was performed to confirm the complete reaction of the amines. Excess solvent and unreacted methyl acrylate were removed using a rotary evaporator and high vacuum, yielding NS201 as a slightly yellowish oil.

[0134] The same two-step process was repeated twice to obtain subsequent NS-polymers, including NS212, NS202, and NS203 as shown in FIG. 1.

[0135] Methylation of bPEI

[0136] The synthetic scheme is shown in FIG. 2. One g bPEI (Mw 800 or 25,000 referred as NS101 and NS103, respectively, for formulation coding) was mixed with 14 mL formic acid and 12 mL of 38% formaldehyde aqueous solution in a round bottom flask under N2 (g). The mixture was heated at 120 °C for overnight, followed by cooling to 25 °C. Approximately 20 mL ofdiethyl ether was added to the reaction mixture, and KOH pellets were added until the organic layer turned yellow and the product (methylated PEI referred as NS 102 and NS 104, respectively) was extracted in the organic layer. This extraction procedure was repeated three times. The product, an orange-colored organic liquid, was then dried over anhydrous magnesium sulfate, and the solvent was removed using a rotary evaporator to yield NS 102 or NS 104. The NS 102 (Mw 1086.84 g / mol) and NS 104 (Mw -25000 g / mol) polymers were characterized by 1H-NMR, and mass spectra to confirm their identities (FIG. 3-5). Structures of NS101-104 are shown in Table 1.

[0137] LNP preparation and characterization

[0138] The polyamine-modified LNPs (p-LNPs) were synthesized by varying the molar ratio of NS-polymer in the standard LNP composition. The formulations in our study contained an ionizable lipid, an NS-polymer, DSPC, cholesterol, and PEG-DMG at a mole ratio of 50-x: x: 10: 38.5: 1.5, wherein x was between 0 and 25. When x = 0, the formulation refers to the standard LNP. The lipid / polymer components were dissolved in ethanol at a concentration of -2.5 mg / mL. For the aqueous phase, the nucleic acid (mRNA or siRNA) was dissolved in 25 mM acetate buffer (pH 4.0) at 0.1-0.2 mg / mL. The organic and aqueous phases were mixed using the NanoAssemblr Benchtop (Precision Nanosystems, Vancouver, BC, Canada) at a flow ratio of 1:3 with a total flow rate of 15 mL / min. The N (from ionizable lipid) / P ratios were 3 and 6 for siRNA and mRNA, respectively. The resulting formulation was dialyzed against phosphate- buffered saline (PBS, pH 7.4) using a 10 kD MWCO dialysis tubing (Spectrum Labs) for 24 h at room temperature. The LNP was then filtered through a 0.2 pm filter and concentrated using the Amicon Ultra 100K centrifugal filter (Millipore, Billerica, MA). All experimental procedures were carried out at room temperature. Empty formulations containing no payload were prepared using the same method without including RNA. Particle size, polydispersity index (PDI), and zeta potential of formulations were measured by dynamic light scattering using Malvern Zetasizer NanoZS (Worcestershire, UK). RNA concentration and encapsulation efficiency were determined using the Quant-iT Ribogreen RNA assay from Thermo Fisher Scientific (Waltham, MA) using the manufacturer's protocol.

[0139] Measurement of lipids using UPLC

[0140] Lipid concentrations in formulations were measured by Waters ACQUITY ultra-high- performance liquid chromatography (UPLC) (Milford, MA). Briefly, 10 pL of the formulation was mixed with 490 uL organic solvent (ethanol: methanol, 3:4, v / v), and 10 pL of the mixturewas injected into the UPLC system. The mobile phase was comprised of solvent A (0.1% formic acid and 0.1 % ammonium hydroxide in water, v / v) and solvent B (0.1 % formic aid and 0.1 % ammonium hydroxide in 75% acetonitrile and 25 % isopropyl alcohol v / v), with the following gradient applied: 1.5 min: A / B (10 / 90), 2.0 min: A / B (7 / 93), 3.6 min: A / B (2 / 98), 8.5 min: A / B (2 / 98), 8.6 min: A / B / (10 / 90), 11 min: A / B (10 / 90). The sample was separated in a Waters Acquity BEH-C18 column (particle size: 1.7 pm, inner diameter: 2.1 mm length: 50 mm) at a flow rate of 0.3 mL / min and was detected by an evaporative light scattering detector (ELSD). Data were analyzed using the Empower 3.0 software (Waters). Standard UPLC chromatograms of cholesterol and DSPC are shown in FIG. 4.

[0141] Cryo electron microscopy

[0142] LNPs and p-LNP formulations were concentrated to a total lipid concentration of 10 mg / mL using Amicon centrifugal filter units (Millipore). For imaging, 3-5 pl of each sample was applied to a copper grid and rapidly frozen with a Leica Vitrobot. The grid was then transferred to a pre-equilibrated transfer station set at or below -180 °C before being loaded into an FEI Tecnai G2 Twin transmission electron microscope (FEI, Hillsboro, OR) operating at 200 kV under low-dose conditions. Sample freezing, grid preparation, and imaging were performed at the UBC Bioimaging Facility (Vancouver, BC) [9].

[0143] pH buffering effect

[0144] To measure the pH buffering effect of different formulations, acid titration was performed. pH values were measured using a pH meter (Mettler Toledo) each time when 0.5-5 pL of 1 M HC1 was added into empty formulation (100 pg / mL of total lipids) in PBS until the pH reached 2 or 3.

[0145] Synthesis of fluorescein labeled luciferase mRNA

[0146] The reaction mixture containing 50 mM ATP, GTP, CTP and UTP / fluorescein 12-UTP, 40 mM CleanCap Reagent AG, 2 U / pL murine RNase inhibitor, 0.005 U / pL pyrophosphatase, 50 ng / pL linear Luciferase DNA template (Supplementary Sequence No. 1), 50 U / pLT7 RNA Polymerase, 4 mM MgC12, 1 mM DTT, 2 mM Spermidine, 50 mM NaCl, and 40 mM Tris-HCl (pH 8.0) was incubated for 3 h at 37 °C using Thermal Cycler 2720 (Thermo Fisher Scientific, Waltham, MA). The linear DNA template was removed by incubating the mixture with DNase I at a final concentration of 0.1 U / pL for 10 min. The mRNA product was purified using LiCl precipitation (New England Biolabs, Ipswich, MA), and the mRNA yield was measured using a Nanodrop 2000C spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

[0147] Cell culture

[0148] HEK293 human embryonic kidney cells were maintained in Dulbecco’s Modified Eagle’s Medium with 10% fetal bovine serum (FBS) supplemented with 1% penicillin-streptomycin. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.

[0149] In vitro cytotoxicity

[0150] The cells were seeded in a 96-well plate at a density of 1 x 104cells per well and maintained for 24 h. Cells were then treated with different formulations at a range of concentrations up to 1000 mg / mL of total lipid for 24 h. The cell viability was then determined by the MTT assay as described previously

[0010] .

[0151] Cellular uptake and intracellular trafficking of particles

[0152] Fluorescein-tagged RNA (siRNA and mRNA) was loaded into different nanoparticle formulations and incubated with HEK 293 cells at 2 pg / mL RNA for 4 h at 37 °C. To quantify the cellular uptake, flow cytometry was performed and the results were analyzed by FlowJo. To visualise the intracellular trafficking, cells were washed with PBS trice and treated with Lysotracker Red for the labeling of endosomes and lysosomes. The cells were then fixed and mounted onto a slide using Fluoro shield™. The stained cells were imaged by confocal laser scanning microscopy (CLSM) and quantitatively analyzed with ZEN software (Carl Zeiss, Oberkochen, Germany) and ImageJ. Endosomal release % from the confocal images were quantified using the following equation; Endosomal release (%) = (Non-overlayed fluorescence intensity of intracellular RNA ) / (Total fluorescence intensity of intracellular RNA ) X 100%.

[0153] Endocytosis inhibitor treatments

[0154] To investigate the internalization mechanism of our novel p-LNP formulations, several endocytosis inhibitors were used. HEK293 cells were seeded in 12-well plates and pretreated with 30 uM Clathrin-mediated endocytosis inhibitor Chlorpromazine (CPM), 25 uM phagocytosis inhibitor Amiloride (EIPA) and 100 uM caveolin-mediated endocytosis inhibitor Genistein (GEN), respectively, for 30 minutes. Subsequently, Dil labelled p-LNP formulations were introduced into the wells for a 4-hour internalization period. Following the incubation, the cells were harvested and analyzed for intracellular Dil fluorescence intensity using flow cytometry. Bafilomycin Al, a proton pump inhibitor, was used to confirm that the improvement of endosome escape efficiency was due to the enhancement of proton sponge effect. HEK293 cells were seeded in 12-well plates and pretreated with 200 nM bafilomycin Al for 1 hour. Subsequently, cGFP-mRNA-loadcd p-LNP formulations were introduced into the wells for a 12-hour transfection period. Following the incubation, the cells were harvested and analyzed for eGFP expression using flow cytometry.

[0155] In vitro eGFPmRNA transfection study

[0156] HEK 293 cells were incubated with different formulations loaded with eGFP mRNA at 2 pg / mL mRNA in cell culture media containing 10 % FBS at 37 °C for 4 h. Cells were collected and analyzed for eGFP expression by flow cytometry. To examine the effect of proton-pump inhibition on the transfection effect, cells were pre-treated with 200 nM bafilomycin Al for 1 hour before LNP and p-LNP treatment.

[0157] mRNA serum stability study

[0158] eGFP mRNA-loaded LNPs and p-LNPs were diluted in a 1:1 ratio (v / v) with sterile fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) or mouse serum (Innovative Research, Inc, Novi, MI) and incubated at 37 °C for up to 24 hours. At specific time points, 25 L of the sample was collected, and Triton X-100 was added to achieve a final concentration of 2%. Ten pL of the sample was mixed with 10 pL of the loading buffer, analyzed by gel electrophoresis, and the images were captured using BioRad Gel Doc Documentation System. Relative mRNA content (%) was normalized to that at the 0 hour time point.

[0159] In vivo factor VII silencing study

[0160] LNPs and p-LNPs were formulated with anti-Factor VII siRNA (siFVII) following our previously mentioned protocol, with an N / P ratio of 3. Mice received a tail vein injection of LNPs or p-LNPs at siFVII doses ranging from 0.05 to 0.5 mg / kg. Twenty-four hours postinjection, plasma FVII was quantified by ELISA (IDEXX Mukilteo, WA).

[0161] In vivo immunotoxicity study

[0162] The empty LNP formulations were injected into mice at a dose of total lipid 20 mg / kg, and the serum of mice was collected after 6 hours, and the concentrations of IFN-y, ILl-p, IL6 and TNF-a cytokines in the serum were determined according to the protocols provided by the ELISA kit manufacturers.

[0163] In vivo Luc-mRNA transfection study

[0164] Luc-mRNA was encapsulated into different formulations and intravenously (IV) or intramuscularly (IM) injected at a dose of 5-300 pg / kg into CD1 mice (Charles River Laboratories, Wilmington, MA). At different time points (6, 24, 48 h), around 120 pL of d- luciferin (30 mg / ml) was injected intraperitoneally (i.p) injected, and 15 min later, biolumincsccncc within mice was imaged by the IVIS Lumina III In Vivo Imaging System(Spectral Instruments Imaging, Tucson, AZ). Quantitative image analysis was performed using Aura Image analysis Software 4.0.8 (Spectral Instruments Imaging, Tucson, AZ).

