A room-temperature stable lyophilized lipid nanoparticle composition based on universal base lipids and its application

Abstract

This invention relates to a room-temperature stable lyophilized lipid nanoparticle composition based on universal base lipids and its applications. Specifically, this invention discloses a universal base-modified ionizable lipid comprising (i) an ionizable lipid backbone and (ii) universal base groups covalently linked to the ionizable lipid backbone. This invention also discloses the use of universal bases in modifying ionizable lipids to endow them with the ability to assemble nucleic acids in a neutral aqueous phase, and lipid nanoparticles comprising the universal base-modified ionizable lipid.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to universally modified ionizable lipids and their uses. Background Technology

[0002] The success of mRNA-lipid nanoparticle (mRNA-LNP) vaccines has revolutionized the landscape of modern medicine. 1,2 However, the inherent instability of these formulations limits their shelf life and necessitates cryogenic storage³. For example, two FDA-approved mRNA vaccines remain stable for only a few days at 2–8°C (Spikevax for 30 days and Comirnay for 5 days) and require long-term storage at -20°C or -70°C. This creates significant cold chain barriers and limits equitable global accessibility, particularly in resource-constrained regions. 4 The thermal instability of mRNA-LNP is driven by several degradation pathways in the aquatic environment. 5 Although mRNA molecules themselves are susceptible to hydrolysis, recent studies have identified ionizable lipid-induced chemical degradation as a major contributor to potency loss. 6,7,8 Specifically, the tertiary amines and unsaturated tails of ionizable lipids are readily oxidized, producing reactive electrophilic species such as aldehydes. These degradation products react with mRNA nucleobases to form lipid-mRNA adducts, which can reduce mRNA translation efficiency even if the nanoparticles remain structurally intact. In addition to chemical fragility, the dynamic colloidal nature of LNPs makes them prone to physical instability, often leading to particle aggregation or phase separation during long-term storage. 9 .

[0003] Lyophilization (freeze-drying) represents a common strategy for enhancing the thermal stability of pharmaceutical formulations by removing water to prevent hydrolysis, slow down oxidation, and reduce molecular migration. 10,11 However, freeze-drying pre-assembled mRNA-LNPs is complex and destructive. During freeze-drying, ice crystal growth and changes in salt concentration impose mechanical stress on the LNPs¹². Furthermore, the pH of biological buffers such as phosphate-buffered saline (PBS) changes with decreasing temperature, altering the surface charge of the nanoparticles¹³. These stresses collectively force the LNPs to reassemble, leading to structural damage and subsequent leakage of the mRNA load. Moreover, even in the dried state, the close proximity of ionizable lipids to the mRNA in the co-lyophilized cake does not eliminate the risk of long-term chemical degradation due to lipid-derived impurities. 6,7 While incorporating glycosyl lyophilization protectants into LNP formulations and optimizing buffer components can mitigate these risks during lyophilization to some extent, achieving long-term room-temperature storage without affecting in vivo efficacy remains a significant challenge. 14,15 .

[0004] To circumvent the limitations of co-lyophilization, physically separating mRNA from lipid carriers offers a potential alternative¹ 6 In this strategy, mRNA loading can be achieved via a post-encapsulation method, where lyophilized empty LNPs and mRNA are stored separately to minimize the lipid-mRNA interaction window, and then assembled immediately upon rehydration before use. However, current post-encapsulation strategies are limited by their high dependence on electrostatic interactions. Forcing polyanionic mRNA into pre-formed intact LNPs requires overcoming significant thermodynamic barriers, necessitating stringent preparation conditions or specific formulation equipment. Typically, these methods require acidic buffers to protonate ionizable lipids and external heating to increase membrane fluidity. 16,17 Attempts to circumvent these conditions by using permanently charged cationic lipids (e.g., 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) inevitably introduce severe cytotoxicity and undesirable inflammatory responses. 18,19,20 Overall, these limitations complicate on-site LNP preparation and eliminate the immediate-use practicality of current mRNA-LNP therapeutics.

[0005] Here, the inventors report a platform called Assemblable lyophilized lipid nanoparticles for long-term storage and express therapy (AllSet), which enables the on-site formulation of functional mRNA-LNPs from individually lyophilized components using pure water. Figure 1 Post-assembly is completed within seconds in a neutral aqueous environment at ambient temperature, bypassing the need for acidic buffer solutions, external heating, and specialized equipment. Figure 2This rapid and simplified assembly is driven by a novel class of universally base-modified peptide ionizable lipids (UB-PILs), which mediate multiple non-electrostatic interactions, including π-π stacking between the universal base and the mRNA nucleobase, and hydrogen bonding between the UB-PIL amide backbone and the mRNA phosphate group. Furthermore, the non-electrostatic assembly mechanism endows the AllSet platform with broad versatility, allowing for the encapsulation of a variety of charged and uncharged nucleic acids and integration into various established LNP systems. After six rounds of continuous chemical evolution, the inventors identified a 5-nitroindole (Nii)-modified Nii-C4-LD-a10a12K2 UB-PIL that exhibited significantly higher mRNA delivery efficiency (approximately 4-fold) compared to the FDA-approved ALC-0315 formulation, and achieved highly efficient mRNA expression in mouse liver at nanogram-level mRNA doses (250 ng). Notably, the corresponding AllSet LNP maintained significant physicochemical integrity and in vivo efficacy for at least 8 months at both 4°C and room temperature (25°C). In contrast, the conventional co-lyophilized ALC-0315 formulation exhibited particle aggregation and mRNA degradation issues, losing its biological activity within one week. The excellent thermostability of AllSet LNP directly translates into broad applicability; after 6 months of storage at room temperature, AllSet LNP successfully maintained strong responses in mice across a wide range of clinically relevant applications, including prophylactic mRNA vaccines (respiratory syncytial virus, RSV; influenza A virus H1N1 subtype; and rabies virus glycoprotein, RVG), CRISPR-based PCSK9 gene editing (Cas9-mediated knockout and adenine base editing), and siRNA-mediated TTR gene silencing. Integrating a universal base and separation lyophilization strategy into the AllSet platform offers significant potential for extending next-generation genetic therapeutics to resource-constrained communities. Summary of the Invention

[0006] In a first aspect, the present invention provides a universally base-modified ionizable lipid comprising...

[0007] (i) Ionizable lipid backbone, and

[0008] (ii) A universal base group covalently linked to the ionizable lipid backbone, The universal base group is directly attached to the ionizable lipid backbone, or attached to the ionizable lipid backbone via a spacer group.

[0009] In some embodiments, the universal base group is derived from one or more universal bases selected from the following or derivatives thereof: hypoxanthines, purines, isoquinolinones, pyrimidines, etc. Azides, nitropyrroles, nitroindoles, nitroimidazolides, formylpyrroles, nitrobenzodioxanepentenes, benzotriazoles, cyanuric acids, diaminotriazines, acylaminopyridines, pyrimidines, pteridine analogs, urea-based pyrimidinones, and general bases or their derivatives.

[0010] In some embodiments, the universal base group is derived from a universal base or a derivative thereof that has a reactive functional group. In some embodiments, the reactive functional group is capable of reacting with reactive sites in ionizable lipids to form covalent bonds.

[0011] In some embodiments, the reactive functional group is selected from one or more of the following: carboxyl, amino, hydroxyl, mercapto, activated ester, acyl chloride, acid anhydride, chloroformate, activated carbonate, isocyanate, isothiocyanate, sulfonyl chloride, haloalkyl, methanesulfonate, p-toluenesulfonate, haloacetyl, epoxy, vinyl sulfone, acryloyl, methacryloyl, maleimide, aldehyde, ketone, aminooxy, hydrazyl, acylhydrazine, azide, alkynyl, cycloalkynyl, tetrazinyl, transcyclooctenyl, norbornyl, cyclopropenyl, and combinations thereof.

[0012] In some embodiments, the universal base group is derived from a universal base derivative having a carboxyl group, preferably a carboxylated universal base.

[0013] In some embodiments, the carboxylation universal base is selected from: 1-carboxymethyl-3-nitropyrrole (Nip), 1-carboxymethyl-5-nitroindole (Nii), (2,4,6-trioxo-1,3,5-triazin-1-yl)acetic acid (Cya), 2,6-bis(acetamido)-4-pyridinecarboxylic acid (Dapy), 2-(4-nitroimidazol-1-yl)acetic acid (Nipu), 2-(2,4,5,7-tetraoxo-8H-pyrimidino[4,5-d Pyrimidin-1-yl)acetic acid (Ppt), N-[[(1,4-dihydro-6-methyl-4-oxo-2-pyrimidinyl)amino]carbonyl]-glycine (Upy), inosine-9-acetic acid (Ino), 2-(9H-purin-9-yl)acetic acid (Pur), 2-(8-methyl-1-oxo-1,2-isoquinoline-2-yl)acetic acid (MICS), 1,3,4,7-tetrahydro-7-oxo-6H-pyrimidino[4,5-c][1,2] P-imino, (2-formyl-1-pyrrolidinyl)acetic acid (Fp), (6-nitro-1,3-benzodioxane-5-yl)acetic acid (Nipi), benzotriazol-1-acetic acid (Tap), 2-(4,6-diamino-1,3,5-triazin-2-yl)acetic acid (Dat), or any combination thereof.

[0014] In some embodiments, the number of universal base groups covalently linked to the ionizable lipid backbone is 1 to 10, for example, 1, 2, 3, 4 or 5.

[0015] In some embodiments, the attachment position of the universal base group to the ionizable lipid backbone is not particularly limited. In some embodiments, the universal base group can be attached to any covalently bondable site within the ionizable lipid backbone. In some embodiments, the universal base group can be attached to the head region, central backbone region, side chain region, and / or hydrophobic tail region of the ionizable lipid backbone. In some embodiments, the universal base group can be attached to the terminal, internal, or side chain position of the ionizable lipid backbone.

[0016] In some embodiments, the ionizable lipid backbone is derived from ionizable lipids having reactive sites.

[0017] In some embodiments, the reactive site is selected from one or more of the following: carboxyl, amino, hydroxyl, mercapto, activated ester, acyl chloride, acid anhydride, chloroformate, activated carbonate, isocyanate, isothiocyanate, sulfonyl chloride, haloalkyl, methanesulfonate, p-toluenesulfonate, haloacetyl, epoxy, vinyl sulfone, acryloyl, methacryloyl, maleimide, aldehyde, ketone, aminooxy, hydrazyl, acylhydrazine, azide, alkynyl, cycloalkynyl, tetrazinyl, transcyclooctenyl, norbornyl, cyclopropenyl, and combinations thereof.

[0018] Preferably, the reactive functional group and the reactive site constitute a complementary reaction pair, wherein the complementary reaction pair is selected from carboxyl / amino, activated ester / amino, acyl chloride / amino, acid anhydride / amino, carboxyl / hydroxy, activated ester / hydroxy, acyl chloride / hydroxy, acid anhydride / hydroxy, chloroformate / amino, chloroformate / hydroxy, activated carbonate / amino, activated carbonate / hydroxy, isocyanate / amino, isocyanate / hydroxy, isothiocyanate / amino, sulfonyl chloride / amino, sulfonyl chloride / hydroxy, haloalkyl / amino, haloalkyl / mercapto, methanesulfonate / amino, sulfonyl chloride / hydroxy, sulfonyl chloride / amino, sulfonyl chloride / hydroxy, sulfoalkyl / amino, haloalkyl / mercapto, methanesulfonate / amino, sulfoalkyl / mercapto, sulfonyl chloride ... The group comprises one or more of the following: amino, methanesulfonate / mercapto, p-toluenesulfonate / amino, p-toluenesulfonate / mercapto, haloacetyl / mercapto, epoxy / amino, epoxy / mercapto, vinyl sulfone / mercapto, acryloyl / mercapto, methacryloyl / mercapto, maleimide / mercapto, aldehyde / amino, ketone / amino, aldehyde / aminooxy, ketone / aminooxy, aldehyde / hydrazine, ketone / hydrazine, aldehyde / acylhydrazine, ketone / acylhydrazine, azide / alkynyl, azide / cycloalkynyl, tetrazinyl / trans-cyclooctenyl, tetrazinyl / norbornenyl, and tetrazinyl / cyclopropenyl. Either group in the above complementary reaction pair may be located on the universal base or a derivative thereof, and the other group may be located on the ionizable lipid, and the two react to form a covalent bond. The reaction may be carried out optionally under conditions of coupling agent, activator, catalyst, reducing agent, base, light, or heat.

[0019] In some embodiments, the ionizable lipid with reactive sites is an ionizable lipid containing a protonable amino group and one or more hydrophobic tails, preferably the ionizable lipid with reactive sites is SM-102, cKK-E12 or ALC-0315.

[0020] In some embodiments, the ionizable lipid having a reactive site is cKK-E12 (3,6-bis[4-[bis(2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione). In some embodiments, the universal base group is covalently attached to a hydroxyl group on the hydrophobic tail of cKK-E12.

[0021] In some embodiments, the ionizable lipid having a reactive site is a peptide-based ionizable lipid (PIL) having the structure of Formula II.

[0022] Formula II, in, A4 represents a hydrogen atom or an alkyl group. n is an integer from 1 to 30. X is O or S. R1 represents the N-terminus of the PIL, and R1 is a hydrogen atom or modified with an acetyl group, amino acid, and / or other functional groups. R2 represents the side group contained in the PIL, and R2 is independent each time it appears. Natural or non-natural amino acid side groups, and at least one R2 in the PIL is , R3 represents the C-terminus of the PIL, and R3 is a hydroxyl group or has an amino group, an amino acid group, and / or other functional group modification. m is an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. A1 and A2 are hydrophobic tails and are independently substituted saturated or unsaturated, linear or branched alkyl chains having 4 to 25 carbon atoms, wherein the alkyl chains optionally contain one or more linking groups L selected from amide bonds, ester bonds, disulfide bonds, ketethiocarbamate bonds, ether bonds or combinations thereof.

[0023] In some implementations, n is 2.

[0024] In some embodiments, adjacent amino acid building blocks in the PIL have an ectopic configuration, for example... LD configuration or DL Configuration. As used herein, the term "heterotopic configuration" refers to a configuration in which two adjacent amino acid building blocks in a PIL have different D / L chiral configurations. Specifically, when the preceding amino acid building block is L-type and the following amino acid building block is D-type, it is called a heterotopic configuration. LD Configuration; when the preceding amino acid building block is D-type and the following amino acid building block is L-type, it is called configuration. DL Configuration.

[0025] In some implementations, m is 4.

[0026] In some embodiments, the universal base group is covalently attached to any covalently attached site in the PIL, such as the universal base group being covalently attached to the N-terminus, C-terminus, central backbone, side group R2, A1 hydrophobic tail, and / or A2 hydrophobic tail of the PIL.

[0027] In some embodiments, the universal base group is covalently linked to the N-terminal amino group of the PIL, optionally only one universal base group is covalently linked to the N-terminal amino group of the PIL; or, The universal base group is covalently attached to the A1 hydrophobic tail and / or A2 hydrophobic tail of the PIL, preferably to the hydroxyl group on the A1 hydrophobic tail and / or A2 hydrophobic tail.

[0028] In some embodiments, the reactive site is the N-terminal amino group of the PIL, and more preferably, only one universal base group is covalently attached to the N-terminal amino group of the PIL.

[0029] In some embodiments, the reactive sites are located at the A1 and / or A2 hydrophobic tails of the PIL.

[0030] In some embodiments, the universal base group is linked to the N-terminal amino group of the PIL via a spacer group.

[0031] In some embodiments, the spacer group is a four-carbon spacer group (C4), most preferably -NH-(CH2)4-CO-. In some embodiments, the spacer group is linked to a universal base group via an amino group and to the N-terminal amino group of the PIL via a carbonyl group. In some embodiments, the universal base group forms a linkage structure with the PIL as shown in UB-C(O)-NH-(CH2)4-C(O)-NH-PIL.

[0032] In some embodiments, A1 and A2 are saturated alkyl chains, and more preferably A1 and A2 are -CH2CH2(CH2). q CH2CH3, where q is an integer from 0 to 21 (e.g., 4, 5, 6, 7, 8, 9, 10, 11 or 12), preferably q is different for A1 and A2, for example, 6 for A1 and 8 for A2.

[0033] In some embodiments, the universally modified ionizable lipid is Nii-C4- LD -a10a12K2: .

[0034] In some embodiments, the universally modified ionizable lipids are organ-targeting ionizable lipids, such as liver-targeting peptide-based ionizable lipids (Nip-a12Dab4), lung-targeting peptide-based ionizable lipids (Am-K2K(Nii)a12K4), and spleen-targeting peptide-based ionizable lipids (a12K4K(Nii)E-Ca). , ,

[0036] In some embodiments, the universal base modification preserves the organ targeting mediated by ionizable lipids. In some embodiments, the organ targeting mediated by ionizable lipids is reduced by no more than 50%, 40%, 30%, 20%, 10%, or 5% after the universal base modification.

[0037] In a second aspect, the present invention provides the use of a universal base in modifying ionizable lipids to endow the ionizable lipids with the ability to assemble nucleic acids in a neutral aqueous phase. Preferably, the universal base is a universal base having a reactive functional group or a derivative thereof, such as a carboxylated universal base, such as the carboxylated universal base defined in the first aspect. More preferably, the ionizable lipid is an ionizable lipid having a reactive site, such as the ionizable lipid having a reactive site defined in the first aspect.

[0038] In some embodiments, the neutral aqueous phase is an aqueous medium with a pH of 6.0 to 8.0, preferably 6.5 to 7.5, and more preferably about 6.8.

[0039] In some embodiments, the aqueous medium includes water (such as nuclease-free water) or an aqueous solution.

[0040] In some embodiments, the aqueous solution is a buffer solution containing a buffer.

[0041] In some embodiments, the buffer concentration of the aqueous medium is 0 to 500 mM.

[0042] In some embodiments, the buffer is selected from Tris, HEPES, phosphates, citrates, acetates, and any combination thereof.

[0043] Thirdly, the present invention provides a lipid nanoparticle comprising an ionizable lipid with universal base modification as described in the first aspect.

[0044] In some embodiments, the lipid nanoparticles are used to deliver active agents such as nucleic acids.

[0045] In some embodiments, the active agent of the present invention is a charged (e.g., mRNA, siRNA, sgRNA, pegRNA, pDNA, ssDNA, Cas9 RNP, cGAMP, PE mRNA or epigRNA) or an uncharged (e.g., PNA or PMO) nucleic acid.

[0046] Fourthly, the present invention provides a method for preparing lipid nanoparticles encapsulating nucleic acids, wherein the lipid nanoparticles comprise ionizable lipids modified with universal bases according to the first aspect, the method comprising: (a) A lipid phase containing the universally modified ionizable lipid is mixed with an aqueous phase containing the nucleic acid via microfluidics to form lipid nanoparticles encapsulating the nucleic acid, wherein the aqueous phase is an acidic or neutral medium. (b) Empty lipid nanoparticles without nucleic acid are prepared from a lipid composition containing the universally modified ionizable lipids, and the empty lipid nanoparticles are then incubated with the nucleic acid to form lipid nanoparticles encapsulated with nucleic acid, wherein the empty lipid nanoparticles are prepared in an acidic or neutral medium. (c) Empty lipid nanoparticles without nucleic acid are prepared from a lipid composition comprising the universally modified ionizable lipids, and the empty lipid nanoparticles and the nucleic acid are lyophilized separately to obtain separated lyophilized empty lipid nanoparticles and lyophilized nucleic acid; before use, the separated lyophilized empty lipid nanoparticles and lyophilized nucleic acid are post-assembled to form lipid nanoparticles encapsulating nucleic acid, wherein the post-assembly is performed in an acidic or neutral medium; or (d) After forming nucleic acid-containing lipid nanoparticles, the nucleic acid-containing lipid nanoparticles are freeze-dried as a whole and reconstituted before use to obtain reconstituted nucleic acid-encapsulated lipid nanoparticles.

[0047] In some embodiments, in (c), the empty lipid nanoparticles or the nucleic acid are mixed with a freeze-drying protectant before freeze-drying.

[0048] In some embodiments, the freeze-drying protectant is selected from monosaccharides and disaccharides (trehalose, sucrose, fructose, and glucose), sugar alcohols (mannitol), polysaccharides (glucan), cyclic oligosaccharides (hydroxypropyl-β-cyclodextrin, HP-β-CD), synthetic polymers (polyethylene glycol 1500, PEG1500; polyvinyl alcohol, PVA; and polyvinylpyrrolidone K12, PVP-K12) or combinations thereof.

[0049] In some embodiments, the neutral medium is an aqueous medium with a pH of 6.0 to 8.0, preferably 6.5 to 7.5, and more preferably about 6.8; Preferably, the aqueous medium includes water (such as nuclease-free water) or an aqueous solution; Preferably, the aqueous solution is a buffer solution containing a buffering agent; Optionally, the buffer concentration of the aqueous medium is 0 to 500 mM, for example 1 to 100 mM, 5 to 50 mM, about 10 mM to about 20 mM; Optionally, the buffer is selected from Tris, HEPES, phosphates, citrates, acetates, and any combination thereof.

[0050] In some embodiments, the acidic medium is an aqueous medium with a pH less than 6.0; Preferably, the acidic medium is an aqueous medium with a pH of 3.0 to less than 6.0, more preferably 3.5 to 5.5, and more preferably about 4.0; Preferably, the aqueous medium comprises an aqueous solution; preferably, the aqueous solution is an acidic aqueous buffer solution. Optionally, the aqueous medium includes a buffer; Optionally, the buffer is a buffer capable of maintaining the pH of the aqueous medium within the acidic range, such as phosphate, citrate, acetate, or any combination thereof; Optionally, when the aqueous medium contains a buffer, the concentration of the buffer is 1 to 500 mM, for example 1 to 100 mM, 5 to 50 mM, about 10 mM to about 20 mM; For example, the acidic medium is a citrate buffer with a pH of about 4.0; for example, the acidic medium is a citrate buffer of about 10 mM to about 20 mM with a pH of about 4.0.

[0051] In some embodiments, the lyophilization protectant is selected from disaccharides, HP-β-CD, PVA, or combinations thereof. In some embodiments, the concentration of the lyophilization protectant is 40 to 320 mg / mL. - ¹, for example, about 40 mg / mL - ¹, Approximately 80 mg / mL - ¹, Approximately 160 mg / mL - ¹ or approximately 320 mg / mL - ¹.

[0052] In some embodiments, the lyophilization protectant is sucrose. In some embodiments, the concentration of the sucrose is about 80 to about 320 mg / mL. - ¹, for example, about 100 mg / mL - ¹, approximately 120 mg / mL - ¹, approximately 140 mg / mL - ¹, approximately 160 mg / mL - ¹, approximately 180 mg / mL - ¹, approximately 200 mg / mL - ¹, approximately 220 mg / mL - ¹, approximately 240 mg / mL - ¹, approximately 260 mg / mL - ¹, approximately 280 mg / mL - ¹, approximately 300 mg / mL - ¹, preferably about 160 mg / mL - ¹.

[0053] In some embodiments, the lyophilization protectant is selected from trehalose, sucrose, fructose, glucose, or combinations thereof. In some embodiments, the concentration of the lyophilization protectant is 40-160 mg / mL. - ¹, for example, about 40 mg / mL - ¹, approximately 60 mg / mL - ¹, approximately 80 mg / mL - ¹, approximately 100 mg / mL - ¹, approximately 120 mg / mL - ¹, approximately 140 mg / mL - ¹, approximately 160 mg / mL - ¹.

[0054] In some embodiments, the LNP to sucrose mass ratio (160 mg / mL) - ¹Sucrose) between 1% and 5%, for example, about 1%, about 2%, about 3%, about 4% or about 5%.

[0055] In some embodiments, the lyophilization protectant causes the lyophilized empty lipid nanoparticles and / or nucleic acids to form a white, uniform, porous or sponge-like lyophilized cake.

[0056] In some embodiments, the residual moisture content of the resulting freeze-dried cake is less than about 2%, preferably less than about 1%.

[0057] In some embodiments, post-assembly includes dissolving the lyophilized nucleic acid in a neutral aqueous solution, such as pure water with a pH between 6 and 8, preferably around pH 7, to form an aqueous nucleic acid solution. In some embodiments, the pure water does not contain nucleases.

[0058] In some embodiments, post-assembly further includes mixing an aqueous nucleic acid solution with lyophilized empty lipid nanoparticles to form lipid nanoparticles encapsulating nucleic acids.

[0059] In some embodiments, the post-assembly is performed at a temperature of about 4°C to about 40°C, such as about 10°C, about 15°C, about 18°C, about 20°C, about 25°C, about 28°C, about 30°C, or about 35°C.

[0060] In some implementations, the post-assembly includes a brief vortex of 5-10 seconds.

[0061] In some embodiments, the lyophilized empty lipid nanoparticles are reconstructed within about 6 to about 10 seconds, about 7 to about 10 seconds, or no more than about 20 seconds after being mixed with an aqueous nucleic acid solution.

[0062] In some embodiments, the nucleic acid encapsulation rate of the lipid nanoparticles assembled from the lyophilized empty lipid nanoparticles and lyophilized nucleic acids is at least about 80%, at least about 90%, at least about 95%, or more than about 95%.

[0063] Fifthly, the present invention provides a kit for preparing lipid nanoparticles encapsulating nucleic acids, comprising: (i) a first container comprising lyophilized empty lipid nanoparticles, said empty lipid nanoparticles comprising ionizable lipids with universal base modification according to the first aspect; and (ii) A second container containing lyophilized nucleic acids; The components in the first and second containers are adapted for post-assembly before use to form lipid nanoparticles encapsulating nucleic acids.

[0064] In some embodiments, the first or second container further contains a lyophilization protectant, such as the lyophilization protectant defined in the fourth aspect. In some embodiments, the post-assembly is as defined in the fourth aspect.

[0065] In a sixth aspect, the present invention provides a pharmaceutical composition comprising lipid nanoparticles as defined in the third aspect or lipid nanoparticles encapsulating nucleic acids as defined in the fourth or fifth aspect, and a pharmaceutically acceptable carrier.

[0066] In some embodiments, the pharmaceutical composition is a vaccine composition, such as a prophylactic mRNA vaccine (e.g., respiratory syncytial virus (RSV) vaccine; influenza A virus H1N1 subtype vaccine; and rabies virus glycoprotein RVG vaccine).

[0067] In a seventh aspect, the present invention provides the use of lipid nanoparticles encapsulating nucleic acids (as described in the third aspect), reagent kits (as described in the fifth aspect), or pharmaceutical compositions (as described in the sixth aspect) in the preparation of pharmaceuticals, said pharmaceuticals being used for vaccine therapy, protein replacement therapy, gene editing therapy, gene silencing therapy, or in vivo cell engineering therapy.

[0068] In some embodiments, the drug is used to treat or prevent diseases such as respiratory syncytial virus infection, H1N1 influenza A, rabies virus infection / rabies, familial hypercholesterolemia, cardiovascular disease, hereditary or wild-type transthyretin amyloidosis.

[0069] Eighthly, the present invention provides a method for delivering an active agent to a target organ or target cell or for preventing or treating a disease, the method comprising administering to a desired object or ex vivo cells or tissue an effective amount of a third aspect of encapsulated nucleic acid lipid nanoparticles, a fifth aspect of a kit, or a sixth aspect of a pharmaceutical composition. In some embodiments, the method is used for vaccine therapy, protein replacement therapy, gene editing therapy, gene silencing therapy, or in vivo cell engineering therapy. In some embodiments, the disease is, for example, respiratory syncytial virus infection, H1N1 influenza A, rabies virus infection / rabies, familial hypercholesterolemia, cardiovascular disease, or hereditary or wild-type transthyretin amyloidosis. Attached Figure Description

[0070] Figure 1 Solid-phase supported synthesis (SPSS) of universally base-modified peptide-ionizable lipids (UB-PIL) and the construction of an assemblable lyophilized lipid nanoparticle (AllSet) for long-term storage and rapid use of therapeutic agents. AllSet mRNA-LNPs were rapidly post-assembled using lyophilized empty lipid nanoparticles and mRNA in pure water via rehydration and vortexing. The appearance of empty AllSet LNPs in water, lyophilized empty AllSet LNPs, and assembled AllSet mRNA-LNPs are shown. Representative scanning electron microscopy (SEM) images (scale bar, 40 μm) of the lyophilized empty LNPs and representative transmission electron microscopy (TEM) images (scale bar, 50 nm) of the assembled AllSet mRNA-LNPs are presented.

[0071] Figure 2 AllSet mRNA-LNP was prepared in-situ using lyophilized empty AllSet LNPs and mRNA via rehydration and vortexing.

[0072] Figure 3 Chemical structures of a12Dab4 modified with the universal base 1-carboxymethyl-3-nitropyrrole (Nip) (Nip-a12Dab4), unmodified a12Dab4, non-ionized Nip-modified c12Dab4 (Nip-c12Dab4), and the benchmark ALC-0315 lipid.

[0073] Figure 4 Structural characteristics of Nip-a12Dab4, a12Dab4, Nip-c12Dab4, and ALC-0315.

[0074] Figure 5 Schematic diagram of microfluidic assembly of mRNA-LNP.

[0075] Figure 6Encapsulation efficiency (EE) of mRNA-LNPs prepared with different aqueous solutions ( n =3 biologically independent samples).

[0076] Figure 7 Record bioluminescent images of mRNA expression in various organs, and quantify mRNA expression in the liver (0.1 mg / kg) 6 h after treatment with mRNA-LNP in mice. -1 Fluc mRNA, n =3 biologically independent samples).

[0077] Figure 8 mRNA-LNPs were assembled by rehydrating and lyophilizing empty LNPs using mRNA solutions with different pH values ​​at different temperatures.

[0078] Figure 9 Using lyophilized empty LNPs under different conditions (pH and temperature, n Encapsulation efficiency (EE) of a12Dab4 and Nip-a12Dab4-based mRNA-LNPs prepared from 3 biologically independent samples.

[0079] Figure 10 : Schematic diagram of mRNA bridging LNP fusion.

[0080] Figure 11 The concentration and hydrodynamic particle size of rehydrated Nip-a12Dab4 LNP with or without mRNA were detected by NTA. n =6 or 3 biologically independent samples).

[0081] Figure 12 : Fluorescence resonance energy transfer (FRET) elimination percentage of rehydrated Nip-a12Dab4 LNP with or without mRNA ( n =3 biologically independent samples).

[0082] Figure 13 Nip-a12Dab4 LNP loaded with mRNA was quantified by nanoflow cytometry using FITC-labeled mRNA and DiA-labeled empty LNP.

[0083] Figure 14 : Molecular dynamics (MD) simulation snapshots of Nip-a12Dab4 with 19-nt polyA, polyG, polyC, or polyU, and π-π stacking and hydrogen bond (HB) counts during the simulation.

[0084] Figure 15 : A schematic diagram of mRNA fusion-mediated loading into a universal base-modified LNP via non-electrostatic interactions (π-π stacking and hydrogen bonding).

[0085] Figure 16 Record bioluminescent images of mRNA expression in various organs, and quantify mRNA expression in the liver (0.1 mg / kg) 6 h after treatment with standard-prepared and post-assembled Nip-a12Dab4 AllSet mRNA-LNP in mice. -1 FlucmRNA, n =3 biologically independent samples).

[0086] Figure 17 : Schematic diagram of the UB-PIL in vivo guided continuous chemical evolution process.

[0087] Figure 18 Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with mRNA-LNP containing different AIFA building blocks, and quantitative analysis of mRNA expression in the liver (0.1 mg / kg). -1 Fluc mRNA).

[0088] Figure 19 Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with mRNA-LNP containing different amounts of AIFA building blocks, and quantitative analysis of mRNA expression in the liver (0.1 mg / kg). -1 Fluc mRNA).

[0089] Figure 20 Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with mRNA-LNP containing different types of natural or universal bases, and quantitative analysis of mRNA expression in the liver (0.1 mg / kg). -1 Fluc mRNA).

[0090] Figure 21 Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with mRNA-LNP containing AIFA building blocks D / L stereoisomers or different amounts of universal bases, and quantitative data on mRNA expression in the liver (0.05 mg / kg). -1 FlucmRNA).

[0091] Figure 22 Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with mRNA-LNP containing different types of spacers, and quantitative analysis of mRNA expression in the liver (0.025 mg / kg). -1 Fluc mRNA).

[0092] Figure 23Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with mRNA-LNP containing asymmetric tails, and quantitative analysis of mRNA expression in the liver (0.0125 mg / kg). -1 Fluc mRNA).

[0093] Figure 24 Nii-C4- was obtained after six rounds of continuous chemical evolution. LD The chemical structure of -a10a12K2 UB-PIL.

[0094] Figure 25A : ALC-0315, Nip-a12Dab4, Nii-C4- LD Comparison of in vivo delivery efficacy of -a10a12K2 and its lyophilized AllSet form (0.0125 mg kg) -1 Fluc mRNA). Nii, 1-Carboxymethyl-5-nitroindole.

[0095] Figure 25B Based on Nii-C4 LD In vivo delivery efficiency of intact mRNA-LNP of -a10a12K2 lipid after lyophilization and reconstitution.

[0096] Figure 26 The chemical evolution process of UB-PIL.

[0097] Figure 27 : Can be encapsulated in empty Nii-C4- LD -a10a12K2 (Nii-lipid) AllSet LNP is a general term for nucleic acid (NA) cargoes, including charged and uncharged NAs. PNA, peptide nucleic acid; PMO, phosphoryldiamine morpholine oligomer.

[0098] Figure 28 : Various NA encapsulation efficiencies (EE) in rehydrated freeze-dried air ALC-0315 and Nii-lipid LNP using AllSet technology ( n =3 biologically independent samples).

[0099] Figure 29 CLSM images of HeLa cells treated with Nii-lipid LNPs encapsulated with various Cy5-labeled NA (red). Cell nuclei were stained with DAPI (blue). Scale bar, 10 μm. The experiment was repeated three times, yielding identical results.

[0100] Figure 30 The universality of LNP systems prepared using the AllSet technique.

[0101] Figure 31Using AllSet technology, a multifunctional mRNA-LNP system can be rapidly assembled in the field from rehydrated lyophilized empty LNPs and mRNA.

[0102] Figure 32 The chemical structures of Nii-modified organ-targeting PILs include Am-K2K(Nii)a12K4 (lung PILOT) and a12K4K(Nii)E-Ca (spleen PILOT)UB-PIL.

[0103] Figure 33 Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with Nii-modified PILOT LNPs in the lungs and spleen, and relative expression (0.1 mg / kg) in the liver, spleen, lungs, heart, and kidneys. -1 Luc mRNA, n =3 biologically independent samples).

[0104] Figure 34 Chemical structures of Nii-modified commercial lipids, including Nii-SM-102 and Nii-ALC-0315 lipids.

[0105] Figure 35 mRNA encapsulation efficiency (EE) in SM-102, Nii-SM-102, ALC-0315, and Nii-ALC-0315 LNPs using AllSet technology ( n =3 biologically independent samples).

[0106] Figure 36 The AllSet technique was used to quantify mRNA expression in the liver of mice 6 h after treatment with SM-102, Nii-SM-102, ALC-0315, and Nii-ALC-0315 mRNA-LNP, and to obtain bioluminescent images of mRNA expression in various organs (0.1 mg / kg). -1 Luc mRNA, n =3 biologically independent samples).

[0107] Figure 37 Nii-C4- LD The chemical structure of -a10a12K2.

[0108] Figure 38 mRNA encapsulation efficiency (EE) in MC3 and LP-01 LNPs containing various molar ratios of Nii-lipid using AllSet technology ( n =3 biologically independent samples).

[0109] Figure 39Quantitative analysis of mRNA expression in the liver of mice 6 h after treatment with standard MC3 and LP-01 mRNA-LNP and their lyophilized AllSet form containing 5% Nii-lipid, and bioluminescent images of mRNA expression in various organs (0.1 mg / kg). -1 Luc mRNA, n =3 biologically independent samples).

[0110] Figure 40 Key quality attributes of freeze-dried AllSet LNPs.

[0111] Figure 41 It contains various types of lyophilization protectants (80 mg / mL). -1 ) freeze-dried air Nii-C4- LD The appearance of -a10a12K2AllSet LNP.

[0112] Figure 42 : Lyophilized empty Nii-C4- with different LNP concentrations and multiple lyophilization protectants LD -a10a12K2 Residual moisture content of AllSetLNP.

[0113] Figure 43 : Lyophilized Nii-C4- with different LNP concentrations and various lyophilization protectants LD Reconstruction time of -a10a12K2 AllSetmRNA-LNP.

[0114] Figure 44 Hydrodynamic particle size, polydispersity index (PDI), and encapsulation efficiency (EE) of assembled AllSet mRNA-LNPs with different lyophilization protectant concentrations and types.

[0115] Figure 45 Heatmap of Fluc expression in MDA-MB-231 cells treated with assembled AllSet mRNA-LNP ( n =4 biologically independent samples). Relative light units (RLU) were normalized to mRNA-LNPs lyophilized with 80 mg mL⁻¹ trehalose.

[0116] Figure 46 Bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with assembled AllSet mRNA-LNPs prepared at various LNP / sucrose weight ratios, and quantitative analysis of mRNA expression in muscle and right lymph node (0.2 mg / kg). -1 FlucmRNA, n =3 biologically independent samples).

[0117] Figure 47 Schematic diagram of the long-term stability evaluation of AllSet mRNA-LNP stored at 4°C and room temperature (RT) for up to 240 days (8 months).

[0118] Figure 48 Long-term stability evaluation of EE and particle size of post-assembled AllSet mRNA-LNP after storage at RT for up to 240 days. n =3 biologically independent samples).

[0119] Figure 49 Bioluminescent images of mRNA expression in the whole body and various organs of mice 6 h after treatment with post-assembled AllSet mRNA-LNP, and quantitative analysis of mRNA expression in the liver and right lymph node (0.1 mg / kg IV injection), after storage at RT for up to 240 days. -1 Fluc mRNA, 0.2 mg / kg, intramuscular injection. -1 Fluc mRNA, n =3 biologically independent samples).

[0120] Figure 50 Schematic diagram of ready-to-use lyophilized empty LNPs and mRNA / sgRNA stored at RT.

[0121] Figure 51 Rapid on-site assembly of AllSet mRNA-LNPs using lyophilized empty LNPs and mRNA in pure water for use in mRNA vaccines and gene-editing therapeutics.

[0122] Figure 52 A schematic diagram of AllSet mRNA-LNP vaccination using a 3-week interval primary-booster strategy (0.25 mg kg-1 antigen mRNA). n =5 biologically independent samples).

[0123] Figure 53 : Detection of antigen-specific (RSV preF, H1N1, and RVG) IgG antibody titers by ELISA ( n =5 biologically independent samples). 6M, 6 months; Lyo., lyophilized.

[0124] Figure 54 Liver function evaluation after AllSet mRNA-LNP vaccination ( n =5 biologically independent samples). ALT, alanine aminotransferase; AST, aspartate aminotransferase.

[0125] Figure 55 Serum cytokine evaluation after AllSet mRNA-LNP vaccination (n =5 biologically independent samples). IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; CXCL1, CXC motif chemokine ligand 1.