[0165] Intracellular distribution of mRNA and particles within the liver

[0166] Dil-labeled particles encapsulating FITC-mRNA were injected intravenously to mice. Six hours post-injection, mice were euthanized, and the liver was collected, embedded in OCT, and sectioned at 10 pm on a Leica Cryostat. The tissue section was fixed in 10% formalin for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with 1% BSA in PBST (PBS + 0.1% Tween 20) for 60 minutes. Following washes, the section was incubated with a primary antibody (5 pg / mL APPL1, 2 pg / mL EEA1, 1:100 diluted Rabl l, or 1: 100 diluted Anti-LBPA) for 1 hour, followed by 3 times of PBS washes. The secondary antibody (Goat anti-Rabbit / Mouse IgG Alexa 647-conjugated, 1 pg / mL) was applied on the section for 1 hour at room temperature, which was washed for 3 times and stained with DAPI for CLSM.

[0167] In vivo toxicity test

[0168] Approximately 150 pL of serum was collected from the mice IV-injected with ~0.3 mg / kg formulation 3-24 h after injection for analysis of inflammatory cytokines by ELISA or safety biomarkers, including urea (BUN), ALT, AST, ALP, creatine kinase and osmolality.

[0169] Statistical analysis

[0170] The data are expressed as mean ± standard deviation (SD). Statistical analysis for comparing two experimental groups was performed using the Student’s t-tests. In experiments with multiple groups, one- or two-way analysis of variance (ANOVA) with Dunnet’s post test was performed using Prism 7 (GraphPad Software). The p value less than 0.05 was considered statistically significant.

[0171] Results

[0172] FIG. 6 illustrates an embodiment of the method of preparation of the p-LNP of the present invention and the formulation coding logics of the p-LNP of the present invention.

[0173] Example 1 - PAMAM dendrimer

[0174] Synthesis of NS polymers

[0175] In this study, we designed three pH-sensitive ionizable polymers NS201, NS202, and NS203, containing only tertiary amine groups without primary or secondary amines. These polymers were built upon 1,4-diaminobutane, and the primary amine groups were converted to tertiary amine groups via the Michael addition of methyl acrylate. Following by an activation step where ethylene diamine substituted outer methyl esters and formed amides, a molecule(NS211) which has primary amines at the outer layer was yielded. NS201 was synthesized from NS211 by repeating the Micheal addition with methyl acrylate to convert the outer layer primary amines to tertiary amines. The same processes were repeated to synthesize NS202 and NS203 that contained only tertiary amines and NS212 that contained a mixture of primary and tertiary amines (FIG. 1). Nuclear Magnetic Resonance (NMR) spectroscopy was employed to confirm structures of the synthesized polymers. As shown in FIG. 7-9, the peak at 5 = 1.47 ppm corresponded to the CH2 protons in the core, while the sharp singlet at 3 - 3.67 ppm indicated the presence of methyl esters at the branch termini of the polymers. Comparative integration of these characteristic peaks revealed that the CH2 protons in the core and the branch-terminal esters were in ratios of 1:6, 1:12, and 1:24 for NS201, NS202, and NS203, respectively. This result demonstrated the successful synthesis of the NS polymers.

[0176] Synthesis and Characterization of LNP and p-LNP formulations

[0177] We then introduced different NS polymers into the standard LNP formulation, creating multiple p-LNP variants with varying mole ratios of the polymers using the microfluidic system. The formulation coding logics is described in FIG. 6. For example, the formulation MC-201-103 was prepared using MC3 ionizable lipid (MC) and NS201 polymer (201), with an mRNA payload (1) and a specific lipid / polymer formula (03): 47.62 mol% MC3, 10 mol% DSPC, 38.5 mol% Cholesterol, 1.5 mol% DMG-PEG and 2.38 mol% of NS201. The MC3 standard LNP formulation encapsulating mRNA was represented as MC-000-101. Subsequently, we utilized a Zetasizer to evaluate the particle properties of these formulations. As illustrated in FIG. 10, the incorporation of 2-15 mol% NS201 or NS202 did not significantly alter the particle size of the p- LNPs when compared to the standard MC3-LNP formulation (MC-000-101). Zeta potential measurements also demonstrated that p-LNPs containing NS201 and NS202 maintained a nearneutral charge, with zeta-potentials within ±5 mV. Additionally, the p-LNPs prepared with NS201 and NS202 exhibited high mRNA encapsulation efficiencies, exceeding 90%. In prior studies involving LNP modification through the addition of supplementary components, the encapsulation efficiency was often compromised. [ 11 , 12] This phenomenon was, however, observed with p-LNPs prepared with NS203, showing significantly increased particle size (-200 nm) and decreased mRNA encapsulation efficiency (35-60%). This could be due to increased aggregation between mRNA and the high molecular weight NS203. It has been shown that electrostatic interaction between two high molecular weight polymers is difficult to control, which leads to formation of large aggregates, compared to polymers with smaller molecularweights.

[0013] Formation of large aggregates between mRNA and NS203 could also impact the lipid assembly, resulting in decreased mRNA encapsulation. Based on these characterizations of p-LNP formulations, we decided to focus on formulations prepared with NS201 and NS202.

[0178] In vitro eGFP mRNA transfection

[0179] We then tested our hypothesis that NS polymers that contained only tertiary amine groups, such as NS201 and NS202, would be more efficient in promoting LNP delivery compared to pH-sensitive polymers consisting of a mixture of primary and tertiary amines, such as NS211 and NS212. The p-LNP formulations prepared with NS211 and NS212 showed comparable particle properties as those prepared with NS201 and NS202 (FIG. 11), although the mRNA encapsulation efficiency was slightly lower. NS211 and NS212 containing primary amines at the termini and were therefore, more hydrophilic compared to NS201 and NS202, decreasing their incorporation into the hydrophobic core of LNPs. As shown in FIG. 12A, compared to the standard formulation MC-000-101, the cells treated with MC-201-106 and MC- 202-106 LNP formulations exhibited 1.4-fold and 1.6-fold stronger eGFP fluorescence intensity, respectively, indicating higher in vitro transfection efficiency. In contrast, the two p-LNP formulations incorporating NS211 and 212 did not show any improvement but instead exhibited slight decreases in the cellular transfection compared to MC-000-101. Specifically, the transfection efficiency of MC-212-106 was significantly lower than that of MC-000-101. The data supported our hypothesis and validated the effectiveness of using NS201 and NS202, which contained only tertiary amine groups, to improve LNP delivery efficiency.

[0180] Quantification of endosomal escape

[0181] Following cellular entry, LNPs and p-LNPs would be entrapped in the endosomes, wherein proton pumps actively transport protons into the endosomal lumen, progressively reducing its pH from neutral to acidic.

[0014] Particle-enclosed endosomes could recycle to the plasma membrane and exeytose LNPs and p-LNPs, and the pH of these recycling endosomes is often around 6.4-6.8. [15-19] The endosomes could also stay intracellularly and progress to early endosomes (pH -6.0-6.5), late endosomes (pH -5.0-6.0), and then lysosomes (pH ~4.5-5.0). [18, 20-22] We hypothesized that under these acidic conditions, the tertiary amine groups in the NS polymers within the p-LNP formulations would absorb hydrogen ions and get protonated, resulting pH buffering. In response, the proton pumps would continue to operate to maintain the acidic environment, leading to an accumulation of protons and chloride ions. This increase in ion concentration would significantly raise the osmotic pressure, driving water influx into theendosomes. Consequently, the endosome would swell and rupture, releasing the p-LNPs into the cytoplasm.

[0182] To test our hypothesis that NS201 and NS202 could introduce the proton sponge effect to the LNP formulation, facilitating the endosomal disruption and release of the internalized formulation, we spiked Dil, a non-diffusive hydrophobic fluorescent dye, into LNP and p-LNP formulations and incubated the particles with HEK293 cells for 1 h. The intracellular trafficking of Dil labeled LNPs and p-LNPs was then analyzed using confocal macroscopy to quantify the cellular uptake and endosomal escape efficiency. As depicted in FIG. 13 A. The regions where Dil (red: particles) and Lysotracker (green: endosome and lysosome) signals overlapped appeared in yellow, indicating particles that remained trapped within endosomes and lysosomes. The images indicated that: First, all p-LNP formulations containing NS201 displayed increased intracellular uptake compared to the standard LNP formulation, MC-000-101, as more Dil fluorescence was detected intracellularly. Second, a decrease in the fluorescence intensity of Lysotracker was observed in the p-LNP-treated cells, indicating a reduction in the number of endosomes, supporting our hypothesis about the proton sponge effect. Third, in the merged images, increasing NS201 content in p-LNPs led to increases in red fluorescence (Dil particles) that did not overlap with green (endosome / lysosome), signifying that more p-LNPs had successfully escaped from the endosomes and lysosomes into the cytoplasm. The fluorescence intensity distribution along the red arrows in the magnified images are presented in the histogram on the right. In the cells treated with the standard MC3-LNP formulation (MC-000-101) and p- LNPs with a low NS polymer content (MC3-201-103), the distribution of red and green fluorescence was highly overlapping, indicating that most of these particles remained trapped within the endosomes and lysosomes. In contrast, cells treated with MC-201-105 and MC-201- 106 that contained higher amounts of NS201 showed significant dissociation of red fluorescence from green fluorescence, indicating endosomal escape of the formulations. It is worth noting that the MC-201-107 formulation, which contained the highest NS201 content, did not exhibit the anticipated increase in dissociation between red and green fluorescence. Instead, most of the observed Dil red fluorescence spots still overlapped with the green LysoTracker signals. This indicated that the endosomal escape efficiency of p-LNP formulations was not strictly positively correlated with the NS polymer content.

[0183] We then quantified the images using ImageJ. The total red fluorescence intensity corresponded to the total particle uptake by the cells, while the red fluorescence not overlappingwith the green fluorescence indicated the amount of particles released from the endosomes into the cytoplasm. As shown in FIG. 13B, p-LNPs containing 2-12 mol% NS201 exhibited up to 1.6 times increased total cellular uptake compared to the standard LNPs. However, MC-201-107 containing -14 mol% NS201 did not show increased overall uptake compared to MC-000-101. The increased overall cellular uptake of the optimal NS201 -based p-LNP formulations could be due to the enhanced cellular internalization and / or retention (i.e., reduced endosomal recycling). Quantitative analysis of Dil fluorescence intensity not colocalized with LysoTracker signals in FIG. 13C revealed a notable increase in the Dil fluorescence in most p-LNP formulations (except MC-201-103) compared to the standard MC-000-101 formulation. This observation indicated that a significant proportion of p-LNPs containing NS201 successfully escaped the endosomal entrapment and were released into the cytoplasm. Among the tested formulations, MC- 201-106 demonstrated the most pronounced released Dil fluorescence signal, exhibiting an approximately 2.5-fold increase relative to the standard formulation. The fluorescence intensity of LysoTracker Green in the confocal images served as an indicator of the amount of remaining endosomes within the cells. As shown in FIG. 13D, a significant reduction in the amount of endosomes and lysosomes was observed in cells treated with all p-LNP formulations, supporting our hypothesis that p-LNPs promoted endosomal rupture and enhanced endosomal escape. The amount of remaining endosomes was inversely associated with the NS201 content (0-12 mol%) in the formulations. Notably, the minimum endosomal fluorescence signal was observed in the cells treated with the MC-201-106 formulation, displaying a reduction to one-fourth of the signal detected in the cells treated with MC-000-101. However, further increasing the NS201 content to -14 mol% (MC-201-107) resulted in a rebound of endosomal fluorescence intensity in the cells. This indicated that MC-201-106 was the optimal formulation exhibiting the strongest proton sponge effect. FIG. 13E showed the calculated endosomal escape efficiency for different formulations. The endosomal escape percentage mean value of NS201-based p-LNPs increased with increasing NS201 content, plateauing at a maximum escape percentage of 60% when -12 mol% NS201 was incorporated. Statistical analysis showed that there was a significant 3-fold increase in the endosomal escape efficiency of MC-201-106 relative to MC-000-101. Although the mean endosomal escape percentages of the rest NS201 -based p-LNP formulations were higher than the standard LNP formulation, the differences were statistically insignificant. Overall, FIG. 13 data indicated that MC-201-106 was the optimal formulation, deliveringincreased bioavailable (i.e., released) particles into the cells compared to LNPs and other NS201- based p-LNPs.