[0126] Figure 56 : Cas9 and ABE-mediated in vivo processes via AllSet mRNA-LNP PCSK9 A schematic diagram of gene editing (1.0 mg kg-1 total RNA, Cas9 or ABEmax mRNA:sgRNA=2:1, wt / wt).

[0127] Figure 57 Liver treated with AllSet and standard-prepared mRNA-LNP PCSK9 Knockout efficiency ( n =3 biologically independent samples).

[0128] Figure 58 Cas9-mediated PCSK9 Serum after gene knockout PCSK9 Protein, LDL-C and triglyceride levels ( n =3 biologically independent samples). LDL-C, low-density lipoprotein cholesterol.

[0129] Figure 59 Liver treated with AllSet and standard-prepared mRNA-LNP PCSK9 Base editing efficiency.

[0130] Figure 60 ABE-mediated PCSK9 Serum after base editing PCSK9 Protein, LDL-C and triglyceride levels ( n =3 biologically independent samples).

[0131] Figure 61 PE-mediated in vivo via AllSet mRNA-LNP PCSK9 A schematic diagram of gene editing (4.0 mg kg-1 total RNA, PEmax mRNA:epegRNA=1:2, wt / wt).

[0132] Figure 62 Immunofluorescence (IF) images of liver cells treated with AllSet mRNA-LNP. Scale bar, 50 μm.

[0133] Figure 63 Liver treated with AllSet and standard-prepared mRNA-LNP EGFP Improve editing efficiency.

[0134] Figure 64 The contrasting self-assembly processes between conventional mRNA-LNPs and universally modified AllSet mRNA-LNPs. The self-assembly of conventional mRNA-LNPs is driven by pH-dependent electrostatic interactions during rapid mixing. When the ethanol lipid phase encounters mRNA in an acidic aqueous buffer (typically pH 4.0), the ionizable lipids are protonated and positively charged, enabling them to bind to the negatively charged phosphate backbone of the mRNA. As the solvent polarity changes during mixing, these components self-assemble into small lipid nanoparticles, which further fuse to form larger particles during buffer exchange to physiological pH (7.4). In contrast, the preparation of AllSet mRNA-LNPs follows a simplified assembly mechanism distinct from conventional electrostatic methods. In this process, pre-formed empty LNPs (in liquid or lyophilized form) are directly rehydrated with an aqueous mRNA solution in a neutral aqueous environment. The mRNA encapsulation is driven by the synergistic combination of π-π stacking interactions between the universal lipid bases and the mRNA bases, and hydrogen bonds between the lipid amide groups and the mRNA phosphate backbone. This molecule recognizes that triggers the spontaneous fusion of LNPs and mRNA loading, enabling rapid one-step assembly and bypassing the need for acidic buffers, external heating, and specialized equipment.

[0135] Figure 65 Physicochemical properties of UB-PIL and UB-PIL-based mRNA-LNPs. A heatmap shows the physicochemical properties of UB-PIL and UB-PIL-based mRNA-LNPs, including the solubility of UB-PIL in ethanol (mg / mL). -1 The following parameters were evaluated: aromatic ring number, HB donor number, hydrodynamic particle size (nm), PDI, zeta potential (mV), EE, pKa value, colloidal stability (%), serum stability (%), LNP dissociation rate (%), hemolytic ability (%), and endosome escape ability (Gal8 spot count). Endosome escape ability was evaluated in HeLa-mRuby3-Gal8 reporter cells using the Galectin-8 (Gal8) recruitment assay, as described in the method.

[0136] Figure 66 Longer AIFA side chains increase the pKa value, dissociation rate, hemolytic activity, and endosome escape ability of LNPs, directly promoting in vivo mRNA delivery.

[0137] Figure 67 The number of AIFA building blocks determines the number of HB donors. Nip-a12K2 with 2 AIFA and 3 HB donors is most efficient for mRNA delivery.

[0138] Figure 68Universal base modifications in PIL significantly outperformed native base modifications, achieving a higher hit rate (60% vs. 0%). Specific base types affected lipid solubility in ethanol, with values ​​>33 mg mL⁻¹ being necessary for efficient mRNA delivery. Furthermore, base type determined the number of HB donors, with three bases showing optimal in vivo efficacy. Higher colloidal stability, LNP dissociation rate, hemolytic activity, and endosome escape ability (all influenced by base type) were directly correlated with superior delivery efficiency. Among all tested bases, the universal base Nii-modified a12K2 exhibited the highest in vivo mRNA delivery efficiency.

[0139] Figure 69 UB-PIL with 2 or 3 Nii units exhibits lower solubility but more aromatic rings and hydrogen bonds. This leads to excessive colloidal and serum stability, thereby reducing in vivo mRNA delivery efficiency. In contrast, the LD conformation enhances endosome escape of LNPs, resulting in superior delivery performance.

[0140] Figure 70 Different spacer groups in UB-PIL affect the colloidal stability of the resulting LNPs. Unlike the stability issues observed in the Nii unit, the stronger colloidal stability here leads to higher mRNA delivery efficiency.

[0141] Figure 71 The asymmetric tail chain in UB-PIL affects LNP dissociation rate, hemolytic activity, and endosome escape ability, all of which collectively determine in vivo mRNA delivery efficiency. The a10a12 asymmetric tail chain showed the highest efficiency among all tested variants.

[0142] Figure 72 Chemical structures of UB-PIL from the first to the seventh generation during six consecutive rounds of evolution.

[0143] Figure 73 Summary of SAR for UB-PIL and structural criteria for the rational design of UB-PIL to generate AllSet mRNA-LNP.

[0144] Figure 74 Nii-C4- LD -a10a12K2(Nii-lipid)AllSet LNP encapsulates mRNAs of various lengths universally.

[0145] Figure 75 Encapsulation efficiency of mRNA in Nii-lipid LNPs prepared using AllSet technology or pure water standard method ( n =3 biologically independent samples).

[0146] Figure 76The universality of the AllSet strategy across different LNP systems, including SORT and cKK-E12 mRNA-LNP.

[0147] Figure 77 Nii-modified SORT and cKK-E12 mRNA-LNP were prepared using the AllSet technique.

[0148] Figure 78 Chemical structures of DOTAP (lung SORT lipids) and DOPS (spleen SORT lipids).

[0149] Figure 79 Molar ratio of DOTAP to DOPS in Nii Lung SORT (top) and Nii Spleen SORT (bottom) LNP formulations.

[0150] Figure 80 The encapsulation efficiency of mRNA in Nii lung SORT and Nii spleen SORT LNPs prepared using AllSet technology, bioluminescent images of mRNA expression in various organs 6 h after mouse treatment with Nii lung SORT and Nii spleen SORT LNPs, and relative mRNA expression (0.1 mg / kg) in liver, spleen, lung, heart, and kidney. -1 Fluc mRNA, n =3 biologically independent samples).

[0151] Figure 81 Chemical structure of Nii-modified cKK-E12 lipid.

[0152] Figure 82 Encapsulation efficiency of mRNA in cKK-E12 or Nii-cKK-E12 LNP, quantification of mRNA expression in liver 6 h after treatment, and bioluminescent images of mRNA expression in various organs of mice 6 h after treatment with cKK-E12 or Nii-cKK-E12 mRNA-LNP.

[0153] Figure 83 : Schematic diagram of the long-term stability evaluation of AllSet mRNA-LNP stored at RT for up to 240 days. AllSet mRNA-LNP was prepared using lyophilized empty LNPs and mRNA, and its stability was evaluated by measuring in vivo mRNA delivery efficiency after ip and sc injection.

[0154] Figure 84 Bioluminescent images of mRNA expression in various organs 6 h after AllSet mRNA-LNP treatment, and liver (ip, 0.1 mg kg). -1 Fluc mRNA) and lymph nodes (sc, 0.2 mg kg) -1Quantification of mRNA expression in Fluc mRNA ( n =3 biologically independent samples).

[0155] Figure 85 Schematic diagram of the long-term stability evaluation of ALC-0315 mRNA-LNP formed by cryotherapy stored at 4°C and RT for up to 30 days. Stability was evaluated by measuring in vivo mRNA delivery efficiency after IV and IM injections.

[0156] Figure 86 Bioluminescent images of mRNA expression in various organs 6 h after treatment with reconstructed ALC-0315 mRNA-LNP, liver (iv, 0.1 mg kg). -1 Fluc mRNA) and right lymph node (im, 0.2 mg kg) -1 Quantification of mRNA expression in Fluc mRNA ( n =3 biologically independent samples).

[0157] Figure 87 Evaluation of batch-to-batch reproducibility of AllSet mRNA-LNP.

[0158] Figure 88 Physicochemical properties and mRNA encapsulation efficiency of AllSet mRNA-LNP across 4 independent batches.

[0159] Figure 89 In vivo mRNA delivery efficiency across 4 independent batches of AllSet mRNA-LNP ( n =3 biologically independent samples).

[0160] Figure 90 Temperature tolerance during assembly of AllSet mRNA-LNP.

[0161] Figure 91 Physicochemical properties and mRNA encapsulation efficiency of AllSet mRNA-LNP assembled in the range of 4°C to 35°C.

[0162] Figure 92 In vivo mRNA delivery efficiency of AllSet mRNA-LNP assembled in the range of 4°C to 35°C ( n =3 biologically independent samples).

[0163] Figure 93 Storage stability of the assembled AllSet mRNA-LNP at 4°C after preparation for use.

[0164] Figure 94Physicochemical properties and mRNA encapsulation efficiency of assembled AllSet mRNA-LNP stored at 4°C for up to 15 days.

[0165] Figure 95 In vivo mRNA delivery efficiency of assembled AllSet mRNA-LNP stored at 4°C for up to 15 days ( n =3 biologically independent samples).

[0166] Figure 96 Schematic diagram of ready-to-use lyophilized empty LNPs and siRNA stored at RT.

[0167] Figure 97 AllSet siRNA-LNPs were rapidly assembled in-situ using pure water for in vivo gene silencing.

[0168] Figure 98 Lipid composition of AllSet and MC3 siRNA-LNP. Ionizable lipids: Nii-C4- LD -a10a12K2.

[0169] Figure 99 Physicochemical properties of AllSet and MC3 siRNA-LNPs. AllSet siRNA-LNPs were prepared using lyophilized empty AllSet LNPs stored at 4°C and RT for 6 months (6M), and then compared with freshly lyophilized and standard prepared samples. n =3 biologically independent samples).

[0170] Figure 100 In vivo testing using AllSet siRNA-LNP TTR Experimental protocol for gene knockdown.

[0171] Figure 101 Liver cells treated with AllSet and MC3 siRNA-LNP for 72 h TTR Evaluation of mRNA levels (normalized to the siCtrl group) (0.1, 0.3, and 1.0 mg / kg) -1 siRNA, n =3 biologically independent samples).

[0172] Figure 102 Serum samples treated with AllSet and MC3 siRNA-LNP at 3, 24, and 72 h post-treatment TTR Evaluation of concentrations (normalized to the siCtrl group) (0.1, 0.3, and 1.0 mg kg) -1 siRNA, n =3 biologically independent samples).

[0173] Figure 103 :Fmoc- L Synthetic route of -Dab(a12)-OH.

[0174] Figure 104 The route for synthesizing Nip-a12Dab4 via solid-phase supported synthesis (SPSS).

[0175] Figure 105 : The route for synthesizing a12Dab4 via SPSS.

[0176] Figure 106 : The route for synthesizing Nip-c12Dab4 via SPSS.

[0177] Figure 107 Characterization of mRNA-LNPs prepared in different aqueous solutions. A, B, and C represent the hydrodynamic particle size (A), PDI (B), and zeta potential (C) of the mRNA-LNPs prepared in sodium citrate buffer (10 mM, pH 4.0), HEPES buffer (20 mM, pH 7.4), and pure water (pH 6.8). Data are expressed as mean ± SD. n = 3 biologically independent samples).

[0178] Figure 108 Mechanistic study of the different transfection performances of Nip-a12Dab4, a12Dab4, Nip-c12Dab4, and ALC-0315 mRNA-LNP. A, TNS fluorescence curves of mRNA-LNP. The apparent pKa of LNP was calculated at pH at which 50% TNS fluorescence was measured. Total lipid concentration: 60 μM; TNS concentration: 4 μM. Nip-c12Dab4 mRNA-LNP could not be ionized due to the lack of ionizable amines. na, unavailable. B, Percentage of lipid dissociation of mRNA-LNP at pH 5.5. For Nip-c12Dab4 mRNA-LNP, negligible lipid dissociation was observed ( n = 3 biologically independent samples). C, the percentage of hemolysis of mRNA-LNP at pH 5.5, pH 6.5 and pH 7.4 at a total lipid concentration of 1.2 mM. The lowest hemolysis was observed for Nip-c12Dab4 mRNA-LNP ( n= 3 biologically independent samples). D, CLSM image of HeLa cells after 4 h of treatment with mRNA-LNP containing Cy5-mRNA (red). Cell nuclei stained with DAPI (blue). Scale bar: 10 μm. All tested mRNA-LNPs showed strong cellular uptake in HeLa cells. Experiments were repeated 3 times, and the same results were obtained. E, CLSM image of HeLa-mRuby3-Gal8 reporter cells after 4 h of treatment with mRNA-LNP. Cell nuclei stained with DAPI (blue). Yellow punctate spots indicate endosomes damaged due to the binding of mRuby3-Gal8 to the ruptured endosome membrane. Scale bar: 10 μm. Experiments were repeated 3 times, and the same results were obtained. The endosome escape ability of Nip-c12Dab4 mRNA-LNP was significantly lower than that of other mRNA-LNPs. Data are expressed as mean ± sd; statistical significance was analyzed using one-way ANOVA. P < 0.0001.

[0179] Figure 109 mRNA was encapsulated into aqueous a12Dab4 or Nip-a12Dab4 empty LNPs under various conditions (gradient pH and external heating).

[0180] Figure 110 The freeze-drying cycle used in this invention consists of three main stages: pre-freezing (to solidify the LNP), primary drying (sublimation of ice under vacuum), and secondary drying (removal of bound water by desorption) to ensure low residual moisture and stability.

[0181] Figure 111 Encapsulation efficiency (EE) of a12Dab4- and Nip-a12Dab4-based mRNA-LNPs prepared using aqueous empty LNPs under different conditions (pH and temperature). Nip-a12Dab4 mRNA-LNPs were prepared using empty LNPs in pure water at RT. Data are expressed as mean ± SD; n = 3 biologically independent samples). Unmodified empty a12Dab4 LNP requires both acidic conditions and external heating (>60 °C) to achieve a maximum EE of approximately 80%, with single treatment achieving only moderate mRNA encapsulation (approximately 15%–60%). In contrast, Nip-a12Dab4 LNP efficiently encapsulates mRNA in pure water at RT, achieving >90% EE in just seconds.

[0182] Figure 112Characterization of rehydrated lyophilized Nip-a12Dab4 LNPs with or without mRNA. A, Hydrodynamic particle size increase of rehydrated lyophilized LNPs induced by mRNA addition, detected by dynamic laser scattering. B, PDI of rehydrated lyophilized LNPs with or without mRNA. C, Zeta potential of rehydrated lyophilized LNPs with or without mRNA. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0183] Figure 113 : Fluorescence resonance energy transfer (FRET) cancellation study. A, Schematic diagram of the preparation of lyophilized FRET-labeled empty LNPs and FRET cancellation mediated by LNP fusion after mRNA addition. B, Percentage of FRET cancellation in lyophilized Nip-a12Dab4 LNPs after mRNA addition at various total lipid / mRNA weight ratios. Significant FRET cancellation was observed in Nip-a12Dab4 LNPs at all tested total lipid / mRNA weight ratios. C, Percentage of FRET cancellation in lyophilized a12Dab4 and ALC-0315 LNPs after mRNA addition at a total lipid / mRNA weight ratio of 40 / 1. Negligible FRET cancellation was observed in a12Dab4 and ALC-0315 LNPs due to the lack of the universal base Nip. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0184] Figure 114 Evaluation of mRNA loading into LNPs using nanoflow cytometry. A, Schematic diagram of DiA-labeled empty Nip-a12Dab4 LNPs and FITC-labeled mRNA loading into LNPs mediated by LNP fusion. B, Quantification of the percentage of FITC-mRNA-loaded DiA-labeled ALC-0315 LNPs and DiA-labeled mRNA-free ALC-0315 LNPs in the standard ALC-0315 mRNA-LNP formulation (prepared in sodium citrate buffer, 20 mM, pH 4.0). The FITC / DiA double-positive population represents mRNA-loaded LNPs, while the DiA single-positive population represents mRNA-free empty LNPs. C, Percentage of mRNA-loaded and mRNA-free LNPs and hydrodynamic particle size in AllSet Nip-a12Dab4 (prepared using lyophilized LNPs) and the standard ALC-0315 LNP formulation (prepared in pH 4.0 citrate buffer). The number of mRNA-free LNPs in the AllSet Nip-a12Dab4 LNP formulation was less than that in the conventional ALC-0315 LNP formulation. For both formulations, larger hydrodynamic particle sizes were observed in the mRNA-encapsulated LNPs. Data are presented as mean ± SD;n = 3 biologically independent samples).

[0185] Figure 115 AllSet mRNA-LNP is assembled from lyophilized empty LNPs and mRNA for direct in vivo administration.

[0186] Figure 116 Chemical mutation sites of UB-PIL. As shown in the Nip-a12Dab4 structure, the chemical mutation sites of UB-PIL include AIFA type, AIFA number, base type, base number, AIFA stereoisomer, spacer type, spacer length, tail chain symmetry and length.

[0187] Figure 117 :Fmoc- L -Dap(a12)-OH, Fmoc- L -Orn(a12)-OH and Fmoc- L Synthetic route of -Lys(a12)-OH.

[0188] Figure 118 The symmetric AIFA building block used in this invention.

[0189] Figure 119 : Routes for synthesizing Nip-a12Dap4, Nip-a12Orn4 and Nip-a12K4 using SPSS.

[0190] Figure 120 The first UB-PIL library containing different types of AIFA building blocks.

[0191] Figure 121 TNS fluorescence curves of LNPs prepared with different UB-PILs. The apparent pKa of LNPs was calculated based on the pH at which 50% TNS fluorescence was measured. Total lipid concentration: 60 μM; TNS concentration: 4 μM.

[0192] Figure 122 Structure-activity relationship (SAR) analysis of UB-PIL containing different types of AIFA building blocks. The correlations between mRNA-LNPs based on UB-PIL with different AIFAs in ethanol solubility, number of aromatic rings, number of HB donors, particle size, zeta potential, PDI, encapsulation efficiency (EE), colloidal stability, serum stability, and mean radiance of bioluminescent signals in liver were analyzed.

[0193] Figure 123 Synthesis routes of Nip-a12K1, Nip-a12K2, Nip-a12K3, Nip-a12K4 and Nip-a12K5.

[0194] Figure 124 : A second UB-PIL library containing varying numbers of AIFA building blocks.

[0195] Figure 125 SAR analysis of UB-PIL containing different numbers of AIFA building blocks. The correlations between mRNA-LNPs based on UB-PIL with different AIFA numbers and their solubility in ethanol, number of aromatic rings, number of HB donors, particle size, zeta potential, PDI, encapsulation efficiency (EE), apparent pKa, colloidal stability, serum stability, membrane fusion capacity, hemolysis, endosome escape capacity, and mean radiance of liver bioluminescence signal were analyzed.

[0196] Figure 126 Synthetic route for carboxylation of the universal base Cya.

[0197] Figure 127 Synthetic route for carboxylated general base Dapy.

[0198] Figure 128 Synthetic route for carboxylated general base Nipu.

[0199] Figure 129 Synthetic route for carboxylated general base Ppt.

[0200] Figure 130 Synthetic route for carboxylation of the universal base Upy.

[0201] Figure 131 : A library of base-derived carboxylic acids for the synthesis of UB-PIL via SPSS.

[0202] Figure 132 : The route for synthesizing Base-PIL using SPSS.

[0203] Figure 133 : A third Base-PIL library containing different bases.

[0204] Figure 134 SAR analysis of Base-PILs containing different bases. The correlations between aromatic ring number, particle size, zeta potential, PDI, encapsulation efficiency (EE), apparent pKa, serum stability, and mean radiance of liver bioluminescence signals were analyzed for each Base-PIL mRNA-LNP containing different bases.

[0205] Figure 135 Nii- LL Synthetic route of the -a12K2 stereoisomer.

[0206] Figure 136 Synthetic routes for UB-PIL containing different numbers of universal bases.

[0207] Figure 137 : A fourth UB-PIL library containing AIFA stereoisomers and varying numbers of universal bases.

[0208] Figure 138 SAR analysis of UB-PIL containing different AIFA stereoisomers and varying numbers of universal bases. The correlations between particle size, zeta potential, PDI, encapsulation efficiency (EE), apparent pKa, and mean radiance of liver bioluminescence signals were analyzed for UB-PIL mRNA-LNPs containing different bases.

[0209] Figure 139 Synthetic routes for UB-PIL containing different spacer groups.

[0210] Figure 140 : The fifth UB-PIL library containing different spacer bases.

[0211] Figure 141 SAR analysis of UB-PIL containing different spacer groups. The correlations between the solubility of UB-PIL mRNA-LNP in ethanol, number of aromatic rings, number of HB donors, particle size, zeta potential, PDI, encapsulation efficiency (EE), apparent pKa, serum stability, membrane fusion ability, hemolysis, endosome escape ability, and mean radiance of liver bioluminescence signal were analyzed.

[0212] Figure 142 Synthetic route for AIFA building blocks with asymmetric tail chains.

[0213] Figure 143 The asymmetric AIFA building block used in this invention.

[0214] Figure 144 Synthetic route of UB-PIL with asymmetric tail chain.

[0215] Figure 145 : The sixth UB-PIL library with an asymmetric tail chain.

[0216] Figure 146 SAR analysis of UB-PIL with asymmetric tail chains. The correlations between the solubility of UB-PIL mRNA-LNP in ethanol, number of aromatic rings, number of HB donors, particle size, zeta potential, PDI, encapsulation efficiency (EE), apparent pKa, colloidal stability, serum stability, and mean radiance of bioluminescent signals in the liver were analyzed.

[0217] Figure 147 : Contains various charged and uncharged nucleic acids based on Nii-C4- LDCharacterization of AllSet LNPs of -a10a12K2. A, Encapsulation efficiency (EE) of AllSet LNPs for various charged and uncharged nucleic acids prepared by standard microfluidic mixing in pure water. B, Hydrodynamic particle size (B), zeta potential (C), and PDI (D) of AllSet LNPs encapsulating various charged (mRNA, siRNA, sgRNA, pDNA, ssDNA, Cas9 RNP, and cGAMP) and uncharged (PNA and PMO) nucleic acids. AllSet LNPs were prepared using lyophilized empty LNPs or by standard microfluidic mixing of aqueous / ethanol phases in pure water. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0218] Figure 148 Nii-C4-based encapsulation of mRNAs of varying lengths, ranging from approximately 1,000 nt to approximately 7,000 nt. LD Characterization of AllSet LNPs of -a10a12K2. Hydrodynamic particle size, PDI, and zeta potential of AllSet LNPs encapsulating mRNAs of different lengths. AllSet LNPs were prepared using lyophilized empty LNPs or by standard microfluidic mixing of aqueous / ethanol phases in pure water. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0219] Figure 149 Encapsulation efficiency (EE) of ALC-0315 LNP for various charged and uncharged nucleic acids.

[0220] Figure 150 Hydrodynamic particle size, zeta potential, and PDI of ALC-0315 LNPs encapsulating various charged and uncharged nucleic acids. ALC-0315 LNPs were prepared using lyophilized empty LNPs with sodium citrate buffer (10 mM, pH 4.0), or using a standard microfluidic mixture of aqueous / ethanolic phase with pure water and sodium citrate buffer (10 mM, pH 4.0). Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0221] Figure 151 Cellular uptake was studied using CLSM. CLSM images of HeLa cells after 4 h of treatment with AllSet LNPs containing Cy5-labeled charged and uncharged nucleic acids. Cell nuclei were stained with DAPI (blue). Scale bar: 30 μm. Cy5-labeled sgRNA was used to prepare Cas9 / sgRNA ribonucleoproteins (Cas9 / sgRNA RNPs). The experiment was repeated three times, yielding identical results.

[0222] Figure 152 The route for synthesizing Am-K2K(Nii)a12K4 via SPSS.

[0223] Figure 153 : The route for synthesizing a12K4K(Nii)E-Ca via SPSS.

[0224] Figure 154 Characterization and in vivo properties of Nii-modified lung and spleen PILOT mRNA-LNP. A, Hydrodynamic particle size, zeta potential, PDI, and encapsulation efficiency (EE) of Nii-modified lung and spleen PILOT LNP prepared using lyophilized empty LNP and pure water. BC, Quantification of Fluc mRNA expression in major organs (including heart, lung, liver, spleen, and kidney) of mice treated with Nii-modified lung (B) PILOT and Nii-modified spleen (C) PILOT LNP at 6 h. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0225] Figure 155 Hydrodynamic particle size, zeta potential, and PDI of Nii-modified SORT LNPs for lung and spleen prepared using lyophilized empty LNPs and pure water.

[0226] Figure 156 Quantitative analysis of Luc mRNA expression at 6 h in major organs (heart, lung, liver, spleen, and kidney) of mice treated with Nii-modified lung SORT and Nii-modified spleen SORT LNPs. Data are expressed as mean ± SD. n = 3 biologically independent samples).

[0227] Figure 157 Synthetic route of Nii-ALC-0315.

[0228] Figure 158 Synthetic route of Nii-SM-102.

[0229] Figure 159 Characterization of SM-102, Nii-SM-102, ALC-0315, and Nii-ALC-0315 mRNA-LNPs. Hydrodynamic particle size (A), zeta potential (B), and PDI (C) of mRNA-LNPs. SM-102 and ALC-0315 mRNA-LNPs were prepared using standard microfluidic mixing with sodium citrate buffer (10 mM, pH 4.0), while Nii-SM-102 and Nii-ALC-0315 mRNA-LNPs were prepared using lyophilized empty LNPs with pure water. Data are expressed as mean ± SD;n = 3 biologically independent samples).

[0230] Figure 160 Synthetic route of Nii-cKK-E12.

[0231] Figure 161 Characterization of cKK-E12 and Nii-cKK-E12 mRNA-LNPs. Hydrodynamic particle size (A), PDI (B), and zeta potential (C) of mRNA-LNPs. cKK-E12 mRNA-LNPs were prepared using standard microfluidic mixing with sodium citrate buffer (10 mM, pH 4.0), while Nii-cKK-E12 mRNA-LNPs were prepared using lyophilized empty LNPs with pure water. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0232] Figure 162 : Contains different molar ratios of Ni-C4- LD Characterization of MC3 and LP-01 mRNA-LNP of -a10a12K2. AC, containing Nii-C4- in molar ratios of 0%, 0.25%, 0.5%, 1%, 2.5%, and 5%. LD Hydrodynamic particle size (A), PDI (B), and zeta potential (C) of MC3 and LP-01 mRNA-LNPs from -a10a12K2. MC3 and LP-01 mRNA-LNPs were prepared using lyophilized empty LNPs with pure water. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0233] Figure 163 Representative appearance of freeze-dried mRNA cake. Fluc mRNA was freeze-dried using 160 mg mL⁻¹ sucrose as a freeze-drying protectant.

[0234] Figure 164 Residual moisture content of lyophilized Fluc mRNA. A and B represent the residual moisture content of lyophilized Luc mRNA containing different types / concentrations of lyophilization protectants (A) and different amounts of mRNA (B). Residual moisture content is calculated and expressed as a weight percentage (%, w / w) relative to the total mass of the lyophilized cake.

[0235] Figure 165ζ-potentials of assembled AllSet mRNA-LNPs with different concentrations and types of lyophilization protectants. AllSet LNPs were prepared using lyophilized empty LNPs with a UB-PIL / DSPC / cholesterol / DMG-PEG2000 molar ratio of 46.3:9.4:42.7:1.6 and a total lipid / mRNA weight ratio of 40:1. Data are presented as averages in the heatmap. n = 3 biologically independent samples).

[0236] Figure 166 Cell viability of MDA-MB-231 cells treated with assembled AllSet mRNA-LNPs containing different concentrations and types of lyophilization protectants. MDA-MB-231 cells were treated with AllSet LNPs containing 80 ng Luc mRNA, and cell viability was assessed by Calcein-AM assay 24 h post-treatment. AllSet LNPs were prepared using lyophilized empty LNPs with a UB-PIL / DSPC / cholesterol / DMG-PEG2000 molar ratio of 46.3:9.4:42.7:1.6 and a total lipid / mRNA weight ratio of 40:1. Data are presented as averages in the heatmap. n = 6 biologically independent samples).

[0237] Figure 167 Long-term stability assessment of mRNA integrity. mRNA integrity was measured by capillary electrophoresis of individually lyophilized mRNA and mRNA extracted from lyophilized conventional ALC-0315 mRNA-LNP (pre-formulated formulation) during storage at 4°C and RT for up to 240 days. Data are expressed as mean ± SD. n = 3 biologically independent samples).

[0238] Figure 168 Long-term stability evaluation of AllSet mRNA-LNPs stored at RT for up to 240 days. PDI (A) and ζ-potential (B) of assembled AllSet mRNA-LNPs stored at RT for up to 240 days. Data are expressed as mean ± SD. n = 3 biologically independent samples).

[0239] Figure 169 Long-term stability evaluation of AllSet mRNA-LNP stored at 4°C for up to 240 days. Hydrodynamic particle size, mRNA encapsulation efficiency (A), PDI (B), and zeta potential (C) of assembled AllSet mRNA-LNP stored at 4°C for up to 240 days were evaluated. Data are expressed as mean ± SD. n= 3 biologically independent samples).

[0240] Figure 170 Long-term stability evaluation of AllSet mRNA-LNP in vivo performance at 4°C. A, Schematic diagram of the long-term stability evaluation of AllSet mRNA-LNP in vivo performance at 4°C for up to 240 days. BF, Bioluminescent images of mRNA expression in mice throughout the body and in various organs 6 h after treatment with AllSet mRNA-LNP at 4°C for up to 240 days (B), and quantitative mRNA expression in the liver (C, IV injection; E, IP injection), right lymph node (D, IM injection), and left and right lymph nodes (F, SC injection) (0.1 mg / kg for IV and IP injections). -1 Luc mRNA; 0.2 mg / kg for both intramuscular and subcutaneous injections. -1 Luc mRNA). Data are expressed as mean ± sd; n = 3 biologically independent samples).

[0241] Figure 171 Based on Nii-C4 LD Characterization of the AllSet mRNA vaccine based on -a10a12K2. Assembly of Nii-C4- LD Hydrodynamic particle size, PDI, zeta potential, and encapsulation efficiency (EE) of the first and second doses of the AllSet mRNA vaccines (RSV, H1N1, and RVG) of type -a10a12K2. AllSet mRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at 4°C or RT for 6 months (6M). Freshly prepared lyophilized AllSet LNPs, standard-prepared AllSet LNPs, and standard-prepared SM-102 LNPs were formulated and evaluated as control groups. Data are expressed as mean ± SD; n = 3 biologically independent samples).

[0242] Figure 172 Weight monitoring during vaccination. Changes in body weight in mice receiving AllSet RSV preF (A), H1N1 (B), and RVG (C) mRNA vaccines (AC). AllSet mRNA vaccines were prepared using lyophilized (Lyo.) empty LNPs stored at 4°C or RT for 6 months (6M). Freshly prepared lyophilized AllSet LNPs, standard-prepared AllSet LNPs, and standard-prepared SM-102 LNPs were formulated and evaluated as control groups. Data are expressed as mean ± SD; n = 5 biologically independent samples).

[0243] Figure 173 Safety characteristics of the AllSet RSV mRNA vaccine. Mice were vaccinated with the AllSet RSV mRNA vaccine at a dose of 0.25 mg kg⁻¹ using a primiparous-booster strategy at 3-week intervals. Serum was separated at 3 h, 24 h, and 48 h post-administration to evaluate liver function (AST and ALT) and kidney function (CREA and UREA). AC, AST, ALT, CREA, and UREA levels in mRNA vaccine-treated mice at 3 h (A), 24 h (B), and 48 h (C). The AllSet mRNA vaccine was prepared using lyophilized (Lyo.) empty LNPs stored at 4°C or RT for 6 months (6M). Freshly prepared lyophilized AllSet LNPs, standard-prepared AllSet LNPs, and standard-prepared SM-102 LNPs were formulated and evaluated as control groups. Data are presented as mean ± SD; n = 5 biologically independent samples). Statistical significance was determined using one-way ANOVA analysis; ns, no significance.

[0244] Figure 174 Evaluation of serum cytokine levels after vaccination. Mice were vaccinated with AllSet RSV mRNA vaccine at a dose of 0.25 mg kg⁻¹ using a primiparous-booster strategy at 3-week intervals. Serum was separated at 3 h, 24 h, and 48 h post-administration to evaluate serum cytokines (IFN-α, IFN-γ, IL-1β, IL-6, IL-10, TNF-α, and CXCL1). AB, serum cytokine levels of mRNA vaccine-treated mice at 24 h (A) and 48 h (B). AllSet mRNA vaccine was prepared using lyophilized (Lyo.) empty LNPs stored at 4°C or RT for 6 months (6M). Freshly prepared lyophilized AllSet LNPs, standard-prepared AllSet LNPs, and standard-prepared SM-102 LNPs were formulated and evaluated as control groups. Data are expressed as mean ± SD; n = 5 biologically independent samples). Statistical significance was determined using one-way ANOVA analysis; ns, no significance.

[0245] Figure 175 Based on Nii-C4 LD Characterization of AllSet Cas9 mRNA gene editing drugs based on -a10a12K2. Assembly of Nii-C4- LDHydrodynamic particle size, PDI, zeta potential, and encapsulation efficiency (EE) of AllSet Cas9 mRNA gene editing LNPs of -a10a12K2 were evaluated. AllSet mRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at RT for 6 months. Standard-prepared AllSet LNPs were formulated and evaluated as a control group. Data are presented as mean ± SD; n = 3 biologically independent samples).

[0246] Figure 176 Based on Nii-C4 LD Characterization of the AllSet ABEmax mRNA gene editing drug based on -a10a12K2. Assembly of Nii-C4- LD Hydrodynamic particle size, PDI, zeta potential, and encapsulation efficiency (EE) of AllSet ABEmax mRNA gene editing LNPs from -a10a12K2 were evaluated. AllSet mRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at RT for 6 months. Standard-prepared AllSet LNPs were formulated and evaluated as a control group. Data are presented as mean ± SD; n = 3 biologically independent samples).

[0247] Figure 177 Based on Nii-C4 LD Safety characteristics of the AllSet gene-editing drug -a10a12K2. Mice were administered a total RNA dose of 1.0 mg / kg via intravenous injection. -1 AllSet gene-edited (Cas9 and ABEmax) LNPs (mRNA / sgRNA = 1:1, wt / wt) were used. Serum was separated at 3 h, 24 h, and 48 h post-drug administration to evaluate liver function (AST and ALT) and kidney function (CREA and UREA). AC, AST, ALT, CREA, and UREA levels in mice treated with AllSet gene-edited LNPs at 3 h (A), 24 h (B), and 48 h (C). AllSet mRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at RT for 6 months. Standard-prepared AllSet LNPs were formulated and evaluated as a control group. Data are presented as mean ± sd; n = 3 biologically independent samples). Statistical significance was determined using one-way ANOVA analysis; p < 0.001; p < 0.01; p <0.05.

[0248] Figure 178 Evaluation of serum cytokine levels after gene editing. Mice were administered total RNA at a dose of 1.0 mg / kg via intravenous injection. -1 AllSet gene-edited (Cas9 and ABEmax) LNPs (mRNA / sgRNA = 1:1, wt / wt) were used. Serum was separated at 3 h, 24 h, and 48 h post-drug administration to evaluate serum cytokines (IFN-α, IFN-γ, IL-1β, IL-6, IL-10, TNF-α, and CXCL1). AC, serum cytokine levels in mice treated with AllSet gene-editing drugs at 3 h (A), 24 h (B), and 48 h (C). AllSet mRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at RT for 6 months. Standard-prepared AllSet LNPs were formulated and evaluated as a control group. Data are presented as mean ± sd; n = 3 biologically independent samples). Statistical significance was determined using one-way ANOVA analysis; p < 0.0001; p < 0.001.

[0249] Figure 179 Based on Nii-C4 LD Characterization of the AllSet PEmax mRNA gene editing formulation based on -a10a12K2. Assembly of the Nii-C4- LD Hydrodynamic particle size, PDI, zeta potential, and encapsulation efficiency (EE) of the AllSet PEmax mRNA gene editing formulation of -a10a12K2 were evaluated. AllSet mRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at RT for 6 months. Standard-prepared AllSet LNPs were formulated and evaluated as a control group. Data are presented as mean ± SD; n = 3 biologically independent samples).

[0250] Figure 180 Nii-C4- prepared using PBS and standard LD -a10a12K2 mRNA-LNP and Nii-C4- LD Immunofluorescence (IF) image of liver cells treated with AllSet mRNA-LNP of -a10a12K2. Cell nuclei (blue) stained with DAPI. Scale bar, 50 μm.

[0251] Figure 181 Nii-C4- prepared using PBS and standard methods LD -a10a12K2 mRNA-LNP and Nii-C4- LD IF images of heart, spleen, lung, and kidney treated with AllSet mRNA-LNP of -a10a12K2. No leader editing was observed in major extrahepatic organs. Cell nuclei (blue) stained with DAPI. Scale bar, 50 μm.

[0252] Figure 182 Based on Nii-C4 LD Safety characteristics of the -a10a12K2 AllSet siRNA drug. Mice were administered an intravenous injection of 1.0 mg / kg. -1 AllSet siRNA LNPs were prepared. Serum was separated at 3 h, 24 h, and 48 h post-drug administration to evaluate liver function (AST and ALT) and kidney function (CREA and UREA). AC, AST, ALT, CREA, and UREA levels in mice treated with AllSet siRNA at 3 h (A), 24 h (B), and 48 h (C). AllSet siRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at RT for 6 months. Standard-prepared AllSet LNPs were formulated and evaluated as a control group. Data are presented as mean ± sd; n = 3 biologically independent samples). Statistical significance was determined using one-way ANOVA analysis; P < 0.001; P < 0.01; ns, no significance.

[0253] Figure 183 Evaluation of serum cytokine levels after gene silencing. Mice were administered 1.0 mg / kg via intravenous injection. -1 AllSet siRNA LNPs were prepared. Serum was separated at 3 h, 24 h, and 48 h post-drug administration to evaluate serum cytokines (IFN-α, IFN-γ, IL-1β, IL-6, IL-10, TNF-α, and CXCL1). AC represents serum cytokine levels in mice treated with AllSet siRNA at 3 h (A), 24 h (B), and 48 h (C). AllSet siRNA-LNPs were prepared using lyophilized (Lyo.) empty LNPs stored at RT for 6 months. Standard-prepared AllSet LNPs were formulated and evaluated as a control group. Data are presented as mean ± SD; n = 3 biologically independent samples). Statistical significance was determined using one-way ANOVA analysis. P <0.001; P < 0.05; ns, no significance.