[0184] Comparable confocal imaging results were observed with NS202-based p-LNPs (FIG. 14A). Notably, the optimal NS202-based p-LNP formulations (i.e. MC-202-105 and MC-202- 106) was 2.5-fold (FIG. 14B) and about 8-fold (FIG. 14C) more efficient in delivering and releasing nanoparticles compared to standard LNPs, respectively. In FIG. 14C, quantitative analysis of the fluorescence intensity of total intracellular Dil-labeled particles and Dil-labeled particles not overlapping with LysoTracker revealed that both the cellular uptake or retention of p-LNPs and their release from the endosomes increased with rising NS202 content in the formulations. This trend plateaued when the NS202 content reached 8-12 mol% (MC-202-105 and MC-202-106). However, a decline was observed when the NS202 content exceeded 14 mol% (MC-202-107). Statistical analysis further confirmed this trend, showing no significant difference in cndosomal release amount of Dil particles between the two optimal formulations, MC-202-105 and MC-202-106, while a significant reduction was noted in MC-202-107 compared to MC-202-106. Moreover, the optimal NS202-based p-LNPs (MC-202-105 and MC- 202-106) were more efficient in endosomal escape compared to the NS201-based formulation (MC-201-106) (FIG. 14E vs. FIG. 13E; 80% vs 60%). The endosomal escape percentage of all NS202-containing p-LNP formulations was significantly higher than that of the standard MC3 formulation, consistently exceeding 60%. Notably, the MC-202-106 formulation achieved a maximum endosomal escape efficiency of 83%. Statistical analysis showed no significant difference between MC-202-105 and MC-202-106 in their endosomal release efficiency, while a significant decrease was observed in MC-202-107. In both cases of NS201- and NS202-based p- LNP formulations, when the polymer content exceeded 14 mol%, the intracellular delivery efficiency started to decline compared to the optimal formulations. To elucidate the underlying mechanism, we compared the pH buffering effects of different formulations using the acid titration method. Formulations were titrated with aqueous HC1 to mimic the endosomal acidification process, and the pH was monitored. Prior to titration, the LNPs or p-LNPs were dispersed in neutral PBS (pH ~7), where the tertiary amines in the formulations remained uncharged. As HC1 was added during the titration, the tertiary amine groups in the formulations absorbed protons from the solution, reaching ionization equilibrium, which resulted in a gradual decrease in pH, thereby demonstrating the pH buffering effect. First, we compared the standard MC3 LNP formulation with the two p-LNP formulations prepared with NS201 and NS202,respectively. As shown in FIG. 15A, upon the addition of HC1, the standard MC3 formulation (MC-000-001) reached the titration jump first, followed by MC-201-006 and then MC-202-006. This supported our hypothesis that the inclusion of NS polymers containing tertiary amine groups enhanced the buffering capacity of p-LNPs. Notably, NS202 exhibited superior buffering capacity compared to NS201, resulting in increased endosomal release efficiency (66% vs. 83%) and overall intracellular delivery (FIG. 13E and FIG. 14E).

[0185] Next, we compared the pH buffering effect of p-LNP formulations containing 4-15 mol% NS202. As shown in FIG. 15B, the titration curve shifted to the right as the content of NS202 increased from 4.5 mol% to 11.5 mol%, indicating that the buffering capacity of the p-LNPs was positively associated with NS202 amount incorporated in the formulation. However, when the NS202 content reached a mol% of 14.3%, the titration curve of the MC-202-007 formulation shifted to the left, indicating a decrease in buffering capacity. The results confirmed our cellular uptake data that the optimal mol% of NS202 was 11.54 mol%, achieving the highest pH buffering effect among the p-LNP formulations. A further increase of the NS202 content might make the formulation too hydrophobic as unprotonated NS202 was water insoluble, impeding proton penetration into the formulation. Overall, the pH buffering results supported the findings in the cellular uptake study that p-LNPs containing 11.54 mol% NS polymer was optimal and NS202 was superior to NS201 in promoting the endosomal release and intracellular delivery due to the enhanced pH buffering effect. To further confirm that the enhanced proton sponge effect was the primary mechanism driving the improved endosomal escape efficiency of the formulations containing NS201 or NS202, we pretreated cells with the proton pump inhibitor Bafilomycin Al prior to the incubation with the p-LNP formulations encapsulated with eGFP mRNA. Bafilomycin Al would inhibit the endosomal acidification, thereby suppressing the proton sponge effect. As shown in FIG. 12B, the transfection efficiencies of both MC-201-106 and MC-202-106 were completely reduced to the baseline levels following Bafilomycin Al pretreatment. This result validated that the enhanced pH buffering and proton sponge effect were the major mechanisms contributing to the improved endosomal escape efficiency. Taken together, these data supported our hypothesis that incorporating the NS-polymers containing only tertiary amines equipped the formulation with the proton sponge effect, which resulted in increased endosomal rupture and cytosolic release, leading to reduced endosomal recycling and overall increased uptake / retention of p-LNPs.

[0186] Internalization mechanism

[0187] We also compared the cellular internalization mechanisms of the standard LNPs (MC- 000-101) and the two optimal p-LNPs (MC-202-105 and MC-202-106). Flow cytometry was used to detect fluorescence signals of intracellular Dil labeled nanop articles. As illustrated in FIG. 16 A, at 37°C in complete medium, the cellular uptake of the optimized p-LNP formulations was 3- to 4-fold greater than that of the MC3 standard formulation. When the incubation temperature was dropped to 4°C, the cellular internalization of all three formulations was inhibited to the basal level (FIG. 16B), indicating that the uptake mechanism was energy dependent. Similarly, when the incubation medium was deprived of FBS, the cellular' uptake of all three formulations was inhibited to the basal level as untreated cell control (FIG. 16C). Overall, these results indicated that LNPs and p-LNPs utilized energy- and serum proteindependent pathways for cellular- entry. Next, we treated the cells with specific inhibitors, including the macropinocytosis inhibitor EIPA (Amiloride), the clathrin-mediated endocytosis inhibitor Chlorpromazine (CPM), and the cavcolin-mcdiatcd endocytosis inhibitor Gcnistcin (GEN), to examine the internalization mechanism.[23-25] The results demonstrated that treatment with EIPA did not significantly affect the cellular internalization of either LNPs or p- LNPs compared to the normal conditions (FIG. 16D), indicating that macropinocytosis played an insignificant role for the uptake of LNPs and p-LNPs. In contrast, the presence of the caveolin inhibitor GEN completely inhibited the internalization of all three nanoparticle formulations (FIG. 16ETreatment with the clathrin inhibitor CPM resulted in complete inhibition of the standard MC3 LNP uptake, whereas the uptake of NS202 p-LNP formulations was inhibited by approximately 50% (FIG. 16F). These findings aligned with previous reports showing that the LDLR pathway that has been associated with the cellular uptake of LNPs can facilitate endocytosis via either caveolae- or clathrin-mediated pathways. Our results also revealed that p- LNPs exhibited a preferential reliance on caveolin-mediated endocytosis. Collectively, these data suggested that MC-202-105 and MC-202-106 adsorbed serum proteins to the particle surface, which could be recognized by cell surface receptors, leading to caveolae-mediated endocytosis. This internalization pathway was likely different from that for the standard LNPs.

[0188] In vivo mRNA delivery

[0189] To compare in vivo mRNA delivery efficiency of different LNP and p-LNP formulations, 5-week-old female CD-I mice were intravenously (IV) administered these formulations containing luciferase mRNA at a dose of 5 pg / kg. After 6 hours, 300 pg luciferin was intraperitoneally (IP) injected into the mice, and biolumincsccncc was detected using an in vivoimaging system (IVIS). FIG. 17A and FIG. 17B display the imaging results of mice injected with NS201- and NS202-based p-LNP preparations, and the whole-body images of mice treated with the standard MC3 formulation (MC-000-101) are shown in FIG. 17C. The data revealed that all p-LNP formulations achieved enhanced mRNA transfection in the liver compared to the standard MC3 LNP formulation. As the molar ratio of NS201 or NS202 increased, the bioluminescence signal intensity in the liver was enhanced, peaking at the formulations containing 11.5 mol% polymers. Quantitative analysis of total flux in the thoracic cavity (liver region) confirmed these observations (FIG. 17D,E). The bioluminescence signal intensity of all p-LNP preparations exceeded that of the MC3 standard LNP. The in vivo results were consistent with the in vitro data (FIG. 13E), indicating that MC-201-106 and MC-202-106 were the optimal formulations that increased the mean total bioluminescence flux by 75- to 120-fold relative to MC-000-101. Moreover, when comparing the two optimal p-LNP formulations, MC-202-106 displayed slightly improved transfection efficiency, which was again consistent with the in vitro results. Similar to the in vitro data, when the NS201 or NS202 content further increased to 14.29 mol%, the in vivo transfection efficiency of MC-201-107 and MC-202-107 started to declined.

[0190] We next investigated whether the optimal NS polymer, NS202, was compatible with LNPs prepared with other ionizable lipids, such as SM-102 and ALC-0315. Characterization results of LNP and p-LNPs containing SM-102 or ALC-0315 are shown in FIG. 18, and all these formulations exhibited mean diameters <100 nm, neutral potential, and high encapsulation efficiency of around 90%. As shown in FIG. 19A, the liver bio luminescence of mice injected with SM-202-104 containing 4.5 mol% NS202 was significantly enhanced compared to the standard SM LNP. Quantitative analysis revealed that the total flux reached 1.8xl09p / s, which was 100 times that of SM-000-101. However, SM-202-105, which contained 8.33 mol% NS202, did not show any further enhancement, indicating that SM-202-104 represented the optimal formulation among the SM-based p-LNPs. In contrast, FIG. 19B illustrates that incorporating NS202 into LNPs composed of ALC-0315 lipid resulted in only a modest, approximately a 2- fold increase in the liver bioluminescence. No clear trend of improved in vivo delivery efficiency was observed with increasing NS202 content in the ALC-based formulations. Furthermore, the enhancement mediated by NS202 in the ALC series p-LNPs was moderate compared to that observed in the MC3- and SM-based formulations. Notably, even the standard ALC formulation, ALC-000-101 , which lacked NS polymers, achieved a total liver flux of approximately IxlO8p / s in the injected mice. This finding suggested that the ALC formulation inherently exhibited strongin vivo transfection efficiency, potentially limiting the relative gains from incorporating NS202. To explore this hypothesis, we tracked the intracellular trafficking of ALC formulations using confocal microscopy. As shown in FIG. 20A, cells treated with the optimal NS202-based p-LNP formulation (SM-202-104) showed no increase in intracellular Dil fluorescence compared to the standard LNP formulation (SM-000-101), indicating no significant enhancement in cellular uptake or retention. However, a reduction in LysoTracker fluorescence intensity was observed in cells treated with p-LNPs, suggesting a decrease in endosome numbers, likely due to an enhanced proton sponge effect. Additionally, in the merged images, cells treated with NS202- containing p-LNPs exhibited a greater amount of Dil particles not overlapping with green fluorescence (LysoTracker), indicating that more p-LNPs had escaped from the endosomes. The histogram on the right displays the distribution of Dil and LysoTracker fluorescence signals along the red arrows in the magnified images. In cells treated with the standard LNP (SM-000- 101), the red and green fluorescence showed a high degree of colocalization. In contrast, cells treated with the optimized p-LNP formulation (SM-202-104) demonstrated a clear separation of red and green fluorescence, signifying successful endosomal escape of the p-LNPs. Quantitative analysis of the confocal images using ImageJ software corroborated these visual observations. As shown in FIG. 20B, there was no significant difference in the Dil fluorescence signals between cells treated with SM-000-101 and SM-202-104. FIG. 20C and FIG. 20D illustrate that, compared to the standard LNP formulation, cells treated with SM-202-104 exhibited a reduction in green fluorescence intensity (indicative of endosomes / lysosomes) to approximately 20%, while the Dil fluorescence intensity not overlapping with LysoTracker increased by 4.7-fold. The nanoparticle release efficiency of SM-202-104 was 4.5 times higher than that of the standard formulation SM-000-101 (FIG. 20E). Confocal images of ALC -based LNPs and p-LNPs are presented in FIG. 20F. Similar to the SM-based results, there was no significant difference in the total intracellular uptake and retention of Dil particles between cells treated with the standard formulation ALC-000- 101 and the optimized formulation ALC-202- 105 (FIG. 20F,G). Furthermore, LysoTracker fluorescence intensity in cells treated with ALC-202- 105 decreased to 20% (FIG. 20F,H). In the merged images and the accompanying histogram, it was observed that cells treated with the standard formulation ALC-000- 101 already exhibited significant separation of red and green fluorescence, unlike the high colocalization observed in the confocal images of MC-000- 101 or SM-000-101 treated cells. Quantitative analysis revealed that in cells treated with the optimized formulation ALC-202- 105, the Dil fluorescence intensity not overlappingwith LysoTracker was only 1.3 times that of the standard formulation (FIG. 201), while the endosomal escape efficiency increased from 77% to 92% (FIG. 20J). These findings explain the in vivo mRNA delivery results in FIG. 19. The incorporation of NS202 significantly enhanced the endosomal escape efficiency of the SM-102 standard LNP (from 21% to 97%), leading to a nearly 100-fold increase in the in vivo delivery efficiency. In contrast, the ALC-0315 standard LNP already demonstrated a high endosomal escape efficiency of 77% under our experimental conditions. Incorporating NS202 into the optimized formulation ALC-202-105 increased the p- LNP endosomal escape efficiency by only -15%, resulting in a limited 2-fold increase in the in vivo experiments.

[0191] Cytotoxicity and immunotoxicity of NS202

[0192] To assess cytotoxicity and immunotoxicity of the optimal NS polymer NS202 and corresponding optimal p-LNP formulations (MC-202-006 and SM-202-004), we performed XTT and inflammatory cytokine assays on cells and animals after treatment with empty standard LNPs (MC-000-001, SM-000-101) and empty p-LNPs (MC-202-006, SM-202-004). In these safety studies, we examined mRNA-free formulations to eliminate any confounding interference from the payload. The results are shown in FIG. 21A and FIG. 21B that even at a total lipid concentration of 1000 pg / mL (NS202 concentration at 300 pg / mL), none of the LNP or p-LNP formulations exhibited cytotoxicity. Subsequently, we compared the in-vivo immunotoxicity of the standard LNPs and p-LNPs. Mice were intravenously administered with MC-000-001, MC- 202-006, SM-000-002, or SM-202-004 at a dose of 20 mg / kg of total lipid, and untreated mice served as the negative control. Serum was collected 6 hours post-injection, and the levels of IFN- gamma, IL 1 -beta, IL6, and TNF-alpha cytokines in the serum were measured using the enzyme- linked immunosorbent assay (ELISA). As shown in FIG. 21 C-J, no significant differences were observed in the concentrations of these four cytokines in the serum of mice injected with MC- 202-006 and SM-202-004, compared to the untreated mice or standard LNP groups. All cytokine levels remained within the normal ranges, indicating that NS202 was a safe component in p-LNP formulations.

[0193] Conclusion

[0194] Our studies demonstrated that incorporating NS polymers that contained only tertiary amines as a pH-sensitive functional group into standard LNP formulations significantly enhanced mRNA delivery efficiency by improving the endosomal escape. Incorporating an appropriate amount of NS polymer with a MW below 3000 g / mol, such as NS201 or NS202, enhanced thepH buffering effect while maintaining the physicochemical properties relative to the standard LNP formulations. NS202 exhibited an enhanced pH buffering effect due to its higher molecular weight compared to NS201, whereas the inclusion of NS203 with a MW exceeding 3000 g / mol resulted in particle aggregation. The optimal mole ratio of NS202 in the MC3-based p-LNP formulations appeared to be 11.54%, leading to a 4-fold increase in endosomal release and 120- fold increased luciferase bioluminescence intensity in the liver after luciferase mRNA delivery compared to standard MC3 LNP. Additionally, NS202 was compatible with LNP formulations prepared with other ionizable lipids such as SM-102 and ALC-0315, improving the in vivo transfection efficiency up to 100-fold. Importantly, NS202 showed no cytotoxicity and did not elicit immune responses in mice, affirming its high safety and biocompatibility.

[0195] In vitro characteristics of p-LNP formulations comprising dsDNA as a payload

[0196] FIG. 22 illustrates the physicochemical properties of the LNPs and the -LNPs loaded with dsDNA (calf thymus). Data = mean ± SD (n=3).

[0197] Example 2 - bPEI

[0198] Synthesis of NS102 and NS104

[0199] We first compared four different polyamines for their effect in promoting gene delivery of LNP, including two commercial bPEI with different Mw (800 or 25K, also referred to as NS 101 and NS 103, respectively) that contain a mixture of primary, secondary and tertiary amines, and NS 102 and NS 104 that only consist of tertiary amines (Table 1). The pKas of primary, secondary, and tertiary amines within PEI are around 7.5-8.5, 6.5-7.5, and 4.5-6.5, respectively, and only tertiary amine is truly “ionizable” in physiological conditions. At pH 4 or the acidic environment within the endosome and lysosome, the tertiary amine is protonated to interact with negatively charged nucleic acid or exert the proton sponge effect, while at pH 7.4, the tertiary amine remains uncharged for minimal non-specific interaction with biological molecules, leading to improved safety. NS 102 and NS 104 were synthesized using an efficient one-step methylation reaction from NS 101 and NS 103, respectively (FIG. 2). In the ’ H NMR spectrum of NS101, a sharp single peak for protons of CH3 at 5 = 2.24 ppm indicated the presence of three sets of -CH3 protons (FIG. 3). The disappearance of the characteristic singlet at 5 = 1.67 ppm that corresponded to the NH / NH2 protons confirmed methylation of all primary and secondary amines into tertiary amines (FIG. 4). The identity of NS 102 was further evidenced by mass spectrometry, which shows a prominent peak for the molecular ion [M+H]+at m / z 1088(FIG. 5). Similarly, the characteristic singlet at 8 = 1.67 ppm also disappeared in the ’ H NMR spectrum of NS 104, indicating methylation of primary and secondary amines to tertiary (FIG. 5).

[0200] In vitro characteristics of LNP and p-LNP formulations

[0201] We first compared particle characteristics of LNP and p-LNP formulations prepared with different NS-polymers. As shown in FIG. 23, spiking 4.5 mol% of NS 101 (Mw 800) or NS 102 (Mw 1086.84) in the standard MC3-LNP formulation (MC-000-101) did not alter the particle characteristics significantly, and these formulations displayed comparable size (70-80 nm), PDI (<0.2), zeta potential (~0 mV), and mRNA encapsulation efficiency (>90%). However, when larger Mw NS-polymers (NS 103 and NS 104, Mw 25K) were used to fabricate p-LNPs, the resulting particles showed slightly decreased size (60-70 nm), increased PDI (~0.2), and decreased mRNA encapsulation efficiency (70-90%). The reduced particle size might be due to the increased condensation of mRNA by larger Mw NS-polymers. However, this strong complcxation could interfere with the interaction between MC3 ionizable lipid and mRNA, leading to decreased encapsulation efficiency. We then performed the in vitro cell transfection study with these LNP and p-LNP formulations and employed flow cytometry to quantify eGFP expression to compare their transfection efficiency. As shown in FIG. 24A, p-LNPs prepared with low Mw polyamines (MC-101-104 and MC-102-104) exhibited significantly higher eGFP expression compared to the standard MC3-LNP (MC-000-101). Specially, p-LNPs prepared with NS102 that contained only tertiary amines (MC-102-104) demonstrated transfection efficiency about 10-fold greater than MC-000-101 and twice that of MC-101-104. In contrast, p-LNPs prepared with high Mw NS-polymers (MC-103-104 and MC-104-104) showed only marginal increases of eGFP expression relative to MC-000-101 (FIG. 24A). The results indicated that both the Mw and amine group content of NS-polymers were critical in determining the mRNA transfection efficiency. High Mw of poly amines could result in strong interaction with mRNA, reducing cytosolic release of mRNA. Tertiary amine that is truly ionizable in physiological conditions appeared to be more important compared to primary and secondary amines in promoting mRNA transfection. We then compared cytotoxicity of LNP and p-LNP formulations prepared with different NS-polymers at total lipid concentrations of 50 and 500 pg / mL. At 50 pg / mL, all LNP and p-LNP formulations showed no significant toxicity (FIG. 24B). However, at 500 pg / mL, p-LNPs prepared with larger Mw NS-polymers (MC-103-104 and MC-104-104) exhibited marked toxicity, with cell viabilities of only 15% and 4% relative to control, respectively (FIG. 24B). In contrast, p-LNPs composed of low Mw NS-polymers (MC-101-104and MC-102-104) showed no toxicity even at 500 pg / mL. Toxicity of polyamines has been associated with their Mw and amine density [26,27,31], and our data were consistent with the previous reports. Overall, our preliminary findings indicated that p-LNP prepared with NS 102 possessed comparable physicochemical properties and safety, and improved in vitro transfection efficiency compared to the standard MC3-LNP.

[0202] pH-buffering effect of p-LNP formulations incorporated with different amounts of NS102

[0203] Based on the results in FIG. 24, we decided to focus on optimizing the p-LNP formulation by varying the mol% of NS 102. We incorporated 2.4-8.3 mol% NS 102 into the standard MC3-LNP formulation to prepare empty p-LNP (no RNA payload) and compared their pH-buffering effects with empty MC3-LNP (MC -000-001). As shown in FIG. 25, spiking 2.4-8.3 mol% of NS 102 into empty MC3-LNP formulation did not alter any of the particle properties, including size, PDI, and zeta potential. However, p-LNP displayed an increased pH buffering effect compared to LNP (FIG. 26), with the formulation containing 4.5 mol% of NS 102 (MC- 102-004) showing the optimal result. Before the acid titration at pH ~7, tertiary amines in NS 102 were uncharged, but would start absorbing protons from the added HC1 during the titration process to slow down the decrease of the pH in the environment, leading to an increased pH buffering effect. However, when too much NS102 was included (in the case of MC- 102-005), the formulation might become too hydrophobic (uncharged NS 102 is water insoluble), impeding proton ions from penetrating into the formulation, which led to a decreased pH buffering effect.

[0204] Cellular uptake and intracellular localization of siRNA delivered by LNP and p-LNP formulations

[0205] Results in FIG. 26 suggest that NS 102-based p-LNP formulations could effectively decrease pH acidification in the endosome and lysosome after cellular internalization, leading to instability of the vesicles, which would result in increased cytosolic release and reduced endosomal recycling of p-LNP back to the extracellular medium. As such, the overall cellular uptake and retention of bioavailable RNA delivered by p-LNP would be increased. To test this hypothesis, we loaded fluorescein-siRNA into LNP and p-LNP formulations incorporated with 2.4-8.3 mol% of NS 102 and compared their intracellular uptake and trafficking. Again, siRNA- loaded p-LNP formulations displayed comparable particle properties as the standard LNP formulation, MC-000-201 (FIG. 27). HEK 293 cells were incubated with these formulations at 1 pg siRNA / mL for 4 h, and the overall cellular uptake of fluorcsccin-siRNA was analyzed byflow cytometry. As shown in FIG. 28A, all -LNP formulations exhibited increased intracellular delivery of fluorescein- siRNA compared to the standard MC3-LNP, while MC- 102-204 containing 4.5 mol% of NS 102 displayed the highest delivery efficiency that was 2-fold increased relative to MC3-000-201. The results are consistent with the pH buffering data in FIG. 26, suggesting the pH buffering effect was the primary mechanism contributing to the increased siRNA delivery of the -LNP formulations.