[0254] Figure 184 :Fmoc- L -Dab(a12)-OH in CDCl3 1 H NMR spectrum. Fmoc- L The yield of -Dab(a12)-OH was 81.2%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.74 (d, 2H, Ar H -Fmoc), 7.53 (d, 2H, Ar H -Fmoc), 7.28 (t, 2H, Ar H -Fmoc), 7.26 (t, 2H, Ar H -Fmoc), 5.27 (s, 1H, -N H CO-), 4.33 (m, 2H, -N) H COOC H 2-), 4.26 (m, 1H, -C H 2C H - Fmoc), 4.23 (m, 1H, - H OOCC H CON H -), 2.30, 2.25, 2.02 (t, 6H, -C H 2C H 2-Tertiary amine), 1.63 (m, 2H, - H OOCC H (C H 2)CONH-), 1.28 (m, 40H, -C in the alkyl chain) H 2-), 0.88 (s, 6H, -C H 3).

[0255] Figure 185 :Fmoc- L -Dap(a12)-OH in CDCl3 1 H NMR spectrum. Fmoc- L The yield of -Dap(a12)-OH was 78.2%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.73 (t, 2H, ArH -Fmoc), 7.58 (t, 2H, Ar H -Fmoc), 7.33 (t, 2H, Ar H -Fmoc), 7.29 (t, 2H, Ar H -Fmoc), 5.29 (s, 1H, -N H CO-), 4.32 (m, 2H, -N) H COOC H 2-), 4.26 (m, 1H, -C H 2C H - Fmoc), 4.22 (m, 1H, - H OOCC H CON H -), 2.31, 2.25, 2.05 (t, 6H, -C H 2C H 2-Tertiary amine), 1.26 (m, 40H, -C in the alkyl chain) H 2-), 0.88 (s, 6H, -C H 3).

[0256] Figure 186 :Fmoc- L -Orn(a12)-OH in CDCl3 1 H NMR spectrum. Fmoc- L The yield of -Orn(a12)-OH was 84.5%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.77 (d, 2H, Ar H -Fmoc), 7.64 (d, 2H, Ar H -Fmoc), 7.33 (t, 2H, Ar H -Fmoc), 7.29 (t, 2H, Ar H -Fmoc), 5.33 (s, 1H, -N H CO-), 4.28 (s, 2H, -N) H COOC H 2-), 4.24 (s, 1H, -C H 2C H - Fmoc), 4.21 (t, 1H, - H OOCC H CON H-), 2.33, 2.25, 2.06 (m, 6H, -C H 2C H 2-Tertiary amine), 1.78 (m, 2H, - H OOCC H (C H 2)CONH-), 1.26 (m, 42H, -C in the alkyl chain) H 2-), 0.89 (t, 6H, -C H 3).

[0257] Figure 187 :Fmoc- L -K(a12)-OH in CDCl3 1 H NMR spectrum. Fmoc- L The yield of -K(a12)-OH was 83.1%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.73 (t, 2H, Ar H -Fmoc), 7.61 (t, 2H, Ar H -Fmoc), 7.31 (t, 2H, Ar H -Fmoc), 7.28 (t, 2H, Ar H -Fmoc), 6.13 (s, 1H, -N H CO-), 4.33 (m, 2H, -N) H COOC H 2-), 4.26 (m, 1H, -C H 2C H - Fmoc), 4.22 (t, 1H, - H OOCC H CON H -), 2.79 (m, 6H, -C H 2C H 2-Tertiary amine), 1.89 (m, 2H, - H OOCC H (C H 2)CONH-), 1.27 (m, 44H, -C in the alkyl chain) H 2-), 0.87 (t, 6H, -C H 3).

[0258] Figure 188 :Fmoc- L -K(a8a12)-OH in CDCl3 1H NMR spectrum. Fmoc- L The yield of -K(a8a12)-OH was 65.9%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.77 (d, 2H, Ar H -Fmoc), 7.66 (m, 2H, Ar H -Fmoc), 7.35 (m, 2H, Ar H -Fmoc), 7.29 (m, 2H, Ar H -Fmoc), 6.11 (s, 1H, -N H CO-), 4.34 (m, 2H, -N) H COOC H 2-), 4.23 (m, 1H, -C H 2C H - Fmoc), 4.19 (t, 1H, - H OOCC H CON H -), 2.80 (m, 6H, -C H 2C H 2-Tertiary amine), 1.88 (m, 2H, - H OOCC H (C H 2)CONH-), 1.26 (m, 36H, -C in the alkyl chain) H 2-), 0.89 (t, 6H, -C H 3).

[0259] Figure 189 :Fmoc- L -K(a10a12)-OH in CDCl3 1 H NMR spectrum. Fmoc- L The yield of -K(a10a12)-OH was 56.6%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.76 (d, 2H, Ar H -Fmoc), 7.64 (m, 2H,Ar H - Fmoc), 7.38 (t, 2H, Ar H -Fmoc), 7.31 (t, 2H, Ar H -Fmoc), 6.11 (s, 1H, -N H CO-), 4.39 (m, 2H, -NH COOC H 2-), 4.25 (m, 1H, -C H 2C H - Fmoc), 4.19 (t, 1H, - H OOCC H CON H -), 2.90 (m, 6H, -C H 2C H 2-Tertiary amine), 1.89 (m, 2H, - H OOCC H (C H 2)CONH-), 1.27 (m, 40H, -C in the alkyl chain) H 2-), 0.88 (t, 6H, -C H 3).

[0260] Figure 190 :Fmoc- L -K(a14a12)-OH in CDCl3 1 H NMR spectrum. Fmoc- L The yield of -K(a14a12)-OH was 45.9%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.77 (d, 2H, Ar H -Fmoc), 7.64 (t, 2H,Ar H - Fmoc), 7.38 (t, 2H, Ar H -Fmoc), 7.29 (t, 2H, Ar H -Fmoc), 6.11 (s, 1H, -N H CO-), 4.38 (m, 2H, -N H COOC H 2-), 4.25 (t, 1H, -C H 2C H - Fmoc), 4.15 (t, 1H, - H OOCC H CON H -), 2.89 (m, 6H, -C H 2C H 2-Tertiary amine), 1.89 (m, 2H, - H OOCC H (C H2)CONH-), 1.28 (m, 48H, -C in the alkyl chain) H 2-), 0.88 (t, 6H, -C H 3).

[0261] Figure 191 :Fmoc- L -K(a16a12)-OH in CDCl3 1 H NMR spectrum. Fmoc- L The yield of -K(a16a12)-OH was 63.3%. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.77 (d, 2H, Ar H -Fmoc), 7.64 (t, 2H,Ar H - Fmoc), 7.39 (t, 2H, Ar H -Fmoc), 7.29 (t, 2H, Ar H -Fmoc), 6.12 (s, 1H, -N H CO-), 4.32 (m, 2H, -N H COOC H 2-), 4.25 (m, 1H, -C H 2C H - Fmoc), 4.19 (t, 1H, - H OOCC H CON H -), 2.90 (m, 6H, -C H 2C H 2-Tertiary amine), 1.90 (m, 2H, - H OOCC H (C H 2)CONH-), 1.29 (m, 52H, -C in the alkyl chain) H 2-), 0.89 (t, 6H, -C H 3).

[0262] Figure 192 Nip-a12Dab4 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.64 (s, 4H, -CON H -), 6.95-7.04 (m, 4H, -NCH=C-, -CON H2), 6.28 (s, 1H, -CC H =C H -), 5.04 (s, 2H, -C H 2CON H -), 4.51, 4.39, 4.27 (t, 4H, -N H C H CO-), 3.23 (t,2H, -C H C H 2C H 2N-), 3.04 (m, 16H, -C H 2C H 2C H 2N-), 2.38, 2.26, 2.14 (m, 8H, -C H C H 2C H 2N-), 1.64 (s, 16H, -C H 2C H 2C H 2N-), 1.29 (m, 160H, -C in the alkyl chain) H 2-), 0.89(t, 24H, -C H 3).

[0263] Figure 193 Nip-a12K1 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.64 (s, 1H, -C=C H N-), 6.96 (s, 2H, -NC H 2CON H -), 6.78 (s, 2H, -CON H 2), 6.26 (t, 1H,-CC H =C H N-), 4.97 (s, 1H, -CC H =C H N-), 4.50 (t, 2H, -N H C H CO-), 3.05 (t, 2H, -C H C H 2C H 2C H 2C H 2N-), 2.97 (m, 4H, -C H2C H 2C H 2C H 2C H 2N-), 1.90 (m, 2H, -C H C H 2C H 2-), 1.64 (s, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.27 (m, 40H, -C in the alkyl chain) H 2-), 0.89 (t, 6H, -C H 3).

[0264] Figure 194 Nip-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.74 (s, 2H, -CON H -), 6.96 (d, 2H, -CC H =C H N-), 6.74 (s, 2H, -CON H 2), 6.25 (t, 1H, -C=C) H N-), 4.92 (s, 2H, -C H 2CON H -), 4.50 (t, 2H, -N H C H CO-), 3.04 (t, 4H, -C H C H 2C H 2C H 2C H 2N-), 2.90 (m, 8H, -C H 2C H 2C H 2C H 2C H 2N-), 1.90 (m, 4H, -C H C H 2C H 2-), 1.64 (s, 8H, -C H C H 2C H 2C H 2C H2N-), 1.28 (m, 80H, -C in the alkyl chain) H 2-), 0.90 (t, 12H,-C H 3).

[0265] Figure 195 Nip-a12K3 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 6.95 (d, 2H, -C=CHN-, -CC H =C H N-), 6.51 (s, 3H, -CON H -), 6.24 (t, 1H, -CC H =C H N-), 4.89 (s, 2H, -C H 2CO-), 4.46 (t, 3H, -N H C H CO-), 2.66 (s, 18H, -C H 2-Tertiary amine), 1.87 (m, 6H, -C) H C H 2C H 2C H 2C H 2N-), 1.62 (m, 12H, -C H C H 2C H 2C H 2C H 2N-), 1.27 (m, 120H, -C in the alkyl chain) H 2-), 0.88 (t, 18H, -C H 3).

[0266] Figure 196 Nip-a12K4 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 6.97 (d, 2H, -C=CHN-, -CC H =C H N-), 6.42 (s, 2H, -CON H 2), 6.25 (t, 1H, -CC H =C H N-), 4.89 (s, 2H, -C H2CO-), 4.48 (t, 2H, -N H C H CO-), 2.70 (s, 24H, -C H 2-Tertiary amine), 1.88 (m, 8H, -C) H C H 2C H 2C H 2C H 2N-), 1.60 (m, 24H, -C H C H 2C H 2C H 2C H 2N-), 1.27 (m, 160H, -C in the alkyl chain) H 2-), 0.88 (t, 24H, -C H 3).

[0267] Figure 197 Nip-a12K5 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 6.95 (d, 2H, -C=CHN-, -CC H =C H N-), 6.43 (s, 2H, -CON H 2), 6.24 (t, 1H, -CC H =C H N-), 4.90 (s, 2H, -C H 2CO-), 4.48 (t, 5H, -N H C H CO-), 2.60 (s, 30H, -C H 2-Tertiary amine), 1.86 (m, 10H, -C) H C H 2C H 2C H 2C H 2N-), 1.59 (m, 30H, -C H C H 2C H 2C H 2C H 2N-), 1.26 (m, 200H, -C in the alkyl chain) H 2-), 0.89 (t, 30H, -C H 3).

[0268] Figure 198 Ade-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.06(s, 1H, -C(N H 2)=NCH=N-), 7.94 (s, 1H, -NC H =N-), 6.51 (s, 2H, -CON H 2), 6.27 (s,2H, -CON H -), 5.49 (s, 2H, -C H 2CON H -), 4.47 (t, 2H, -N H C H CO-), 2.47 (s, 12H, -C H C H 2C H 2C H 2C H 2N-), 1.88 (m, 2H, -C H C H 2C H 2C H 2C H 2N-), 1.70 (m, 2H, -C H C H 2C H 2C H 2C H 2N-), 1.25-1.50 (m, 80H, -C in the alkyl chain) H 2-), 0.89 (t, 12H, -C H 3).

[0269] Figure 199 Cya-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 6.43 (s, 1H, -CON H -), 6.23 (s, 2H, -CON H 2), 4.96 (s, 2H, -C H 2CON H -), 4.46 (t, 2H, -N H C H CO-), 2.39 (t, 12H, -CH C H 2C H 2C H 2C H 2N-), 1.89 (m, 2H, -C H C H 2C H 2C H 2C H 2N-), 1.69 (m, 2H, -C H C H 2C H 2C H 2C H 2N-), 1.27 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.89 (t, 12H, -C H 3).

[0270] Figure 200 Cyt-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.54(d, 1H, -C H =C H N-), 7.46 (d, 2H, -C H =C H N-), 7.36 (m, 2H, -N=C(C)NH2-), 6.49(s, 2H, -CON H -), 5.53 (s, 1H, -C H 2CON H -), 4.52 (t, 2H, -N H C H CO-), 2.53 (s, 12H, -C H C H 2C H 2C H 2C H 2N-), 1.88 (m, 2H, -C H C H 2C H 2-), 1.71 (m, 2H, -C H C H 2C H 2C H 2CH 2N-), 1.15-1.64 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.89(t, 6H, -C H 3).

[0271] Figure 201 : Dapy-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.04 (s, 2H, -CC H =C-), 6.48 (s, 2H, -CON H -), 6.26 (s, 2H, C H 3CON H -), 5.48 (s,2H, -CON H 2), 4.46 (t, 2H, -N H C H CO-), 2.46 (t, 12H, -C H 2C H 2N-), 2.03 (s64H,C H 3CON H -), 1.86 (m, 2H, -C H C H 2C H 2-), 1.71 (m, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.23-1.54 (m, 40H, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.87 (t, 12H, -C H 3).

[0272] Figure 202 Fp-a12K2 in CDCl3 1 H NMR spectrum. 1H NMR (500 MHz, CDCl3) δ (ppm) = 9.52 (s, 1H, -C H O), 8.02 (s, 1H, -NC H =C H =C H -), 6.96 (d, 1H, -NC H =C H =C H -), 6.64 (s,2H, -CON H -), 6.25 (s, 2H, -CON H 2), 5.30 (s, 1H, -NC H =C H =C H -), 4.89 (s, 2H, -C H 2CON H -), 4.50 (t, 2H, -C H C H 2C H 2C H 2C H 2N-), 2.78 (m, 12H, -C H 2-Tertiary amine), 1.79 (m, 4H, -C) H C H 2C H 2C H 2C H 2N-), 1.73 (m, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.25-1.63 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.89 (t, 12H, -C H 3).

[0273] Figure 203 Gua-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.37(m, 3H, -NCH=N-, -N=C(N HNH2), 6.44 (s, 2H, CON) H 2), 5.52 (s, 2H, -C H 2CON H -), 4.48 (q, 2H, -N H C H CO-), 2.53 (s, 12H, -C H 2C H 2N-), 1.87 (m, 4H, -C H C H 2C H 2-), 1.67 (m, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.20-1.58 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.90 (t, 6H, -C H 3).

[0274] Figure 204 Ino-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 6.47, 6.26 (s, 2H, -N=C H N-), 5.47 (s, 2H, -C H 2CON H -), 4.46 (q, 2H, -N H C H CO-), 2.46 (s, 12H, -C H 2C H 2N-), 1.87 (m, 4H, -C H C H 2C H 2-), 1.68 (ddd, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.12-1.58 (m, 84H, -CH2- in the alkyl chain, -C H CH 2C H 2C H 2C H 2N-), 0.90(t, 12H, -C H 3).

[0275] Figure 205 Nii-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.58, 8.06 (s, 2H, NO2C=C H -), 7.35 (d, 1H, -C H =C H C-), 7.30 (d, 1H, -NC H =C H -),6.66 (d, 1H, -NC H =C H -), 6.38 (s, 2H, -CON H 2), 4.78 (s, 2H, -C H 2CON H -), 4.40(td, 2H, -N H C H CO-), 2.89 (m, 12H, -C H 2C H 2N-), 2.04 (s, 4H, -C H C H 2C H 2-), 1.08-1.98 (m, 88H, -CH2-, -CHCH2CH2CH2CH2N-, -C in the alkyl chain) H C H 2C H 2C H 2C H 2N-), 0.90 (t, 6H, -C H 3).

[0276] Figure 206 Nipi-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.92 (m, 2H, -Ar H ), 7.70 (d, 2H, -OC H2O-), 6.89 (d, 2H, -CON H -), 6.16 (s, 2H,-CON H 2), 5.47 (s, 2H, -C H 2CON H -), 4.47 (q, 2H, -N H C H CO-), 2.52 (s, 12H, -C H 2C H 2N-), 1.87 (dt, 4H, -C H C H 2C H 2-), 1.70 dt, 4H, -CHCH2CH2CH2CH2N-), 1.10-1.61 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.90 (t, 12H, -C H 3).

[0277] Figure 207 Nipu-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 6.97 (p, 1H, -N=C H N-), 6.73 (d, 1H, -C=C H N-), 6.24 (dd, 2H, -CON H -), 5.10 (s,2H, -C H 2CON H -), 4.51 (td, 2H, -N H C H CO-), 2.96 (m, 12H, -C H 2C H 2N-), 1.83 (m,4H, -C H C H 2C H 2-), 1.73 (dd, 4H, -C H C H 2C H 2C H 2C H2N-), 1.22-1.69 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.90 (t, 12H, -C H 3).

[0278] Figure 208 Ppt-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 6.44 (s, 2H, -CON H -), 6.23 (m, 2H, -CON H 2), 5.45 (s, 2H, -C H 2CON H -), 4.45 (q, 2H, -N H C H CO-), 2.45 (m, 12H, -C H 2C H 2N-), 1.88 (m, 4H, -C H C H 2C H 2-), 0.99-1.77 (m, 88H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.90 (t, 12H, -C H 3).

[0279] Figure 209 Pur-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.04 (s, 1H, -N=C H C-), 7.95, 7.88 (s, 2H, -NC H =N-), 6.50 (s, 2H, -CON H -), 6.30 (s,2H, -CON H 2), 5.50 (s, 2H, -C H 2CON H-), 4.46 (q, 2H, -N H C H CO-), 2.46 (m, 12H, -C H 2C H 2N-), 1.87 (m, 4H, -C H C H 2C H 2-), 1.67 (m, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.22-1.55 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.90 (t, 12H, -C H 3).

[0280] Figure 210 Tap-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.04, 7.57, 7.45, 7.35 (s, 4H, -Ar H ), 6.64 (s, 2H, -CON H 2), 4.99 (s, 2H, -C H 2CON H -), 4.42 (td, 2H, -N H C H CO-), 2.85 (m, 12H, -C H 2C H 2N-), 1.86 (d, 4H, -C H C H 2C H 2-), 1.77 (dt, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.10-1.67 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2CH 2N-), 0.90 (t, 12H, -C H 3).

[0281] Figure 211 Thy-a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 7.09 (s, 1H, -CON H CO-), 6.50 (s, 2H, -CON H 2), 5.69 (s, 2H, -C H 2CON H -), 4.49 (q, 2H,-N H C H CO-), 2.60 (s, 12H, -C H 2C H 2N-), 2.04 (s, 3H, -CO-C(=C)CH3-), 1.88 (m, 4H,-C H C H 2C H 2-), 1.70 (m, 4H, -C H C H 2C H 2C H 2C H 2N-), 1.06-1.64 (m, 84H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2N-), 0.90 (t, 12H, -C H 3).

[0282] Figure 212 Nii-C2- LD -a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.58, 8.07, 7.48, 7.34, 6.66 (s, 5H, -Ar H ), 6.42 (s, 2H, -CON H 2), 4.82 (s,2H, -C H 2CON H -), 4.38 (td, 2H, -NH C H CO-), 3.11 (td, 2H, -N H C H 2C H 2CO-), 2.81 (m, 12H, -C H 2C H 2N-), 1.96 (td, 2H, -N H C H 2C H 2CO-), 1.61 (m, 12H, -C H C H 2C H 2C H 2C H 2-), 1.05-1.45 (s, 72H, -C in the alkyl chain) H 2-), 0.91 (t, 12H, -C H 3).

[0283] Figure 213 Nii-C4- LD -a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.58, 8.06, 7.36, 7.30, 6.66 (s, 5H, -Ar H ), 6.38 (s, 2H, -CON H 2), 4.78 (s,2H, -C H 2CON H -), 4.42 (td, 2H, -N H C H CO-), 2.81 (m, 14H, -CH2CH2N-, -N H C H 2C H 2C H 2C H 2CO-), 2.04 (s, 6H, -N H C H 2C H 2C H 2C H 2CO-), 1.79 (m, 4H, -C H C H 2C H 2C H 2C H2-), 1.55 (m, 8H, -C H C H 2C H 2C H 2C H 2-), 1.15-1.41 (s, 80H, -C in the alkyl chain) H 2-), 0.90 (t, 12H, -C H 3).

[0284] Figure 214 Nii-EG2- LD -a12K2 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.58, 8.06, 7.49, 7.35, 6.66 (s, 5H, -Ar H ), 6.49 (d, 2H, -CON H 2), 4.78(s, 2H, -C H 2CON H -), 4.40 (td, 2H, -N H C H CO-), 3.92, 3.78, 3.66, 3.61 (m, 8H, -C H 2O-), 3.05 (m, 2H, -N H C H 2C H 2O-), 2.81 (m, 12H, -C H 2C H 2C H 2C H 2N-), 2.03 (s, 4H,-C H C H 2C H 2C H 2C H 2-), 1.14-1.93 (m, 88H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2-), 0.90(t, 12H, -C H 3).

[0285] Figure 215 Nii-C4- LD -a12K2 in CDCl31 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.58, 8.06, 7.49, 7.36, 6.66 (s, 5H, -Ar H ), 6.41 (s, 2H, -CON H 2), 4.78 (s,2H, -C H 2CON H -), 4.39 (td, 2H, -N H C H CO-), 3.10 (s, 2H, -CON H C H 2C H 2C H 2C H 2CO-), 2.82 (m, 12H, -C H 2C H 2N-), 2.03 (s, 4H, -C H C H 2C H 2C H 2C H 2-), 1.03-1.89 (s, 80H, -CH2- in the alkyl chain, -C H C H 2C H 2C H 2C H 2-), 0.91 (t, 12H, -C H 3).

[0286] Figure 216 Nii-SM-102 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.60, 8.10, 7.35, 7.31, 6.70 (s, 5H, -Ar H ), 4.89 (m, 1H, -OC H (C H 2)-), 4.76(s, 2H, -C H 2CON H -), 4.09 (t, 2H, -COOC H 2C H 2N-), 3.82 (m, 2H, -COOC H 2C H 2CH 2C H 2-), 2.97 (t, 2H, -COOC H 2C H 2N-), 2.85 (m, 4H, -COOC H 2C H 2N(C H 2)CH2-), 2.31 (m, 4H, -C H 2C H 2COO-), 1.60 (m, 12H, -COOCH2CH2-, -COOC H (C H 2) CH2-, -N(CH2CH2CH2)CH2CH2-), 1.30 (m, 50H, -C in the alkyl chain) H 2-), 0.90 (m, 9H, -C H 3).

[0287] Figure 217 Nii-ALC-0315 in CDCl3 1 H NMR spectrum. 1 H NMR (500 MHz, CDCl3) δ (ppm) = 8.60, 8.10, 7.35, 7.31, 6.70 (s, 5H, -Ar H ), 4.75 (s, 2H, -OC H (C H 2)-), 4.07(t, 4H, -C H 2OCO-), 3.61 (t, 2H, -COOC H 2-), 2.96 (m, 2H, -COOC H 2C H 2C H 2C H 2N-), 2.84 (m, 4H, -C H 2N(C H 2)CH2-), 2.33 (td, 2H, -COOC H 2C H 2C H 2C H 2N-), l.77 (q, 2H, -COOC H 2C H 2C H 2C H 2N-), 1.60 (s, 12H, -NC H 2CH 2C H 2C H 2C H 2C H 2OCO-), 1.20-1.50 (m, 54H, -C in the alkyl chain) H 2-), 0.89 (m, 12H, -C H 3).

[0288] Figure 218 :Fmoc- L ESI mass spectrum of -Dap(a12)-OH. Calculated value: 662.5; Measured value of [M+H]+: 663.5; Measured value of [M+2H]2+: 332.3.

[0289] Figure 219 :Fmoc- L HPLC chromatogram of -Dap(a12)-OH.

[0290] Figure 220 :Fmoc- L ESI mass spectrum of -Lys(a14a12)-OH. Calculated value: 732.6; Measured value of [M+H]+: 733.5.

[0291] Figure 221 :Fmoc- L HPLC chromatogram of -Lys(a14a12)-OH.

[0292] Figure 222 :Fmoc- L ESI mass spectrum of -Lys(a16a12)-OH. Calculated value: 760.6; Measured value of [M+H]+: 761.5; Measured value of [M+2H]2+: 381.5.

[0293] Figure 223 :Fmoc- L HPLC chromatogram of -Lys(a16a12)-OH.

[0294] Figure 224 ESI mass spectrum of Nip-a12K1. Calculated value: 633.5; [M+H]+ measured value: 634.5.

[0295] Figure 225 HPLC chromatogram of Nip-a12K1.

[0296] Figure 226 ESI mass spectrum of Cya-a12K2. Calculated value is 1114.9; measured value of [M+H]+ is 1115.7, and measured value of [M+2H]2+ is 558.5.

[0297] Figure 227 HPLC chromatogram of Cya-a12K2.

[0298] Figure 228 ESI mass spectrum of Fp-a12K2. Calculated value is 1081.0; measured value of [M+H]+ is 1082.2, and measured value of [M+K+H]2+ is 560.7.

[0299] Figure 229 HPLC chromatogram of Fp-a12K2.

[0300] Figure 230 ESI mass spectrum of Tap-a12K2. Calculated value is 1105.0; measured value of [M+H]+ is 1106.1, and measured value of [M+2H]2+ is 553.5.

[0301] Figure 231 HPLC chromatogram of Tap-a12K2.

[0302] Figure 232 ESI mass spectrum of Dapy-a12K2. Calculated value is 1165.0; measured value of [M+H]+ is 1166.1, and measured value of [M+2H]2+ is 583.6.

[0303] Figure 233 HPLC chromatogram of Dapy-a12K2.

[0304] Figure 234 ESI mass spectrum of Cyt-a12K2. Calculated value is 1097.0; measured value of [M+H]+ is 1098.1, and measured value of [M+2H]2+ is 550.2.

[0305] Figure 235 HPLC chromatogram of Cyt-a12K2.

[0306] Figure 236 ESI mass spectrum of Pur-a12K2. Calculated value is 1106.0; measured value of [M+H]+ is 1107.1, and measured value of [M+K+H]2+ is 573.1.

[0307] Figure 237 HPLC chromatogram of Pur-a12K2.

[0308] Figure 238 ESI mass spectrum of Thy-a12K2. Calculated value is 1112.0; measured value of [M+H]+ is 1113.1, and measured value of [M+K+H]2+ is 576.1.

[0309] Figure 239 HPLC chromatogram of Thy-a12K2.

[0310] Figure 240 ESI mass spectrum of Ino-a12K2. Calculated value is 1122.0; measured value of [M+H]+ is 1123.1, and measured value of [M+2H]2+ is 562.1.

[0311] Figure 241 HPLC chromatogram of Ino-a12K2.

[0312] Figure 242 ESI mass spectrum of Ppt-a12K2. Calculated value is 1181.9; measured value of [M+H]+ is 1182.7, and measured value of [M+2H]2+ is 592.2.

[0313] Figure 243 HPLC chromatogram of Ppt-a12K2.

[0314] Figure 244 ESI mass spectrum of Nipu-a12K2. Calculated value is 1099.0; measured value of [M+H]+ is 1100.0, and measured value of [M+2H]2+ is 550.6.

[0315] Figure 245 HPLC chromatogram of Nipu-a12K2.

[0316] Figure 246 ESI mass spectrum of Gua-a12K2. Calculated value is 1137.0; measured value of [M+H]+ is 1138.0, and measured value of [M+2H]2+ is 569.5.

[0317] Figure 247 HPLC chromatogram of Gua-a12K2.

[0318] Figure 248 ESI mass spectrum of Nii-a12K2. Calculated value is 1148.0; measured value of [M+H]+ is 1149.0, and measured value of [M+K+H]2+ is 594.1.

[0319] Figure 249 HPLC chromatogram of Nii-a12K2.

[0320] Figure 250 ESI mass spectrum of Nipi-a12K2. Calculated value is 1152.9; measured value of [M+H]+ is 1153.9, measured value of [M+2H]2+ is 577.6, and measured value of [M+3H]3+ is 385.4.

[0321] Figure 251HPLC chromatogram of Nipi-a12K2.

[0322] Figure 252 :3Nii- LL ESI mass spectrum of -a12K2. Calculated value: 1808.3; Measured value of [M+2H]2+: 905.1, [M+3H]3+: 603.7, [M+K+3H]4+: 462.5.

[0323] Figure 253 :3Nii- LL HPLC chromatogram of -a12K2.

[0324] Figure 254 Nii-C2- LD ESI mass spectrum of -a12K2. Calculated value is 1219.0; measured value of [M+H]+ is 1220.1, and measured value of [M+2H]2+ is 610.5.

[0325] Figure 255 Nii-C2- LD HPLC chromatogram of -a12K2.

[0326] Figure 256 Nii-C4- LD ESI mass spectrum of -a12K2. Calculated value is 1247.1; measured value of [M+H]+ is 1248.1, and measured value of [M+2H]2+ is 624.8.

[0327] Figure 257 Nii-C4- LD HPLC chromatogram of -a12K2.

[0328] Figure 258 Nii-EG2- LD ESI mass spectrum of -a12K2. Calculated value is 1293.1; measured value of [M+H]+ is 1294.1, and measured value of [M+2H]2+ is 647.6.

[0329] Figure 259 Nii-EG2- LD HPLC chromatogram of -a12K2.

[0330] Figure 260 ESI mass spectrum of Nii-SM-102. Calculated value: 911.7; Measured value of [M+H]+: 912.7; Measured value of [M+2H]2+: 457.0; Measured value of [M+3H]3+: 305.1.

[0331] Figure 261 HPLC chromatogram of Nii-SM-102.

[0332] Figure 262 ESI mass spectrum of Nii-ALC-0315. Calculated value: 967.8; Measured value of [M+H]+: 968.5.

[0333] Figure 263 HPLC chromatogram of Nii-ALC-0315. Detailed Implementation

[0334] To further understand the present invention, preferred embodiments will be described below with reference to examples. These descriptions are merely illustrative of the features and advantages of the present invention and are not intended to limit the scope of protection of the present invention.

[0335] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0336] In this invention, the singular forms “an,” “a,” and “the” are intended to include the plural forms. In this invention, the term “and / or” includes any and all combinations of one or more of the associated listed items. It should also be understood that when used in this specification, the terms “comprising” or “including” specify the presence of the stated features, steps, and / or elements, but do not exclude the presence or addition of one or more other features, steps, and / or elements. It should also be understood that when used, the terms “comprising” or “including” encompass the meaning of “consistently consisting of” or “comprises of.” The term “or a combination thereof” refers to a combination including at least one of the foregoing elements.

[0337] Unless otherwise stated, all figures used in this specification and claims should be understood to be modified by the term "about" in all cases. When used in this invention, the term "about" or "approximately," when applied to one or more values ​​of interest, refers to a value that is close to or within an acceptable error range of the stated reference value. In one instance, the term "about" refers to any value within a variation range of up to ±10% of the value modified by the term "about," including both integer and fractional components. Alternatively, according to practice in the art, "about" may mean within 3 or more standard deviations, within 5 times, or within 2 times.

[0338] As used herein, the term "alkyl chain" includes saturated and unsaturated alkyl chains, as well as linear, branched, or cyclic alkyl chains. In the definition of this invention, an "alkyl chain" may contain one or more linking groups L in addition to its carbon skeleton. The term "unsaturated alkyl chain" refers to an alkyl chain containing one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unsaturated bonds, such as carbon-carbon double or triple bonds. The term "alkyl chain" as used herein also includes optionally substituted alkyl chains, such as hydroxyl-substituted alkyl chains.

[0339] As used herein, the term "hydrocarbon group" refers to a group formed by removing one or more hydrogen atoms from a hydrocarbon molecule (hydrocarbon compound). In some embodiments, the "hydrocarbon group" of this invention refers to an aliphatic hydrocarbon group. In some embodiments, the "hydrocarbon group" of this invention refers to a linear or branched alkyl, alkenyl, or alkynyl group. For example, "saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms" of this invention refers to a linear or branched alkyl, alkenyl, or alkynyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. Representative "alkyl" groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl. Representative "alkenyl" groups include vinyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and 2,3-dimethyl-2-butenyl. Representative "alkynyl" groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

[0340] As used herein, the term "lower alkyl" refers to an alkyl group containing 10 or fewer, preferably 6 or fewer, carbon atoms, such as 1, 2 or 3 carbon atoms.

[0341] As used herein, the term “branching” or “branching” refers to a given structure, such as an alkyl chain, hydrocarbon group, or alkyl group, containing a tertiary carbon atom bonded to three other carbon atoms or a quaternary carbon atom bonded to four other carbon atoms.

[0342] As used herein, the term "optionally substituted" means that one to six hydrogen groups in a given structure are replaced by a specific substituent, including but not limited to hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, and nitro groups. Preferably, "optionally substituted" means "optionally hydroxyl-substituted." Preferably, 0, 1, 2, or 3 hydrogen groups in the alkyl chain are replaced by hydroxyl groups.

[0343] In this invention, the symbol "-" refers to a single bond, does not indicate any preferred stereochemistry, and covers all stereoisomers and mixtures thereof. When drawn perpendicularly through the bond, for example for ,symbol" "" indicates the connection point of the group.

[0344] Universal bases

[0345] As used herein, the term "universal base" or "UB" refers to a base analog, non-natural base, or derivative thereof that can interact or pair with many or all of the natural bases of adenine (A), thymine (T), uracil (U), guanine (G), and cytosine (C), and exhibits lower base selectivity relative to typical Watson-Crick base pairing. As used herein, the term "universal base derivative" refers to a compound formed by directly introducing a reactive functional group onto a universal base or UB as defined herein, or by introducing a reactive functional group via a linker.

[0346] As used herein, the term "universal base group" refers to a residue formed by the covalent attachment of a universal base or its derivative to an ionizable lipid backbone. This covalent attachment can be formed by the reaction of a reactive functional group in the universal base or its derivative with a reactive site in the ionizable lipid; the reactive functional group and the reactive site are as defined herein. The universal base group can be directly attached to the ionizable lipid backbone or attached to the ionizable lipid backbone via a spacer group. In some embodiments, the universal base group is derived from one or more universal bases or their derivatives selected from: hypoxanthines, purines, isoquinolinones, pyrimidines, etc. Azides, nitropyrroles, nitroindoles, nitroimidazolides, formylpyrroles, nitrobenzodioxanes, benzotriazoles, cyanuric acids, diaminotriazins, amidepyridines, pyrimidines, pteridine analogs, urea-based pyrimidinones, or their derivatives. In some embodiments, the universal base group may be derived from a universal base derivative having a carboxyl group, preferably a carboxylated universal base. The universal base for carboxylation is selected from: 1-carboxymethyl-3-nitropyrrole (Nip), 1-carboxymethyl-5-nitroindole (Nii), (2,4,6-trioxo-1,3,5-triazin-1-yl)acetic acid (Cya), 2,6-bis(acetamido)-4-pyridinecarboxylic acid (Dapy), 2-(4-nitroimidazol-1-yl)acetic acid (Nipu), 2-(2,4,5,7-tetraoxo-8H-pyrimidino[4,5-d]pyrimidin- 1-yl)acetic acid (Ppt), N-[[(1,4-dihydro-6-methyl-4-oxo-2-pyrimidinyl)amino]carbonyl]-glycine (Upy), hypoxanthine-9-acetic acid (Ino), 2-(9H-purin-9-yl)acetic acid (Pur), 2-(8-methyl-1-oxo-1,2-isoquinoline-2-yl)acetic acid (MICS), 1,3,4,7-tetrahydro-7-oxo-6H-pyrimidino[4,5-c][1,2] P-imino, (2-formyl-1-pyrrolidinyl)acetic acid (Fp), (6-nitro-1,3-benzodioxane-5-yl)acetic acid (Nipi), benzotriazol-1-acetic acid (Tap), 2-(4,6-diamino-1,3,5-triazin-2-yl)acetic acid (Dat), or any combination thereof.

[0347] In this invention, the term "reactive functional group" can also be referred to as "covalently coupled functional group," which refers to a functional group located on a universal base nucleus or its linker that can react with reactive sites in ionizable lipids to form a covalent bond. The reactive functional groups include, but are not limited to, carboxyl, amino, hydroxyl, mercapto, activated ester, acyl chloride, acid anhydride, chloroformate, activated carbonate, isocyanate, isothiocyanate, sulfonyl chloride, haloalkyl, methanesulfonate, p-toluenesulfonate, haloacetyl, epoxy, vinyl sulfone, acryloyl, methacryloyl, maleimide, aldehyde, ketone, aminooxy, hydrazyl, acylhydrazine, azide, alkynyl, cycloalkynyl, tetrazinyl, transcyclooctenyl, norbornyl, cyclopropenyl, and combinations thereof.

[0348] In this invention, the term "reactive site" refers to a functional group or structural unit located on an ionizable lipid that is capable of undergoing amidation, esterification, thioesterification, urea formation, thiourea formation, carbamate formation, nucleophilic substitution, Michael addition, epoxy ring opening, click reaction, thiol-ene addition, thiol-alkyne addition, oxime formation, hydrazone formation, reductive amination, borate ester formation, phosphate ester formation, or cycloaddition reactions to form a covalent bond. The reactions may optionally be carried out under conditions of coupling agent, activator, catalyst, reducing agent, base, light, or heat.

[0349] In this invention, the term "universal base group derived from universal base or its derivative" means that the universal base or its derivative exists as a universal base residue in the universal base-modified ionizable lipid after its reactive functional group reacts with a reactive site in the ionizable lipid.

[0350] Amino acid molecular building blocks

[0351] In this invention, the term "amino acid building block" refers to the basic monomers linked by amide bonds to form peptides or proteins, encompassing the amino acid building blocks of Formula I in this invention and any natural or other non-natural amino acid building blocks known in the art.

[0352] In some embodiments, the amino acid building blocks of the present invention are amino acid building blocks of Formula I. In some embodiments, the amino acid building blocks of the present invention are alkylated amino acid building blocks based on Nα-fluorenemethoxycarbonyl (Fmoc) protected primary amino groups (Alkylated iodizable Fmoc-protected Amino acid, AIFA). In some embodiments, the amino acid building blocks of the present invention are amino acid building blocks formed by alkylating the primary amino groups of lysine, ornithine, 2,4-diaminobutyric acid, or 2,3-diaminopropionic acid.