[0206] Confocal microscopy was employed to visualize the intracellular localization and release of fluorescein-siRNA delivered by LNP and p-LNP formulations. Confocal images revealed the fluorescence of fluorescein-siRNA (green), endosome / lysosome (red), and nuclei (blue) in FIG. 28B. Consistent with FIG. 28A, MC-102-204 displayed the highest intracellular delivery of fluorescein-siRNA among all. The merged images showed that cells treated with MC-102-204 displayed enhanced dissociation between siRNA and Lysotracker Red, indicating increased cytosolic release compared to other groups. The histogram data in FIG. 28B included the distribution of endosome / lysosome and fluorescein-siRNA on the yellow lines indicated in the merged images. The data further exemplified that there was the least overlay between siRNA and endosome / lysosome in cells treated with MC-102-204, indicating increased release of siRNA. The confocal images were further quantified by ImageJ. Consistent with FIG. 28 A, FIG. 28C showed that the overall intracellular delivery of siRNA by MC-102-204 was increased by 2-fold compared to the standard LNP (MC-000-201). Furthermore, MC-102-204 increased the delivery of released siRNA (i.e., dissociated from endosome / lysosome) by 4-fold compared to LNP (FIG. 28D), and the endosomal escape percentage of siRNA delivered by MC-102-204 was increased from 30% to 75% when compared with LNP (FIG. 28E). There was a slight reduction in the total fluorescence intensity of lysotracker (red) in the cells treated with MC-102-104 compared to MC-000-101 (FIG. 28F), but not statistically different, suggesting that the increased release of MC-102-104 from the endosomes could be largely due to increased membrane fusion between the -LNP and endosomes or increased endosomal membrane permeability, rather than endosomal rupture. Altogether, our data support the hypothesis that incorporating a t ciliary - amine-containing polymer into LNP enhanced the pH buffering effect during the endosomal and lysosomal acidification process, facilitating endosomal / lysosomal release of the payload and increasing the overall delivery of RNA. To demonstrate the benefit of this -LNP formulation for siRNA delivery in vivo, we loaded siRNA against FVII in standard MC3-LNP (MC-000-201) or -LNP containing 4.5 mol% of NS 102 (MC-102-204) and intravenously delivered to mice at arange of doses. One day later, FVII levels in plasma were measured to compare the effectiveness of these two formulations. As shown in FIG. 28G, MC- 102-204 displayed significantly enhanced gene silencing efficacy compared to MC-000-201, and the EC50 was decreased from 0.1 to 0.06 mg / kg.

[0207] Cellular uptake and intracellular localization of fluorescein-mRNA delivered by LNP and p-LNP formulations

[0208] To examine whether p-LNP formulations could also promote delivery of larger RNA such as mRNA, we encapsulated fluorescein-mRNA in LNPs and p-LNPs containing 2.4-8.3 mol% of NS 102 and compared their intracellular uptake and trafficking. Fluorescein-mRNA-loaded p- LNP formulations displayed comparable particle properties as the standard LNP formulation, MC-000-101 (FIG. 29). HEK 293 cells were incubated with these formulations at 1 pg mRNA / mL for 4 h, and the overall cellular uptake of fluorescein-mRNA was analyzed by flow cytometry. As shown in FIG. 30A, all -LNP formulations exhibited increased intracellular delivery of mRNA compared to the standard LNP (MC-000-101), while MC-102-104 containing 4.5 mol% of NS 102 displayed the highest delivery efficiency that was 1.5-fold increased relative to MC-000-101. The results showed a similar trend as that of the siRNA (FIG. 28A). Confocal microscopy was utilized to examine the intracellular localization and release of fluorescein- mRNA delivered via LNP and -LNP formulations. As shown in FIG. 30B, confocal images captured fluorescence from fluorescein-mRNA (green), Lysotracker (red), and nuclei (blue). Consistent with the findings in FIG. 30A, cells treated with -LNP formulations exhibited substantially higher mRNA uptake than those treated with the standard LNP, with the MC-102- 104 formulation achieving the highest mRNA delivery. Merged images revealed that MC-102- 104-treated cells showed significantly more fluorescein-mRNA separated from Lysotracker, indicating a greater degree of cytosolic release compared to other groups. Histogram analysis of the merged images in FIG. 30B further illustrated this trend, as MC-102-104 treatment displayed the lowest co-localization between mRNA (green) and Lysotracker (red). To quantify these observations, ImageJ analysis was performed, and results in FIG. 30C confirmed that MC-102- 104 achieved a 1.5-fold increase in intracellular mRNA delivery relative to the standard LNP. Additionally, MC-102-104 enhanced the release of fluorescein-mRNA into the cytosol (i.e., mRNA dissociated from the endosome / lysosome) by 3-fold compared to LNP, as shown FIG. 30D. FIG. 30E illustrated that the endosomal escape efficiency of fluorescein-mRNA delivered by MC-102-104 increased from 30% to 60% relative to standard LNP. Furthermore, the totalfluorescence intensity of lysotracker in cells after different treatments showed comparable results as those reported with the siRNA formulations, that the incorporation NS 102 moderately decreased endosomes in the cells but did not reach a statistically different level

[0028] .

[0209] In vitro mRNA transfection efficiency and serum and storage stability

[0210] To compare mRNA transfection efficiency of LNP and -LNP formulations, eGFP mRNA was loaded into these particles and their properties were characterized and transfection efficiency compared in HEK293 cells. As shown in FIG. 31, these LNP and -LNP formulations displayed comparable size, PDI, zeta potential and mRNA encapsulation efficiency. Then HEK 293 cells were incubated with the LNP and p-LNP formulations in the complete cell culture medium for 4 h, and eGFP expression was quantified by flow cytometry. Consistent with the fluorescein-mRNA uptake results, MC- 102- 104 displayed the highest transfection efficiency compared to others (FIG. 32A), showing- 1.5-fold increased mean eGFP fluorescence intensity compared to MC-000-101 (FIG. 32A). Furthermore, Bafilomycin Al was used to validate the mechanism how NS 102 incorporation increased mRNA delivery. Bafilomycin Al is a proton pump inhibitor that selectively inhibits vacuolar H+- adenosine triphosphatase (V-ATPase), blocking endosomal and lysosomal acidification. HEK 293 cells were treated with Bafilomycin Al for an hour, post which the cells were incubated with MC-000-101 and MC- 102- 104 for 4 h. Cells were then lysed and the eGFP expression was quantified by a fluorescence plate reader. Control groups without bafilomycin Al treatment were also monitored for eGFP expression. As shown in FIG. 32B, while Bafilomycin Al had no effect on the mRNA transfection efficiency of MC-000-101, it significantly decreased that of MC- 102- 104, supporting that NS102 enhanced mRNA transfection efficiency through the proton sponge effect. Overall, our systematic in vitro investigation of the NS102-incorporated / ?-LNP formulations consistently showed that NS 102 spiked at 4.5 mol% increased the pH buffering effect, leading to enhanced endosomal and lysosomal release of siRNA and mRNA and overall improved uptake and efficiency of gene silencing and transgene expression. We also investigated whether NS 102 incorporation improved mRNA stability in physiological fluids. eGFP mRNA-loaded LNPs and -LNPs were incubated with fetal bovine serum (FBS) and mouse serum for up to 24 hours. At designated time points, the mRNA was extracted, analyzed by gel electrophoresis, and quantified using ImageJ to determine the relative remaining mRNA content compared to that before incubation. The electrophoresis results revealed a gradual reduction in mRNA band intensity over time for both MC-000-101 and MC-102-104 in both FBS and mouse scrum (FIG. 33), while the mRNA inNS 102 incorporated p-LNPs (MC- 102- 104) decreased at a lower rate compared to that in LNPs (MC-000-101). Quantitative analysis showed that MC-102-104 retained approximately 70-80% of its mRNA after 4-hour incubation with serum, while MC-000-101 retained only 50-60% under the same conditions (FIG. 33). The data indicated that in addition to increasing the endosomal release, the -LNP formulation increased mRNA transfection also through enhancing stability of mRNA.

[0211] In vivo mRNA delivery by LNP and p-LNP formulations

[0212] Based on the in vitro results, incorporating 4.5 mol% NS 102 in LNP significantly enhanced the cellular uptake, endosomal release, mRNA stability and mRNA transfection, and therefore, this formulation was selected for in vivo studies and compared with standard LNP. To test whether NS 102 was compatible with other ionizable lipids, LNP and -LNP formulations prepared with ALC-0315 and SM-102 were also examined. The formulated p-LNPs containing luciferase mRNA, along with the standard four-component LNPs, were first assessed for their physicochemical properties (FIG. 34). Compared to the MC3 particles, formulations prepared with ALC-0315 and SM-102 displayed increases in particle size up to 20 nm, and the formulations prepared with ALC-0315 appeared more heterogenous with increased PDI (>0.2), while the SM-102 formulations showed decreased mRNA encapsulation efficiency (50-80%). Incorporating 4.5 mol% NS 102 did not significantly alter the particle properties, except that AL- 102-104 showed a 15-nm increase in the mean diameter compared to AL-000-101 and that SM- 102-104 displayed decreased mRNA encapsulation efficiency compared to SM-000-101 (56% vs 80%). LNPs prepared with SM-102 have been shown to exhibit decreased encapsulation efficiency by other groups [29,30]. Similar morphology of these LNPs and -LNPs was revealed by cryo-EM (FIG. 35). Overall, the particle characterization data indicated that NS 102 was highly compatible with various LNP formulations prepared with different ionizable lipids, although fine-tuning of the lipid / polymer composition might be required to obtain optimal results. To compare the in vivo mRNA delivery efficiency, these LNPs and p-LNPs were administered to 5-week-old CD-I female mice either intravenously (IV) or intramuscularly (IM) at a dose of 0.3 mg / kg mRNA. After 6-96 h, mice were injected with luciferin at 150 mg / kg and imaged for bioluminescence to quantify the gene expression at different time points (FIG. 36). As shown in FIG. 36A-C, these formulations showed a similar luciferase bioluminescence profile in mice: liver expression dominated after IV administration, while both liver and the injection site showed biolumincsccncc after IM delivery. The high liver tropism of the standardLNP formulations has been reported before

[0031] , and incorporating NS 102 did not alter this phenomenon. These images also indicated that the p-LNP formulations displayed increased gene transfection compared to their LNP counterparts. Among the formulations prepared with different ionizable lipids, NS 102 showed the most enhancing effect with the MC3 formulation, while the improvement with the formulation prepared with SM-103 was only mild to moderate, especially for IM delivery. This moderate improvement in mRNA delivery could be due to the lower mRNA encapsulation efficiency in SM-102-104 compared to SM-000-101. Nevertheless, the data indicated that incorporating NS 102 significantly increased the mRNA delivery efficiency for all formulations prepared with various ionizable lipids. We also quantified the bioluminescence images and reported the total flux (photons / s) in the liver (for IV) and at the injection site of the gastrocnemius muscle (for IM) at different time points. As shown in FIG. 36E-F, the bioluminescence peaked at 6-24 h post IV injection of LNP and p-LNP formulations, followed by a rapid decline over time from 1O8-1O10photons / s at 6 h to 106photons / s at 96 h. p- LNP formulations composed of different ionizable lipids showed increased transgene expression in the liver compared to their LNP counterparts, with the most enhancing effect in the MC3 formulation (up to 100-fold), followed by ALC-103 (up to 10-fold) and then SM-102 (up to 2- fold). Gene expression at the injection site following IM delivery peaked at 6 h ( 108- 109photons / s) and then declined rapidly to 106photons / s at 96 h (FIG. 36D-F). Instead of maintaining the gene expression at 24 h like the IV delivery, the bioluminescence at the IM injection site decreased by approximately 5-10-fold at 24 h, which could be attributed to the rapid hepatic absorption of the particles from the injection site as evidenced in FIG. 36A-C. This observation aligns with reports of COVID- 19 mRNA vaccines (Comirnaty and Spikevax) causing liver damage due to substantial liver accumulation in some patients

[0032] , Similar to the IV results, all p-LNP formulations displayed increased transgene expression at the IM injection site compared to their LNP counterparts, with the most improving effect in the MC3 and ALC- 0315 formulations (up to 10-fold), and only mild enhancement for the SM-102 formulation (up to 2-fold), which could be due to the decreased mRNA encapsulation efficiency for SM-102-104. Overall, the in vivo gene delivery study indicated that NS 102 was compatible with LNPs fabricated with a wide range of ionizable lipids, improving the transfection efficiency via both IV and IM delivery. Our data also suggest that while the -LNP formulation was optimized for the MC3 lipid, formulations composed of ALC-0315 or SM- 102 require further optimization toachieve improved particle homogeneity and mRNA encapsulation, which could lead to further enhanced gene delivery.