[0353] In this invention, the term "containing one or more linking groups L" means that the alkyl chain is interrupted or spaced apart by one or more linking groups L. For example, an alkyl chain containing one linking group L can be represented as "-alkyl or hydrogen-L-alkyl or hydrogen"; an alkyl chain containing two linking groups L can be represented as "-alkyl or hydrogen-L1-L2-alkyl or hydrogen" or "-alkyl or hydrogen-L1-alkyl-L2-alkyl or hydrogen", wherein the linking groups L or L1 and L2 are independently selected from amide bonds, ester bonds, disulfide bonds, ketethiolide bonds, ether bonds, or combinations thereof. In some embodiments, the alkyl chains A1 or A2 of this invention do not contain linking groups or contain 1, 2, 3, or 4 linking groups.

[0354] In this invention, the term "saturated alkyl chain" or "α-alkyl chain" refers to a linear or branched alkyl group having 4 to 25 carbon atoms and without any unsaturated bonds. In some embodiments, the saturated alkyl chain of this invention is an alkyl group that is not substituted with any substituents. In some embodiments, the saturated alkyl chain of this invention is -CH2CH2(CH2). q CH2CH3, where q is an integer from 0 to 21.

[0355] In this invention, the term "unsaturated alkyl chain" refers to a linear or branched hydrocarbon group having 4 to 25 carbon atoms and containing one or more, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unsaturated bonds, such as carbon-carbon double or triple bonds. Unsaturated alkyl chains that can be used as A1 or A2 of this invention are known in the art, see, for example, R1 and R2 in WO2010054406A1.

[0356] In this invention, the term "hydroxyl-containing alkyl chain" or "e-alkyl chain" refers to a linear or branched hydrocarbon group having 4 to 25 carbon atoms and being substituted with at least one hydroxyl group. In some embodiments, the hydroxyl-containing alkyl chain of this invention is a linear or branched alkyl group having 4 to 25 carbon atoms and being substituted with at least one hydroxyl group, for example, 1, 2, 3, 4, 5, or 6 hydroxyl groups. In some embodiments, the hydroxyl-containing alkyl chain of this invention is -CH2CHOH(CH2). q CH2CH3, where q is an integer from 0 to 21.

[0357] The alkyl chain of A1 or A2 of the present invention optionally further includes one or more linking groups L, said linking group L being independently selected from amide bonds, ester bonds, disulfide bonds, ketethiocarbamate bonds, ether bonds, or combinations thereof.

[0358] In some preferred embodiments, the alkyl chain of A1 or A2 of the present invention contains 0, 1, 2, 3, or 4 linking groups L. In some preferred embodiments, the linking group L can be a biodegradable group, such as an ester bond that is stable at physiological pH but is hydrolyzed by enzymes in tissues and cells, a disulfide bond that is sensitive to the reduced intracellular environment, or a ketthioglycol bond that responds to ROS. The effects of adding biodegradable groups to the alkyl chain are known in the art, see, for example, WO2011153493A2 and WO2013086354A1.

[0359] In this invention, the term "amide-bonded alkyl chain" or "aam alkyl chain" refers to an alkyl chain containing at least one amide bond (-C(=O)-NH- or -NH-C(=O)-). In this invention, when A1 or A2 is an amide-bonded alkyl chain, the number of carbon atoms in A1 or A2 refers to the number of carbon atoms of the hydrocarbon group attached to the amide bond. In some embodiments, the amide-bonded alkyl chain of this invention is -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, wherein each B1 is absent or independently a optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms, wherein L is absent or independently a linking group selected from amide bonds, ester bonds, disulfide bonds, ketethiolide bonds, ether bonds, or combinations thereof, and B2 is an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms. In some embodiments, the amide-bonded alkyl chain of this invention is -CH2CH2CONHCH2CH2(CH2). q CH2CH3 or -CH2CH2NHCOCH2CH2(CH2) q CH2CH3, where q is an integer from 0 to 21.

[0360] In this invention, the term "ester-bonded alkyl chain" or "aat alkyl chain" refers to an alkyl chain containing at least one ester bond (-C(=O)-O- or -OC(=O)-). In this invention, when A1 or A2 is an ester-bonded alkyl chain, the number of carbon atoms in A1 or A2 refers to the number of carbon atoms of the hydrocarbon group attached to the ester bond. In some embodiments, the ester-bonded alkyl chain of this invention is -B1-COO-B1-L-B2 or -B1-OOC-B1-L-B2, wherein each B1 is absent or independently an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms, wherein L is absent or independently a linking group selected from amide bonds, ester bonds, disulfide bonds, ketethiolide bonds, ether bonds, or combinations thereof, and B2 is an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms. In some embodiments, the ester-bonded alkyl chain of this invention refers to -CH2CH2COOCH2CH2(CH2). q CH2CH3 or -CH2CH2OOCCH2CH2(CH2) q CH2CH3, where q is an integer from 0 to 21.

[0361] In this invention, the term "alkyl chain containing disulfide bonds" refers to an alkyl chain containing at least one disulfide bond (-SS-), i.e., -(B1-L). s At least one L in -B2 is a disulfide bond. The term "ketothiol-containing alkyl chain" refers to an alkyl chain containing at least one ketothiol bond (-SC(CH3)2-S-), i.e., -(B1-L). sAt least one L in -B2 is a ketthiolated bond. The term "branched alkyl chain" refers to an alkyl chain containing at least one branched hydrocarbon group, such as -(B1-L). s At least one of B1 or B2 in -B2 is a branched hydrocarbon group.

[0362] In this invention, the term "amino protecting group" refers to any chemical group that stabilizes the amino group of amino acid building blocks in chemical reactions such as peptide synthesis and is easily removed. In this invention, any amino protecting group known in the art can be used, including but not limited to alkoxycarbonyl amino protecting groups, acyl amino protecting groups, or alkyl amino protecting groups. Common amino protecting groups include, but are not limited to, fluorenemethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Trt), p-toluenesulfonyl (Tos), phthaloyl (Pht), N-1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl (Dde), allyloxycarbonyl (Alloc), etc. In some embodiments, the "amino protecting group" of this invention is Fmoc.

[0363] The peptide-based ionizable lipids of the present invention may comprise at least one amino acid molecule building block having the structure of Formula I:

[0364] Formula I

[0365] in, m is an integer from 0 to 10, preferably from 1 to 4. A1 and A2 are hydrophobic tails and are independently substituted saturated or unsaturated, linear or branched alkyl chains having 4 to 25 carbon atoms, wherein the alkyl chains optionally contain one or more linking groups L, the linking groups L being selected from amide bonds, ester bonds, disulfide bonds, ketithiolide bonds, ether bonds, or combinations thereof. A3 is an amino protecting group, and A4 represents a hydrogen atom or an alkyl group.

[0366] In this invention, m is an integer from 0 to 10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 1, meaning the amino acid building block of the present invention is an amino acid building block in which the primary amino group of the side chain of 2,3-diaminopropionic acid (Dap) is alkylated. In some embodiments, m is 2, meaning the amino acid building block of the present invention is an amino acid building block in which the primary amino group of the side chain of 2,4-diaminobutyric acid (Dab) is alkylated. In some embodiments, m is 3, meaning the amino acid building block of the present invention is an amino acid building block in which the primary amino group of the side chain of ornithine is alkylated. In some embodiments, m is 4, meaning the amino acid building block of the present invention is an amino acid building block in which the primary amino group of the side chain of lysine is alkylated. In some embodiments, m is preferably 1, 2, 3, or 4, more preferably 2, 3, or 4.

[0367] In some embodiments, A1 or A2 has 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. In some embodiments, when A1 and A2 contain one or more linking groups L, the number of carbon atoms in the alkyl chain refers to the number of carbon atoms contained in the hydrocarbon group following the last linking group. In some embodiments, when A1 and A2 are alkyl chains containing amide bonds or alkyl chains containing ester bonds, the number of carbon atoms refers to the number of carbon atoms contained in the hydrocarbon group following the amide bond or ester bond.

[0368] In some preferred embodiments, the alkyl chain of A1 or A2 of the present invention contains 0, 1, 2, 3, 4, 5, or 6 linking groups L. In some preferred embodiments, the linking group L may be a biodegradable group, such as an ester bond that is stable at physiological pH but is hydrolyzed by enzymes in tissues and cells, a disulfide bond that is sensitive to the reduced intracellular environment, or a ketithiolide bond that responds to ROS.

[0369] In some embodiments, the amino protecting group of the present invention is fluorenyl methoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), p-methoxybenzyl (PMB), benzyl (Bn), triphenylmethyl (Trt), p-toluenesulfonyl (Tos), phthaloyl (Pht), N-1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl (Dde), or allyloxycarbonyl (Alloc).

[0370] In some embodiments, A4 is a hydrogen atom or an alkyl group. Preferably, A4 is a hydrogen atom or a lower alkyl group, such as methyl, ethyl, or propyl.

[0371] In some implementations, A1 and A2 are independently of each other -(B1-L). s-B2, where s is an integer from 0 to 6, for example 0, 1, 2, 3, 4, 5 or 6, where B1 is independently absent in each (B1-L) unit or is an optional substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms, L is independently absent in each (B1-L) unit or is a linking group selected from amide bonds, ester bonds, disulfide bonds, ketethiocarbamate bonds, ether bonds or combinations thereof, and B2 is an optional substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms.

[0372] In some embodiments, B1 is absent in the (B1-L) unit, i.e., the (B1-L) unit is -L-. In some embodiments, B1, each time it appears, is independently a saturated or unsaturated hydrocarbon group with 1 to 25 optionally substituted carbon atoms, preferably B1 is a linear or branched alkyl, alkenyl, or alkynyl group with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 optionally substituted carbon atoms. In some embodiments, B1 is hydroxylated.

[0373] In some embodiments, L is absent in the (B1-L) unit, i.e., the (B1-L) unit is B1. In some embodiments, L, each time it appears, is independently a linking group selected from amide bonds, ester bonds, disulfide bonds, ketethiocarbamate bonds, ether bonds, or combinations thereof.

[0374] In some embodiments, B2 is an optionally substituted saturated or unsaturated hydrocarbon group having 1 to 25 carbon atoms, preferably a linear or branched alkyl, alkenyl, or alkynyl group having substituted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. In some embodiments, B2 is hydroxyl-substituted.

[0375] In some embodiments, A1 and A2 are independently -B1-L-B2, -B1-CONH-B1-L-B2, -B1-NHCO-B1-L-B2, -B1-COO-B1-L-B2 or -B1-OOC-B1-L-B2, wherein each B1 is absent or independently a saturated or unsaturated hydrocarbon group having 1 to 25 optionally substituted carbon atoms, wherein L is absent or independently a linking group selected from amide bonds, ester bonds, disulfide bonds, ketethiolide bonds, ether bonds or combinations thereof, and B2 is a saturated or unsaturated hydrocarbon group having 1 to 25 optionally substituted carbon atoms.

[0376] In some embodiments, A1 and A2 are independently selected from: saturated alkyl chains, unsaturated alkyl chains, hydroxyl-containing alkyl chains, amide-containing alkyl chains, ester-containing alkyl chains, disulfide-containing alkyl chains, ketethiolated alkyl chains, and branched alkyl chains.

[0377] In some implementations, A1 and A2 are independently selected from: -CH2CH2(CH2) q CH2CH3, -CH2CHOH(CH2) q CH2CH3, -CH2CH2CONHCH2CH2(CH2) q CH2CH3, -CH2CH2NHCOCH2CH2(CH2) q CH2CH3, -CH2CH2COOCH2CH2(CH2) q CH2CH3, -CH2CH2OOCCH2CH2(CH2) q CH2CH3, where q is an integer from 0 to 21, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, preferably an integer from 4 to 10.

[0378] In some implementations, A1 and A2 are the same.

[0379] In some embodiments, the amino acid building blocks of the present invention may be any amino acid building blocks disclosed in CN118852328A and CN120040543A, which are incorporated herein by reference.

[0380] Peptide-based ionizable lipids (PILs)

[0381] In this invention, the term "peptide-based ionizable lipid" or "PIL" refers to peptide-based ionizable lipids composed of alkylated amino acid building blocks of this invention and optionally natural or other non-natural amino acid building blocks linked by amide bonds.

[0382] In this invention, the term "amino acid" includes both D-type and L-type amino acids. In this invention, the term "natural amino acid" refers to amino acids naturally occurring in living organisms, including 20 different amino acids commonly found in proteins: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In this invention, the terms "unnatural amino acid" and "non-canonical amino acid" are also used. "Artificial amino acids" refers to amino acids other than the 20 common amino acids mentioned above that are not encoded by the genetic code in living organisms. These include derivatives of phenylalanine, tyrosine, glutamine, alanine, cysteine, serine, and lysine, such as N-ethylβ-alanine (Neb), sarcosine (Sar), α,β-diaminopropionic acid (Adp), β-amino-N-butyric acid (aBut or Bab), β-aminoisobutyric acid (Bai), α-aminoisobutyric acid (Aib), γ-aminobutyric acid (Gab), and α-aminobutyric acid (Anb). ), N-methylalanine (Nma), N-ethylglycine (Neg), allothreonine (Alt), Hse, 4-amino-3-hydroxybutyric acid (Hga), 2,4-diaminobutyric acid (Dab), hydroxyproline (Hyp), isovaleine (Iva), normal valine (Nor), L-cyclopropylglycine (Cpg), N-propylglycine (Npg), homocysteine ​​(Hcy), piperidine acid (Pip), ornithine (Orn), tertiary leucine (Tle), alloisoleucine (Ali), normal leucine (Nle), 2-aminoheptanoic acid (Ahe), citrulline (Cit).

[0383] In this invention, the term "basic amino acid" refers to an amino acid that has a net positive charge at neutral pH, such as lysine, arginine, and histidine.

[0384] The term "acidic amino acid" refers to amino acids that have a net negative charge at neutral pH, such as glutamic acid and aspartic acid.

[0385] The ionizable lipids of the present invention can be peptide-based ionizable lipids (PILs) having the structure of Formula II below.

[0386] Formula II, in, A4 is defined as described in the amino acid building block section above. n is an integer from 1 to 30. X is O or S. R1 represents the N-terminus of the PIL, and R1 is a hydrogen atom or modified with an acetyl group, amino acid, and / or other functional groups. R2 represents the side group contained in the PIL. Each time R2 appears, it is independently a side group of the amino acid building block as defined in the amino acid building block section above. Natural or non-natural amino acid side groups, and at least one R2 in the PIL is , R3 represents the C-terminus of the PIL, and R3 is a hydroxyl group or has an amino group, an amino acid group, and / or other functional group modification.

[0387] In some implementations, R2 represents the side groups contained in the PIL, in each In the unit, R2 is independently selected from the side groups of the amino acid building blocks as defined in the amino acid building block section above. Natural amino acid side groups or non-natural amino acid side groups.

[0388] In some embodiments, at least 1, 2, 3, 4, or 5 R2s in the PIL of the present invention are side groups of the amino acid building blocks as defined in the amino acid building block section above. .

[0389] In some embodiments, the side groups of natural amino acids are, for example, hydrogen atoms, methyl groups, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)CH2CH3, -CH2CH2SCH3, -CH2C6H5, -CH2C6H4OH, -CH2C8H6N, -CH2OH, -CH(OH)CH3, -CH2SH, -CH2COOH, -CH2CH2COOH, -CH2CONH2, -CH2CH2CONH2, -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CH2C3H3N2, or -CH2CH2CH2- (which forms a ring structure with the amino group).

[0390] In some embodiments, non-natural amino acid side groups refer to non-natural amino acid side groups other than those formed by the alkylation of primary amino groups as defined in the amino acid building block portion of this invention, such as N-ethyl-β-alanine (Neb), sarcosine (Sar), α,β-diaminopropionic acid (Adp), β-amino-N-butyric acid (aBut or Bab), β-aminoisobutyric acid (Bai), α-aminoisobutyric acid (Aib), γ-aminobutyric acid (Gab), α-aminobutyric acid (Anb), N-methylalanine (Nma), N-ethyl-β-alanine (Nma), etc. Amino acid (Neg), allothreonine (Alt), Hse, 4-amino-3-hydroxybutyric acid (Hga), 2,4-diaminobutyric acid (Dab), hydroxyproline (Hyp), isovaleine (Iva), normal valine (Nor), L-cyclopropylglycine (Cpg), N-propylglycine (Npg), homocysteine ​​(Hcy), piperidine acid (Pip), ornithine (Orn), tertiary leucine (Tle), alloisoleucine (Ali), normal leucine (Nle), 2-aminoheptanoic acid (Ahe), or citrulline (Cit) amino acid side groups.

[0391] In some implementations, n is an integer from 1 to 30, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably n is 1, 2, 3, 4 or 5.

[0392] In some embodiments, A1 and A2 are identical within the same amino acid building block monomer. In some embodiments, A1 and A2 are both amino acid building block monomers containing hydroxyalkyl chains, with the number being less than or equal to two.

[0393] In some embodiments, A1 and A2 are alkyl chains with 8-14 carbon atoms and n is 3-5. Preferably, A1 and A2 are alkyl chains with 12 carbon atoms and n is 3 or 4. More preferably, n is 3-5, m is 2-4, and A1 and A2 are saturated alkyl chains with 10-14 carbon atoms. More preferably, n is 4, m is 2, and A1 and A2 are saturated alkyl chains with 12 carbon atoms.

[0394] In some embodiments, in the PIL of the present invention: (i) n is 4-5, (ii) n is 3 and A1 and A2 are alkyl chains with 10-14 carbon atoms. (iii) n is 2 and A1 and A2 are alkyl chains with 16-18 carbon atoms. (iv) A1 and A2 are saturated alkyl chains with 12 carbon atoms or hydroxyl-containing alkyl chains, and n is 3-5. (v) Where A1 and A2 are alkyl chains containing amide bonds with 12 carbon atoms, and n is 3-5, or (vi) A1 and A2 are alkyl chains containing ester bonds with 12 carbon atoms.

[0395] In some embodiments, the PIL has a free amino group (R1 represents -H) or an acetylation modification (R1 represents -COCH3) at its N-terminus. In some embodiments, the PIL has both acetylation and amino acid modification (R1 represents -(aa)x-COCH3) or amino acid modification and a free amino group (R1 represents -(aa)x) at its N-terminus, wherein (aa)x is one or more amino acid residues. In some embodiments, the PIL has a free carboxyl group (R3 represents -OH) or an amidation modification (R3 represents -NH2) at its C-terminus. In some embodiments, the PIL has both amidation and amino acid modification (R2 represents -(aa)x-NH2) or amino acid modification and a free carboxyl group (R2 represents -(aa)x) at its C-terminus, wherein (aa)x is one or more amino acid residues.

[0396] In some embodiments, the amino acids modifying the N-terminus and / or C-terminus of the PIL are natural or non-natural amino acids. In some embodiments, the amino acids modifying the N-terminus and / or C-terminus of the PIL are natural amino acids, including but not limited to alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0397] In some embodiments, the PIL has 1 to 10 amino acid modifications at the N-terminus and / or C-terminus, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications.

[0398] In some embodiments, the other functional modifications are any organ-targeting small molecules. In some embodiments, the other functional group modifications refer to phosphorylation modifications, preferably bisphosphonate modifications, more preferably alendronate modifications, such as succinylated alendronate modifications, for example... .