[0213] Finally, we compared safety of LNPs and p-LNPs spiked with 4.5 mol% NS 102 by IV injecting the formulations to mice at a dose of 0.3 mg / kg. One day later, serum was collected from the mice and analyzed for alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin to examine the liver toxicity. As these particles predominantly accumulated in the liver, we focused the safety assay on the liver. As shown in FIG. 37, there were no significant differences for these liver biomarkers among the different treatments, and the levels were all within the normal range, indicating no significant liver toxicity. Together with the cytotoxicity results in FIG. 24, our studies supported that the formulations incorporated with NS 102 were safe. Nevertheless, NS 102 is non-biodegradable, and its safety after long-term use needs to be studied. One factor that could contribute to its high safety is the low Mw of NS 102, which is -1,000 Da that can normally be excreted by human body

[0033] . Additionally, incorporation of NS 102 did not adversely affect the storage stability of the formulation, which exhibited comparable mRNA content (FIG. 38) and transfection efficiency (FIG. 39) after 4-week storage at -80°C as the freshly prepared p-LNP.

[0214] Conclusion

[0215] This study demonstrated that incorporating a polyamine into standard LNPs significantly enhanced the delivery efficiency for siRNA and mRNA. Both Mw and the amine composition of the polymer determined the safety and delivery efficiency, and the optimal polymer, NS 102, was low Mw (-1,000 Da) with only tertiary amines. The optimized polymer-incorporated LNP (p- LNP) formulation contained 4.5 mol% NS 102 and exhibited comparable physicochemical properties, increased pH buffering capacity, 3-fold improved endosomal release, 2.5-fold enhanced cytosolic delivery, and increased stability of RNA compared to its LNP counterpart. These enhancements led to improved RNA delivery in mice via IV and IM delivery. For siRNA delivery, the ED50 of -LNP was decreased by half compared to LNP, and the mRNA transfection efficiency in the liver and at the IM injection site of mice was increased, which was evidenced by up to 100-fold enhancement of luciferase bioluminescence intensity. The -LNP formulations showed minimal cytotoxicity nor any significant liver toxicity. These findings highlight the potential of -LNPs as a promising platform for enhancing delivery of nucleic acids.

[0216] In vitro characteristics of p-LNP formulations comprising dsDNA or pDNA as a payload

[0217] FIG. 40 illustrates the physicochemical properties of the LNPs and the -LNPs loaded with dsDNA (calf thymus) or plasmid DNA (pDNA). Data = mean ± SD (n=3).

[0218] Example 3 - histidine polymer

[0219] In Vivo Screening to Determine the Optimal Length of Polyhistidine for pLNP Formulation

[0220] The polyhistidine and formulation coding logics are indicated in FIG. 6,41. Linear polyhistidines of different lengths (acetyl-FL-amide to acetyl-H4o-amide, coded as NS304 to NS340) (FIG. 41B) were incorporated into standard MC3-based LNP at a range of mol% (2.38- 25 mol%) to prepare different peptide-LNPs (pLNPs). As shown in FIG. 42, pLNPs containing acetyl-FL-amide to acetyl-Hn-amide (NS304 to NS314) maintained an average size of ~85 nm with a poly dispersity index (PDI) <0.3 that were comparable to the standard LNP (MC-000-101). Incorporating longer polyhistidines (NS315 to NS340) resulted in larger particles (>110 nm), likely due to aggregation caused by increased interaction between longer polyhistidines with mRNA. Increasing the polyhistidine molar ratio tended to increase the particle size and decrease the mRNA encapsulation efficiency (EE%). For example, the particle size increased from 100 nm to 245 nm, and the EE% decreased from 83% to 68% when the mol% of NS313 increased from 2.38 mol% to 25 mol%. Notably, only pLNP formulations containing <8.33 mol% NS309 to NS313 displayed EE% greater than 70%, while pLNP formulations prepared outside these conditions exhibited EE% below 50%. Furthermore, pLNP formulations prepared with >8.33 mol% of larger M.W. polyhistidines (NS316 to NS340) displayed aggregations and were excluded from in vivo screening. Since in vitro data do not always correspond to in vivo results, we screened these pLNP formulations directly in mice (N=3). Luciferase mRNA was encapsulated into different LNP and pLNP formulations and injected intravenously (I.V.) into mice. Mice were imaged for luciferase bioluminescence 5 hours post injection. As shown in FIG. 41, only formulations incorporated with NS305 to NS311 exhibited enhanced gene transfection efficiency compared to MC-000-103, and the mRNA delivery was focused to the liver. Among these, pLNPs containing NS309 to NS311 appeared optimal, showing >50-fold increased luciferase bioluminescence in the liver compared to MC-000-101. In particular, the pLNP containing 2.38 mol% NS310 (MC-310-103) was among the most potent formulations, increasing the luciferase biolumincsccncc in the liver by ~90-fold compared to MC-000-101.Interestingly, the transfection efficiency decreased as the mol% of NS310 increased, likely due to the significantly decreased EE%. We randomly selected four LNP and pLNP formulations that displayed a wide range of transfection efficiency (FIG. 41D), and compared their pH buffering effect (FIG. 4 IE). There appeared to be close association between the transfection efficiency and pH buffering effect: formulations with increased pH buffering effect displayed enhanced mRNA delivery efficiency, suggesting that the pH buffering effect contributed from the peptide component was a key driver to the improved transfection. It is noted that both the N and C termini in the polyhistidines were blocked to ensure that the peptide would not carry a negative charge at pH 4 to affect the electrostatic interaction with mRNA nor become charged after pH neutralization to 7.4, which could negatively affect the encapsulation of mRNA and performance of the formulation. Our strategy selectively incorporated only tertiary amines into the material, distinguishing it from other approaches that either utilized primary / secondary amines to complex with mRNA — potentially leading to significant cytotoxicity — or leave free carboxyl groups, which may compromise the binding efficiency with mRNA. Indeed, pLNP prepared with unblocked Hio peptide (NS-310n) was inferior to that prepared with capped Hio peptide (NS- 310) in mRNA encapsulation efficiency (83.9% vs 21.7%, FIG. 43) and the transfection efficiency in mice (decreased by 5.5-fold, FIG. 44).

[0221] pLNPs incorporated with branched Hio displayed increased in vivo mRNA transfection compared to linear Hio

[0222] We hypothesized that conformation of polyhistidine (linear vs branched) would affect the transfection efficiency of the resulting pLNPs. We designed four branched Hio structures NS401-NS404 to test this hypothesis (FIG. 45). DLS analysis confirmed that all 16 pLNP formulations prepared with these branched Hio displayed comparable size (70-100 nm), PDI (<0.25) and zeta potential (close to neutral). However, only five of them displayed mRNA EE >60%, including MC-401-104, MC-402-103, MC-403-103, MC-403-104, and MC-403-105 (FIG. 46Formulations containing NS404 had poor mRNA EE% (10-55%). In vivo screening revealed that the transfection efficiency of the pLNP formulations was associated with the mRNA EE%. Three out of the four branched Hio enhanced mRNA transfection compared to linear Hio, with the NS402 exhibiting the highest potency (FIG. 45B, C). NS404 did not enhance the transfection efficiency, likely due to their low EE%. In particular, MC-402-103 showed comparable particle properties as MC-31O-1O3 in particle size (80 to 90 nm), PDI (<0.15), zeta potential (~0 mV) and mRNA EE (> 80%). Biolumincsccncc quantification in the liver at 5hours post-administration showed that MC-402-103 increased the luciferase bioluminescence by 2-fold and 266-fold compared to MC-310-103 and MC-000-101, respectively (FIG. 45B, C). The acid titration assay demonstrated that MC-402-103 had a superior pH-buffering effect compared to MC-310-103, explaining the mechanism of the improved gene transfection efficiency (FIG. 45D). Similar results were obtained with Hs, showing that pLNPs prepared with branched Hs showed up to 2.3-fold increased transfection efficiency compared to that spiked with linear Hs (FIG. 47). Characteristics of Hs-incorporated formulations were summarized in FIG. 48. Altogether, the data suggest that conformation of the polyhistidine affected the interaction with mRNA and the pH-buffering effect, leading to different mRNA EE% and in vivo transfection efficiency. Consequently, MC-402-103 was selected for further studies for the comparison with standard LNP, MC-000-101.

[0223] In vivo distribution, mRNA release and transfection of LNP and pLNP

[0224] To elucidate the mechanism of the superior gene transfection efficiency of MC-402-103 over MC-000-101, we first compared their in vivo distribution. As shown in FIG. 49A, both MC- 000-101 and MC-402-103 showed significant liver and spleen accumulation with minor uptake in other tissues. While there was no significant difference in the liver uptake, accumulation of MC-402-103 in the spleen and lungs was 2.3-fold and 1.6-fold higher, respectively, than that of MC-000-101 (FIG. 49B). However, only the liver displayed increased mRNA expression with MC-402-103 treatment compared to MC-000-101 (FIG. 41). We then further analyzed the intracellular uptake of DiR-labeled MC-000-101 and MC-402-103 loaded with FITC-mRNA within the liver. FIG. 49C, D revealed 500-fold higher intracellular uptake of MC-402-103 compared to MC-000-101, and MC-402-103 tended to form large aggregates within the cells. Intracellular FITC-mRNA distribution analysis showed that, in the MC-402-103-treated liver, mRNA was evenly distributed within the cells, whereas barely any mRNA signal was detected in the MC-000-101 -treated liver. Quantitative analysis of intracellular FITC-mRNA (FIG. 49E) revealed an 8-fold increase in the MC-402-103-treated liver compared to MC-000-101. Taken together, the data in FIG. 49 suggested that, although effectively accumulating in the liver, MC- 000-101 was largely located extracellularly, which could be washed out during the tissue section preparation. The results were consistent with the low gene transfection efficiency of MC-000- 101. While the liver accumulation was comparable between MC-000-101 and MC-402-103, MC- 402-103 was more effectively internalized by or retained within the liver cells, leading to increased cytosolic release of mRNA and gene expression.

[0225] MC-402-103 Exhibited Reduced Endosomal Association and Enhanced Endosomal Escape Compared to MC-000-101 In Vivo

[0226] We hypothesized that the imidazole groups with a pKa -6 in NS402 would mediate absorption of protons in the acidic endosomes after endocytosis of MC-402-103, leading to increased pH-buffering effect (supported by FIG. 41E and FIG. 45D), which would in turn result in endosomal swelling and rupture via the proton sponge effect. This process facilitated the osmotic influx of water, causing membrane destabilization and subsequent endosomal escape. As a result, MC-402-103 would show decreased association with endosomal markers and improved cytosolic release compared to MC-000-101. To test this hypothesis, we stained four specific endosomal biomarkers and examined their association with Dil-labeled MC-000-101 or MC- 402-103 after I.V. delivery to mice. Livers were collected 5 h post injection, followed by cryosection, Alexa 647-antibody staining, and confocal microscopy. Four endosomal markers were stained, including APPL1 that marks very early endosomes, the first compartment post- endocytosis; EEA1 that labels early endosomes, responsible for sorting internalized materials in a slightly acidic environment; Rabl l that indicates recycling endosomes, which return components to the plasma membrane; and LBPA that identifies late endosomes or multivesicular bodies (MVBs), which are highly acidic and prone to degradation. FIG. 50 show that association percentages between the intracellular particles with different endosomal markers including APPL1, EEA1, Rabl l and LBPA were decreased from 13% to 7%, 26% to 11.9%, 32.3% to 9.5%, and 32% to 14%, respectively, for MC-402-103 compared to MC-000-101. These data indicate that MC-402-103 was less likely to be recycled or degraded after internalization, leading to increased intracellular delivery, release, and gene expression as shown in FIG. 49.

[0227] Intracellular dynamics of particles and endosomes over 45 minutes

[0228] Early stage of cellular uptake

[0229] At 25 minutes, MC-000-101 particles were found being adhered to the cell membrane (FIG. 51A). By 30 minutes, MC-000-101 signals diminished while lysotracker signals increased, indicating endocytosis and digestion / removal of MC-000-101. The increased Dil intensity in the extracellular space indicated by white stars in FIG. 51A suggested recycling of MC-000-101. In contrast, at 25 minutes, most MC-402-103 particles remained outside the cells (yellow arrows, FIG. 5 IB). At 30 minutes, clusters of MC-402-103 particles were found intracellularly without association with endosomes (yellow stars), indicating endocytosis and endosomal release. The data arc consistent with FIG. 49.

[0230] Interactions between particles and endosomes within cells

[0231] We then tracked intracellular endosomes (LysoTracker Green) and Dil-labeled particles separately at different time intervals (30-35 min, 35-40 min, and 40-45 min) (FIG. 52A and B). The videos were analyzed by MATLAB (left panel) and the intracellular trafficking of particles and endosomes are reported separately. The edges of cell clusters are labeled with yellow dashed lines. MATLAB analysis showed that in the MC-000-101 group, both the endosomes and particles showed decreasing mobility over time (from 30 to 45 min). In contrast, mobility of the endosomes and MC-402-103 peaked at 35-40 min but was low at earlier (30-35 min) and later (40-45 min) time intervals. For MC-000-101, endosome trafficking was prominent at 30-35 minutes, indicating active endocytosis (FIG. 52A). After internalization, mobility of the endosomes decreased (40-45 min). Notably, the intracellular particle trajectories decreased, while the extracellular trajectories increased, indicating the internalized MC-000-101 was recycled back to the medium over time. In contrast, cells treated with MC-402-103 exhibited moderate particle and endosome mobility between 30 and 35 minutes, both of which increased at 35-40 minutes (FIG. 52B). This suggests that MC-402-103 started triggering endocytosis at 35 minutes. This was confirmed by the increase of moving velocity of endosomes at 35 min (FIG. 51C). Similarly, mobility of the endosomes and MC-402-103 decreased after cellular internalization (40-45 min). However, very few MC-402-103 particles were detected outside the cells, indicating low recycling activity. We hypothesized that MC-402-103 exhibiting increased pH buffering effect would induce the proton sponge effect, disrupting the endosomes for payload release. Indeed, FIG. 52C shows that the total endosome area within the cells decreased over time after incubation with MC-402-103. In contrast, cells treated with MC-000-101 displayed comparable total endosome area, indicating little endosomal rupture.

[0232] MC-402-103 enabled efficient delivery of adenosine deaminase base editor 9ABE) for in vivo genome editing

[0233] We then compared the efficacy of MC-402-103 and MC-000-103 in delivering RNAs for genome editing in a transgenic mice model (LumA). This reporter mouse model carries the R387X mutation (c.Al 159T) in the luciferase gene within the Rosa26 locus, obtained from Dr. Colin Ross’s lab (UBC). This mutation abolishes luciferase activity, which can be restored through A-to-G correction by a SpCas9 adenine base editor (ABE), as illustrated in FIG. 53. We formulated ABE mRNA and sgRNA into MC-000- 101 and MC-402-103 and I.V. administered them to the LumA mice, followed by monitoring of genome editing over 14 days. As shown inFIG. 54, MC-402-103 had a mean size of 74 nm with a PDI < 0.1, while MC-000-101 were 84 nm with a PDI of 0.2. Both formulations carried a slightly negative charge ( — 2 mV) and exhibited comparable RNA EE% > 90%. As shown in FIG. 53B, the gene editing in both groups became detectable by day 3 for the liver luciferase bioluminescence, which increased in intensity over time and plateaued at day 12. Notably, the MC-402-103 formulation produced an 8-fold stronger bioluminescence signal than MC-000-101, with quantification presented in FIG. 53C. On day 14, mice were euthanized, and major organs were collected and sequenced, revealing that MC-402-103 achieved an 8% T-to-C conversion, whereas MC-000-101 resulted in only -1% (FIG. 53D). Remarkably, this high editing efficiency was achieved with a lower dose (0.14 mg / kg) than typically used in similar studies (0.25 mg / kg). These results demonstrate the high delivery efficiency by MC-402-103.

[0234] Safety evaluation of MC-402-103

[0235] In vitro cytotoxicity studies in HEK 293 cells showed no significant changes in viability, even at a high lipid concentration of 500 pM (FIG. 55A). In vivo immunotoxicity was examined by analyzing the serum collected from mice 3 hours post I.V. injection of MC-402-103 at 0.3 mg / kg. As shown in FIG. 53B and C, TNF-a and IL-6 levels remained undetectable, indicating no immunotoxicity. Given the liver's predominant role in particle accumulation, hepatic toxicity was assessed one day post-injection through serum biomarker analysis. As summarized in FIG. 56, all biomarker levels remained within normal physiological ranges, with no significant differences among the treatment groups, suggesting no overt liver toxicity.

[0236] Machine Learning-Guided Optimization of the conformation of Polyhistidine (Hio) to prepare enhanced pLNPs

[0237] FIG. 45 indicates that conformation of Hio critically affected the transfection efficiency of the resulting pLNPs. However, there are numerous design possibilities for branched Hio. Machine learning (ML) provides a powerful tool for integrating experimental data and predicting promising candidates, thereby reducing the need for extensive screening. To develop an ML- guided optimization strategy, we first compiled experimental data from EIG. 41 and 45, including polyhistidine structures, mol% of peptide in pLNP formulations, and quantitative bioluminescence results at different time points. We then constructed a graph representation where amino acids were treated as nodes rather than atoms, simplifying feature selection to make the data easy to understand by the machine. A Graph Neural Network (GNN) was trained to predict biolumincsccncc data from polyhistidinc structures, followed by global mean pooling andconcatenation with time point and peptide mol%. Finally, a multilayer perceptron (MLP) was used for prediction (FIG. 57 A). Using this model, we designed polyhistidine sequences (Table 2). Model predictions (FIG. 57B) identified a star-like branched configuration as optimal. Notably, the ML model ranked NS537 as 1st, NS506 as 2nd, NS533 as 13th, and NS534 as 31st. Their molecular structures are shown in FIG. 57C. As shown in FIG. 58, pLNPs incorporating NS506 and NS537 had an average size slightly above 100 nm, with a PDI <0.3. LNPs incorporating NS533 and NS534 had a size of approximately 90 nm, with a PDI <0.15 for NS533 and <0.3 for NS534. The encapsulation efficiency (EE%) for all formulations was >70%. In vivo studies showed that NS506 exhibited superior gene transfection potency compared to NS402, resulting in a 2-fold increase in luciferase bioluminescence in the liver. NS533 and NS537 led to 1.9-fold and 1.1-fold increases compared to NS402, respectively, while NS534 was inferior to NS402 (FIG. 57D). To assess the accuracy of this model, the predicted value of NS506 (16.4-17.8) was compared to its experimentally determined value (15.3-18.3), demonstrating that the predicted results align well with the experimental data. We then incorporated the new bioluminescence data from NS506, NS533, NS534, and NS537 to further define the model. Given the strong performance of the star-like branched Hio (NS506 and NS533), we focused on designing the similar configuration (NS535, NS538 to NS549). Their structures are displayed in FIG. 59A. For further validation, we synthesized and tested NS535 in vivo. As shown in FIG. 60, NS535 containing pLNPs displayed comparable size -100 nm with PDI <0.25, but varied EE% (38-93%). All the NS535 incorporated pLNP formulations outperformed those prepared with NS402, the first generation of branched Hio. In particular, mice treated with MC-535-108 displayed 2.2-fold increased bioluminescence in the liver compared to MC-506-102 and 2.9-fold increased compared to MC-NS402-103. These results confirm the ML model’s effectiveness in optimizing polyhistidine conformation. With these optimizations, the luciferase bioluminescence from the mRNA expression in the liver was increased by 705-fold when comparing MC-535-108 and MC-000-101.

[0238] NS308 increases mRNA delivery in vivo

[0239] Control LNP (LNP without the delivery-enhancing polymer of the present invention) or LNP comprising NS308 was intravitreally injected to the mice, and the eGFP mRNA payload was visualized using microscope. FIG. 61 shows that NS308-containing LNP increases mRNA delivery to the ganglion cell layer compared to the control LNP.

[0240] It can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

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Claims

AMENDED CLAIMS received by the International Bureau on 17 September 2025 (17.09.2025)

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28. [Added] A nanoparticle comprising a. nucleic acid molecules; b. cholesterol; c. phospholipid; d. ionizable lipid; and e. delivery-enhancing polymer comprising ionizable functional groups wherein the ionizable functional groups comprise ionizable amines wherein the ionizable amines comprise tertiary amines, heteroaryl nitrogen or other weakly basic moieties that are neutral at physiological pH but protonated at mildly acidic pH (4.5-6.5).

29. [Added] The nanoparticle of claim 28, wherein each of the ionizable functional groups of the delivery-enhancing polymer consists of a tertiary amine and does not comprise any primary or secondary amine.

30. [Added] The nanoparticle of claim 28, wherein the delivery enhancing polymer comprises a histidine polymer wherein the histidine polymer comprises 4 to 15 histidine residues.

31. [Added] The nanoparticle of claim 30, wherein the histidine polymer further comprises one or more lysine residues and does not comprise any primary amines.

32. [Added] The nanoparticle of claim 28, wherein the ionizable functional group of the delivery-enhancing polymer is selected from the group comprising imidazole, substituted imidazole, alkyl tertiary amine, cyclic tertiary amine, heteroaromatic amine, other heterocyclic amines,piperidine, morpholine, piperazine, azepane, diazabicyclo[2.2.2]octane (DABCO), benzimidazole, pyrazole, and triazole.

33. [Added] The nanoparticle of claim 28, wherein the delivery enhancing polymer comprises a linker connecting the ionizable functional groups, wherein the linker is selected from the group comprising alkyl, alkenyl, alkynyl, ether, thioether, ester, amide, carbamate, urea, carbonate, sulfonamide, disulfide, peptide, polyethylene glycol (PEG), and other biocompatible linkers.

34. [Added] The nanoparticle of claim 28, wherein the delivery enhancing polymer comprises a configuration selected from the group comprising linear, branched, star-shaped, dendrimeric, and other multi-arm architectures.

35. [Added] The nanoparticle of claim 28, wherein the delivery-enhancing polymer comprises methylated polyethylenimine (mPEI) and wherein each ionizable functional group of the rnPEI consists of tertiary amines and does not comprise any primary or secondary amines.

36. [Added] The nanoparticle of claim 28, wherein the delivery-enhancing polymer does not comprise primary amine.

37. [Added] The nanoparticle of claim 28, wherein the molecular weight of the delivery-enhancing polymer is from about 500 g / mol to about 5000 g / mol

38. [Added] The nanoparticle of claim 28, wherein the molecular weight of the delivery-enhancing polymer is from about 600 g / mol to about 3500 g / mol.

39. [Added] The nanoparticle of claim 30, wherein the N-terminus of the histidine polymer is chemically modified to block the primary amine and thereby prevent positive charge at neutral pH.

40. [Added] The nanoparticle of claim 30, wherein the N-termini of the histidine polymer is acetylated.

41. [Added] The nanoparticle of claim 30, wherein the C-terminus of the histidine polymer is chemically modified to block the terminal carboxyl group and thereby prevent negative charge at neutral pH.

42. [Added] The nanoparticle of claim 30, wherein the C-terminus of the histidine polymer is amidated.

43. [Added] The nanoparticle of claim 28, wherein the delivery-enhancing

44. [Added] The nanoparticle of claim 28, wherein the delivery-enhancing polymer is selected from: aHH[H]xHn (SEQ ID NO. 1), aHHHHK(HHa)HHHHn (SEQ ID NO. 2), aHHHHK(HHa)K(Ha)HK(Ha)Hn (SEQ ID NO. 3), aHK(Ha)HK(Ha)K(HHa)K(Ha)HK(Ha)Hn (SEQ ID NO. 4), aHHK(HHa)HK(HHa)HHHn (SEQ ID NO. 5), aHHK(HHa)HK(HHHa)HHn (SEQ ID NO. 6),aHHK(HHa)HK(Ha)HHK(Ha)Hn (SEQ ID NO. 7), aHHK(HHa)HHHHHHn (SEQ ID NO. 8), aHHK(HHHHHHa)HHn (SEQ ID NO. 9; SEQ ID NO. 61), aHK(Ha)HK(Ha)HKHK(Ha)HK(Ha)Hn (SEQ ID NO. 10), aHHHHK(Ha)HK(Ha)Hn (SEQ ID NO. 11), aHHK(HHa)K(Ha)HK(Ha)HK(Ha)Hn (SEQ ID NO. 12), aHHHK(HHa)HK(Ha)HHHn (SEQ ID NO. 13), aHHHK(HHa)HK(HHHa)Hn (SEQ ID NO. 14), aHHHHK(Ha)HK(HHa)HHn (SEQ ID NO. 15), aHHK(HHa)K(Ha)HK(HHa)HHn (SEQ ID NO. 16), aHHHK(HHa)HK(HHa)HHn (SEQ ID NO. 17), aHHK(HHa)HHK(HHa)HHn (SEQ ID NO. 18), aHHHK(Ha)HHK(HHa)HHn (SEQ ID NO. 19), aHHK(HHa)HHHK(Ha)HHn (SEQ ID NO. 20), aHHK(HHa)HHHK(HHa)Hn (SEQ ID NO. 21), aHHK(HHa)K(HK(Ha)Ha)HK(Ha)Hn (SEQ ID NO. 22), aHK(Ha)K(HK(Ha)Ha)HK(Ha)HK(Ha)Hn (SEQ ID NO. 23), aHK(Ha)HK(HK(Ha)Ha)HKHK(Ha)Hn (SEQ ID NO. 24), aHK(Ha)K(K(Ha)Ha)HK(Ha)HHK(Ha)Hn (SEQ ID NO. 25), aHK(Ha)K(K(Ha)Ha)K(HK(Ha)Ha)HK(Ha)Hn (SEQ ID NO. 26), aHK(Ha)K(K(Ha)Ha)K(Ha)HK(Ha)HK(Ha)Hn (SEQ ID NO. 27), aHK(HHa)K(K(Ha)Ha)HK(Ha)HHHn (SEQ ID NO. 28), aHK(K(Ha)Ha)K(K(Ha)Ha)HK(Ha)HHHn (SEQ ID NO. 28), aHK(K(Ha)Ha)K(K(Ha)Ha)HK(Ha)K(Ha)HHn (SEQ ID NO. 29), aHK(K(Ha)Ha)K(K(Ha)Ha)HK(Ha)K(HHa)Hn (SEQ ID NO. 30), aHK(Ha)K(K(Ha)Ha)HHK(HHa)HHn (SEQ ID NO. 31),aHK(Ha)K(K(Ha)Ha)HHK(Ha)HHHn (SEQ ID NO. 32), aHK(Ha)K(K(Ha)Ha)HHK(HHHa)Hn (SEQ ID NO. 33), aHK(Ha)K(K(Ha)Ha)HHHK(Ha)K(Ha)Hn (SEQ ID NO. 34), aHK(Ha)K(K(Ha)Ha)HHK(K(Ha)Ha)K(Ha)Hn (SEQ ID NO. 35), aHK(Ha)K(K(Ha)Ha)HHK(Ha)K(Ha)HHn (SEQ ID NO. 36), aHK(Ha)K(K(Ha)Ha)HHK(Ha)K(HHa)Hn (SEQ ID NO. 35), aHK(Ha)K(K(Ha)Ha)HHHK(Ha)HHn (SEQ ID NO. 37), aHK(Ha)K(K(Ha)Ha)HHHK(HHa)Hn (SEQ ID NO. 38), aHK(Ha)K(Ha)HHHHK(Ha)K(Ha)Hn (SEQ ID NO. 39), aHKHK(HKHa)K(HK(Ha)Ha)HK(Ha)Hn (SEQ ID NO. 40), aHK(HK(Ha)HHa)HK(HHa)HHn (SEQ ID NO, 41; SEQ ID NO. 62), aHK(Ha)HK(HK(Ha)Ha)HK(Ha)HHn (SEQ ID NO. 42), aHKHKHKHKHKHKHKHKHKHn (SEQ ID NO. 43), aHHK(Ha)K(HK(Ha)Ha)K(HHa)HHn (SEQ ID NO. 44), aHHK(Ha)K(K(Ha)HHa)K(HHa)HHn (SEQ ID NO. 44), aHK(Ha)K(K(HHa)HHa)K(HHa)HHn (SEQ ID NO. 45), aHK(K(Ha)Ha)K(HHHa)HHHHn (SEQ ID NO. 46), aHK(K(HHa)HHa)K(HHa)HHHn (SEQ ID NO. 47), aHK(K(Ha)Ha)K(Ha)HHHHHHn (SEQ ID NO. 48), aHK(K(Ha)Ha)K(HHa)HHHHHn (SEQ ID NO. 49), aHHHHHHK(K(Ha)Ha)K(Ha)Hn (SEQ ID NO. 50), aHK(Ha)K(K(Ha)Ha)K(Ha)HHHHHn (SEQ ID NO. 51), aHK(Ha)K(K(Ha)Ha)K(HHa)HHHHn (SEQ ID NO. 52), aHK(Ha)K(K(Ha)Ha)K(HHHa)HHHn (SEQ ID NO. 53), aHK(Ha)K(K(Ha)Ha)K(KK(Ha)Ha)K(K(Ha)Ha)K(Ha)Hn (SEQ ID NO. 54), aHHHHK(Ha)K(Ha)HHHHn (SEQ ID NO. 55),aHHHK(HHa)K(HHa)HHHn (SEQ ID NO. 56), aHK(HHa)HHHHK(HHa)Hn (SEQ ID NO. 57), aHHHK(HHHa)HHHHn (SEQ ID NO. 58), aHK(HHa)HK(HHa)HHn (SEQ ID NO. 41), aHHK(HHa)K(HHa)HHn (SEQ ID NO. 59), or aHHHHK(HHa)HHn (SEQ ID NO. 60), wherein in the histidine polymers H is histidine, K is lysine, x is 1-36, a is — COCH3, n is — NH2. and the parenthesis indicates a branch sequence linked to a lysine side chain.

45. [Added] The nanoparticle of claim 28, wherein the delivery-enhancing

46. [Added] The nanoparticle of claim 28, wherein the delivery-enhancing polymer is selected from: aHH[H]xHn (SEQ ID NO. 1), aHHHK(HHa)K(HHa)HHHn (SEQ ID NO. 56), aHHK(HHa)K(HK(Ha)Ha)HK(Ha)Hn (SEQ ID NO. 22), aHKHK(HKHa)K(HK(Ha)Ha)HK(Ha)Hn (SEQ ID NO. 40), aHK(Ha)HK(HK(Ha)Ha)HK(Ha)HHn (SEQ ID NO. 42), aHKHKHKHKHKHKHKHKHKHn (SEQ ID NO. 43),wherein in the histidine polymers H is histidine, K is lysine, x is 6-8, a is — COCH3, n is — NH2, and the parenthesis indicates a branch sequence linked to a lysine side chain.

47. [Added] The nanoparticle of claim 28, wherein the nanoparticle comprises the formulation of: a. about 47.62 mol% MC3, about 10 mol% DSPC, about 38.5 mol% cholesterol, about 1.5 mol% DMG-PEG, about 2.38 mol% NS402 polymer and the nucleic acid molecules comprising mRNA (MC-402- 103); b. about 33.30 mol% MC3, about 10 mol% DSPC, about 38.5 mol% cholesterol, about 1.5 mol% DMG-PEG, about 16.70 mol% NS535 polymer and the nucleic acid molecules comprising mRNA (MC-535- 108); c. about 45.50 mol% MC3, about 10 mol% DSPC, about 38.5 mol% cholesterol, about 1.5 mol% DMG-PEG, about 4.50 mol% NS102 polymer and the nucleic acid molecules comprising siRNA (MC-102- 204); d. about 38.46 mol% MC3, about 10 mol% DSPC, about 38.5 mol% cholesterol, about 1.5 mol% DMG-PEG, about 11.54 mol% NS201 polymer and the nucleic acid molecules comprising mRNA (MC-201- 106); e. about 41.67 mol% MC3, about 10 mol% DSPC, about 38.5 mol% cholesterol, about 1.5 mol% DMG-PEG, about 8.33 mol% NS202 polymer and the nucleic acid molecules comprising mRNA (MC-202- 105); orf. about 38.46 mol% MC3, about 10 mol% DSPC, about 38.5 mol% cholesterol, about 1.5 mol% DMG-PEG, about 11.54 mol% NS202 polymer and the nucleic acid molecules comprising mRNA (MC-202- 106).

48. [Added] The nanoparticle of claim 28, wherein the mol% of the deliveryenhancing polymer in the nanoparticle is about 2% to about 20%.

49. [Added] The nanoparticle of claim 28, wherein the ionizable lipid is selected from the group consisting of DLin-MC3-DMA, SM-102, ALC- 0315, C12-200, OF-02, OF-D13, LP-01, L319, and other amino lipid derivatives comprising tertiary amine headgroups with biodegradable ester, amide, or disulfide linkages.

50. [Added] The nanoparticle of claim 28, wherein the mol% of the ionizable lipid in the nanoparticle is about 20% to about 50%.

51. [Added] The nanoparticle of claim 28, wherein molar ratio of the deliveryenhancing polymer over the ionizable lipid is from about 0.02 to about 1.00.

52. [Added] The nanoparticle of claim 28, wherein the molar ratio of the positively chargeable amine from the ionizable lipid over the nucleic acid molecules (N / P ratio) is from about 2 to about 8.

53. [Added] The nanoparticle of claim 28, further comprising a PEGylated lipid.

54. [Added] The nanoparticle of claim 53, wherein the molar % of ionizable lipid is about 25-50 mol%, phospholipid is about 0-20 mol%, cholesterol is about 25-38.5 mol%, PEG-lipid is about 0.5-5 mol%, and delivery-enhancing polymer is about 1 mol% to about 25 mol%.

55. [Added] A method of treatment of disease suffered by a subject comprising the step of administration of a therapeutically effective amount of the nanoparticle of claim 1 to the subject, wherein the disease comprises any disease that may be treated by expression of the nucleic acid molecules of the nanoparticle.80

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