[0399] In some implementations, (a) The PIL has basic amino acid modification, preferably with basic amino acid modification and acetylation modification at the N-terminus; (b) The PIL has a basic amino acid modification and a free amino group at its N-terminus; (c) The PIL has a proline modification, preferably with a proline modification and a free amino group at the N-terminus; (d) The PIL has an amino acid modification selected from cysteine, histidine, tyrosine, phenylalanine or a combination thereof, preferably having an amino acid modification and acetylation modification selected from cysteine, histidine, tyrosine, phenylalanine or a combination thereof at the N-terminus. (e) The PIL is modified with tryptophan, preferably with tryptophan and acetylation modification at the N-terminus; (f) The PIL has an amino acid modification selected from alanine, asparagine, glutamic acid, glycine, isoleucine, leucine, methionine, proline, serine, threonine, valine or a combination thereof, preferably having an amino acid modification and acetylation modification selected from alanine, asparagine, glutamic acid, glycine, isoleucine, leucine, methionine, proline, serine, threonine, valine or a combination thereof at the N-terminus; (g) The PIL has acidic amino acid modification, preferably with acidic amino acid modification and a free carboxyl group at the C-terminus; or (h) The PIL has phosphorylation modification, for example, phosphorylation modification at the N-terminus or C-terminus, preferably bisphosphonate modification, more preferably alendronate modification.

[0400] In some embodiments, the peptide-based ionizable lipids of the present invention may be any peptide-based ionizable lipids disclosed in CN118852328A and CN120040543A, which are incorporated herein by reference.

[0401] lipid nanoparticles

[0402] In this invention, the term "lipid nanoparticles" refers to particles containing one or more lipids and having a nanoscale size (e.g., 1-1,000 nm).

[0403] In some embodiments, the lipid nanoparticles of the present invention comprise the peptide-based ionizable lipids of the present invention, optional auxiliary lipids, and optional active agents.

[0404] In some embodiments, the lipid nanoparticles of the present invention may have an average particle diameter between about 50 nm and about 200 nm, for example, between about 100 nm and about 120 nm or between about 150 nm and about 190 nm. In some embodiments, the surface charge of the lipid nanoparticles of the present invention may be between about -20 mV and about +20 mV, for example, between about +3 mV and about +7 mV, between about 0 mV and about 5 mV, or between about -3 mV and about +1 mV. In some embodiments, the polydispersity index (PDI) of the lipid nanoparticles of the present invention is between about 0 and about 0.3, for example, between about 0.1 and about 0.2. In some embodiments, the apparent acid dissociation constant of the lipid nanoparticles of the present invention is between about 5 and about 8, for example, greater than about 7.3, between about 6.5 and about 7.3, or between about 6 and about 6.5.

[0405] In this invention, the terms "ionizable lipid" or "cationic lipid" refer to lipids that have a net positive charge at a selected pH, such as physiological pH. In some embodiments, in addition to the peptide-based ionizable lipids of this invention, the lipid nanoparticles of this invention optionally also comprise other ionizable lipids, such as (heptadecane-9-yl-8-((2-hydroxyethyl)(6-keto-6-(undecyloxy)hexyl)amino)octanoate) (SM-102), (4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), (2,3-dioleoylpropyl)trimethylamine (DOTAP), etc.

[0406] As used herein, the term "ionizable lipid backbone" refers to the lipid portion of an ionizable lipid that, after reacting at its reactive sites with reactive functional groups in a universal base or a universal base derivative, remains in a universally modified ionizable lipid. The reactive sites can, after the reaction, be transformed into part of a covalently linked structure connecting the ionizable lipid backbone to the universal base group.

[0407] The ionizable lipids or peptide-based ionizable lipids may be included in the lipid nanoparticles in varying amounts. For example, based on total lipids, the lipid nanoparticles may include about 10 mol% to about 100 mol%, such as about 10 mol% to about 70 mol%, about 20 mol% to about 60 mol%, or about 30 mol% to about 50 mol% of ionizable lipids or peptide-based ionizable lipids.

[0408] In some embodiments, the term "active agent" as used in this invention refers to any active substance intended for delivery via lipid nanoparticles, such as therapeutic agents, immunomodulators, etc. In some embodiments, the active agent is a nucleic acid molecule, such as an antisense oligonucleotide (ASO), mRNA, siRNA, guide RNA (gRNA), viral vector, etc. In some embodiments, the active agent is a gene therapeutic agent. In some embodiments, the gene therapeutic agent is a CRISPR-based gene editing element such as a lead editing element or a chimeric antigen receptor T immunotherapeutic agent. In some embodiments, the active agent is PEmax mRNA and engineered lead editing guide RNA (epegRNA). In some embodiments, the mass ratio of PEmax mRNA to epigRNA in the active agent is 10:1 to 1:10, for example, 1:2 to 2:1, or 1:1. In some embodiments, the mass ratio of total lipids to the active agent in the LNP is about 1-100:1, for example, about 20-80:1, for example, about 40:1. In some embodiments, the amount of active agent in the LNP is from about 1 ng to about 10 μg, for example, from about 400 ng to 1 μg, about 500 ng, or about 600 ng.

[0409] In this invention, the term "auxiliary lipid" refers to lipids in lipid nanoparticles, other than cationic lipids, that contribute to their stability and delivery efficiency. Examples of auxiliary lipids in this invention include, but are not limited to, phospholipids, steroids, and PEG lipids.

[0410] In this invention, examples of phospholipids include, but are not limited to, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal). The phospholipids may be included in the lipid nanoparticles in varying amounts. For example, based on total lipids, the lipid nanoparticles may comprise about 0 mol% to about 30 mol%, for example, about 5 mol% to about 30 mol%, or about 8 mol% to about 15 mol% of phospholipids. In some embodiments, the lipid nanoparticles may comprise a molar ratio of phospholipids to peptide-based ionizable lipids of about 1:10 to about 1:20. In some embodiments, the molar ratio is about 1:5, 2:9, 1:4, 1:2, 8:9, 1:1, 4:3, 2:1, 3:1, 4:1, 6:1, 8:1, to about 10:1.

[0411] In this invention, examples of steroids include, but are not limited to, cholesterol and its derivatives, ergosterol, lanosterol, stigmasterol, and sitosterol. Examples of cholesterol derivatives include, but are not limited to, 5α-cholesterol, 5β-codactyl, cholesterol-(2'-hydroxy)-ethyl ether, cholesterol-(4'-hydroxy)-butyl ether, 6-ketocholesterol, 5α-cholesterol, cholesterolenone, 5α-cholesterolone, and 5β-cholesterolone. The steroids may be included in the lipid nanoparticles in varying amounts. For example, based on total lipids, the lipid nanoparticles may include approximately 0 mol% to approximately 70 mol%, such as approximately 10 mol% to approximately 70 mol%, approximately 20 mol% to approximately 60 mol%, and approximately 30 mol% to approximately 50 mol% of steroids. In some embodiments, the lipid nanoparticles may contain a molar ratio of steroids to peptide-ionizable lipids of approximately 1:4 to approximately 8:1. In some embodiments, the molar ratio is about 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, to about 8:1, or any range thereof. In some embodiments, the molar ratio is about 1:1 to about 6:1, such as 2:1 or 3:1.

[0412] In this invention, PEG lipids refer to any complex of polyethylene glycol (PEG) and lipids, examples of which include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, and PEG-modified diacylglycerol. In some embodiments, the PEG lipid is PEG-modified distearate phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol-methoxy(PEG)MW 2000 (DMG-PEG2000). In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the PEG-modified molecular weight is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. The PEG lipids may be included in the lipid nanoparticles in different amounts. For example, based on total lipids, the lipid nanoparticles may include about 0 mol% to about 10 mol%, such as about 0.01 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 1 mol% to about 2 mol% of PEG lipids. In some embodiments, the lipid nanoparticles may comprise a molar ratio of PEG lipids to peptide-based ionizable lipids of about 1:1 to about 1:100. In some embodiments, the molar ratio is about 1:1, 3:5, 1:2, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, to about 1:100.

[0413] In some embodiments, the LNP of the present invention is an organ or tissue-targeting LNP, such as a spleen-targeting LNP, a liver-targeting LNP, a lung-targeting LNP, or a bone-targeting LNP. "Targeting" or "specificity" in the present invention means that when the LNP is delivered to an organism, its expression rate in a specific organ or tissue exceeds 50%, for example, exceeds 70%, more preferably exceeds 90%. Alternatively, "target organ targeting" in the present invention means that when administered in vivo, the lipid nanoparticles are delivered to a target organ at least 25%, at least 50%, at least 75%, at least 80%, or at least 90% of the administered amount. "Dual targeting" in the present invention means that when administered in vivo, the lipid nanoparticles are delivered to each of two target organs at at least 25%, at least 30%, at least 40%, or at least 45% of the administered amount.

[0414] In some embodiments, the LNP of the present invention is a guided LNP (PILOT LNP, Peptide Ionizable Lipid-driven Organ Targeting lipid nanoparticle). Specifically, a PILOT LNP refers to a PIL-mediated, in vivo organ-targeting LNP that requires no additional ligands or lipid molecules.

[0415] Materials and Methods

[0416] Fmoc- L -Lys-OH, Fmoc- D -Lys-OH, Fmoc- L -Orn-OH, Fmoc- L -Dab-OH, Fmoc- L -Dap-OH, Fmoc-protected natural amino acids, Fmoc-protected spacers, aldehydes, sodium triacetoxyborohydride, natural / universal base monomers, cGAMP (cyclic guanosine monophosphate-adenosine monophosphate), PyBOP (benzotriazine-1-yloxytripyrrolidinylphosphonium hexafluorophosphate), HOBt (hydroxybenzotriazine), triisopropylsilane, ethylenedithiol, Rink Amide AM resin, and all lyophilization protectants were purchased from Aladdin Scientific (Shanghai, China). Cholesterol, 1,2-dimyristoyl- rac -Glyceryl-3-methoxy(poly(ethylene glycol))-2000 (DMG-PEG2000), 1,2-distearate- sn 1,2-Dioleoyl-3-phosphocholine (DSPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl- sn -glycerol-3-phosphate- L - Serine (DOPS), DiA, DLin-MC3-DMA (MC3), cKK-E12, SM-102, and ALC-0315 were obtained from Sinopec (Xiamen, China). D-fluorescein potassium was purchased from Meilunbio (Dalian, China). Triton X-100 was purchased from Innochem (Beijing, China). 9-(2,2-Dicyanovinyl)julonidine (DCVJ), 4',6-diamidinyl-2-phenylindole (DAPI) dihydrochloride, Roswell Park Memorial Institute 1640 (RPMI-1640) medium, Dulbecco modified Eagle medium (DMEM), trypsin-EDTA, fetal bovine serum (FBS), penicillin, streptomycin, 6-( pSodium 2-naphthalenesulfonate (TNS), 1,2-dioleoyl- sn -glycerol-3-phosphate ethanolamine-N-(7-nitro-2-1,3-benzo[] Diazol-4-yl) (ammonium salt) (NBD-PE), 1,2-dioleoyl- sn Glycerol-3-phosphate ethanolamine-N-(Lisylamine Rhodamine B sulfonyl) (ammonium salt) (Rho-PE) and the Pur-A-Lyzer Midi Dialysis Kit (MWCO, 3.5 kDa) were purchased from Sigma-Aldrich (USA). Mouse serum was obtained from Mreda (Beijing, China). The Quant-iT RiboGreen RNA assay kit was purchased from Invitrogen (USA). FITC-UTP was purchased from APExBIO (USA). Luciferase mRNA, RSV antigen mRNA, H1N1 antigen mRNA, RVG antigen mRNA, Cas9 mRNA, and ABEmax mRNA were synthesized using in vitro transcription (IVT). Chemically modified single-guide RNA (sgRNA) was purchased from Integrated DNA Technologies (USA). Chemically modified siRNA was purchased from GenScript (Nanjing, China). Peptide nucleic acid (PNA) was obtained from Sangon Biotech (Shanghai, China), and phosphoryldiamine morpholino oligomer (PMO) was purchased from WuXi TIDES (Shanghai, China). All ELISA kits used for quantification of protein and IgG antibody titers were purchased from Vazyme Biotech (Nanjing, China).

[0417] SPSS for universal base-modified PIL (UB-PIL).

[0418] Universally modified base peptides (UB-PIL) were manually synthesized on Rink Amide AM resin using standard Fmoc-based solid-phase peptide synthesis (SPPS). For the conjugation of the AIFA building block, the coupling step used 4.0 eq AIFA, 4.0 eq PyBOP, 4.0 eq HOBt, and 10.0 eq DIPEA in dichloromethane / dimethylformamide (DCM / DMF, 1:1 v / v, 10 mL g) containing 1% Triton X-100. -1 The resin was subjected to a solvent mixture for 2 h. The resin was incubated with a 20% piperidine DMF solution (3 × 15 min, 10 mL g) for 2 h. -1Fmoc deprotection was achieved using resin. Between each coupling and deprotection step, the resin was washed sequentially with DMF (3×) and DCM (3×), and the reaction completion was monitored by a Kaiser assay. Following final Fmoc deprotection, the N-terminal amino group was conjugated to a universal base (UB) monomer. This final capping step used 4.0 eq UB monomer, 4.0 eq PyBOP, 4.0 eq HOBt, and 8.0 eq DIPEA, in DCM / DMF (1:1 v / v, 10 mL g / ml). -1 The reaction was carried out in the resin for 1 h. Finally, a pre-cooled pyrolysis mixture (TFA / H2O / triisopropylsilane, 95:2.5:2.5 v / v / v; 10 mL g) was used. -1 The synthesized UB-PIL was pyrolyzed from the resin for 90 min. The pyrolysis solution was concentrated under reduced pressure to obtain crude UB-PIL. For in vivo studies, UB-PIL was further purified by column chromatography using DCM / methanol (10:1 v / v) as the eluent.

[0419] mRNA is synthesized through in vitro transcription.

[0420] The coding sequences for luciferase (Luc), RSV antigen, H1N1 antigen, RVG antigen, Cas9 protein, and ABEmax protein were amplified by polymerase chain reaction (PCR) and subsequently cloned into plasmid vectors flanked by optimized 5'- and 3'-untranslated regions (UTRs) and poly(A) tail sequences. The corresponding mRNAs were generated via in vitro transcription (IVT) using the T7 High Yield RNA Transcription Kit (Vazyme). To minimize intrinsic immunogenicity and enhance translation efficiency, native uridine-5'-triphosphate was completely replaced with N1-methylpseudouridine-5'-triphosphate. Furthermore, according to the manufacturer's protocol, co-capping was achieved by incorporating a 3-OH AG cap analog (Glycogene) into the transcription mixture to generate the Cap 1 structure. The coding sequences of the mRNAs used in this invention are provided in Table 2.

[0421] AllSet mRNA-LNP based on UB-PIL was prepared by post-assembly.

[0422] Empty LNPs were initially prepared using standard microfluidic mixing techniques. The aqueous phase consisted only of nuclease-free water without mRNA, while the organic phase contained a mixture of lipids dissolved in ethanol (UB-PIL / cholesterol / DSPC / DMG-PEG2000, molar ratio 46.3:9.4:42.7:1.6). The two phases were rapidly mixed at a flow rate of 3:1 (water:ethanol, v / v). After incubation at RT for 15 min, the resulting empty LNPs were dialyzed against nuclease-free water to remove residual ethanol. For cold chain-free storage, the empty LNPs and mRNA were lyophilized independently. A predetermined amount of lyophilization protectant was added to the dialyzed empty LNPs, followed by supplementation as needed. Figure 8 The freeze-drying cycle was performed as shown. In parallel, the same amount of freeze-drying protectant was added to the aqueous mRNA solution and freeze-dried to produce a stable dry cake. After the freeze-drying process was complete, all vials containing the dry cake were backfilled with high-purity nitrogen to prevent oxidative degradation during storage at ambient temperature.

[0423] The final AllSet mRNA-LNPs are prepared immediately before application via a rapid, user-friendly post-assembly process. In short, the lyophilized mRNA cake is first reconstructed in nuclease-free water. This aqueous mRNA solution is then added to a vial containing empty lyophilized AllSet LNPs. The mixture is briefly vortexed at 4°C, 15°C, 25°C (RT), or 35°C for 5–10 s, which triggers spontaneous self-assembly and encapsulation of the mRNA. For in vivo application, the resulting AllSet mRNA-LNPs are diluted with buffer to produce an isotonic solution suitable for injection.

[0424] A multifunctional Nii-modified AllSet LNP system was prepared by post-assembly.

[0425] To demonstrate the broad applicability of the UB-guided post-assembly strategy, four different types of LNP systems were formulated using the general scheme described above. The molar ratios of the lipid components (ionizable lipids:DSPC:cholesterol:DMG-PEG2000) are as follows: (1) Nii-PILOT system: organ-selective LNPs were formulated using Nii-modified lung PILOT (Am-K2K(Nii)a12K4) or spleen PILOT (a12K4K(Nii)E-Ca) lipids in a molar ratio of 46.3:9.4:42.7:1.6; (2) Nii-modified baseline system: baseline ionizable lipids were chemically modified with Nii. The formulations include Nii-SM-102 (50:10:38.5:1.5), Nii-ALC-0315 (46.3:9.4:42.7:1.6), and Nii-cKK-E12 (35:16:46.5:2.5); (3) UB-lipid doping system (UB-lipid used as the fifth component): Nii-lipid is incorporated as the fifth component into the classic MC3 (50:10:38.5:1.5) and LP-01 (45:9:44:2) LNP formulations. Nii-lipid is added at molar ratios of 0%, 0.25%, 0.5%, 1%, 2.5%, and 5% relative to the total lipid content; (4) UB-based SORT system: For selective organ-targeting (SORT) LNPs, specific SORT molecules are supplemented into the Nii-lipid formulation (46.3:9.4:42.7:1.6). Specifically, lung SORT LNPs include DOTAPs, which account for 25% of the total lipid molars, while spleen SORT LNPs include DOPSs, which account for 15% of the total lipid molars.

[0426] Nanoparticle tracking analysis (NTA).

[0427] The hydrodynamic diameter and particle concentration of Nip-a12Dab4 AllSet LNPs with or without mRNA addition were quantified using a NanoSight NS300 instrument (Malvern Panalytical). To evaluate the post-assembly process, lyophilized empty Nip-a12Dab4 AllSet LNPs were reconstituted with nuclease-free water or aqueous mRNA solution, followed by brief vortexing. All samples were diluted with 1×PBS (1:5,000) to achieve a final concentration of 10⁻⁶ mcg / mL prior to analysis. 7 Up to 10 9 Particles mL -1 The optimal concentration range for measurement was determined. Measurements were performed at 25°C. For each sample, three independent video segments were acquired under constant flow using a syringe pump. Data acquisition and subsequent analysis were performed using NanoSight NTA software (version 3.44), where camera level and detection threshold were optimized and kept constant across all measurements.

[0428] FRET elimination assay.

[0429] To investigate the fundamental mechanisms of mRNA encapsulation, fluorescence resonance energy transfer (FRET) elimination assays were performed. FRET-labeled empty AllSet LNPs were prepared by incorporating lipophilic FRET pairs DiA (donor, 0.5 mol%) and DiD (acceptor, 0.5 mol%) into a lipid mixture before microfluidic mixing. Unlabeled empty LNPs were prepared in parallel and subsequently combined with FRET-labeled LNPs at a 9:1 (unlabeled:labeled) molar ratio. Trehalose was added as a lyophilization protectant (final concentration: 80 mg / mL), and the mixed empty LNPs were lyophilized. For post-triggered assembly, aqueous mRNA solution (pH 6.8) was added to rehydrate and lyophilize the LNPs, followed by brief vortexing at RT for 5–10 s. The fluorescence intensity of DiA was then quantified using a Tecan microplate reader at excitation and emission wavelengths of 545 nm and 585 nm, respectively. To establish a baseline for complete FRET elimination, a simulated complete fusion control was prepared, consisting of empty LNPs containing 0.05 mol% DiA and 0.05 mol% DiD. The degree of LNP fusion was determined by calculating the DiA fluorescence of the experimental group relative to the FRET elimination control.

[0430] Single-particle analysis of mRNA encapsulation was performed using nanoflow cytometry.

[0431] To validate mRNA encapsulation into empty AllSet LNPs at the single-particle level, nanoflow cytometry was employed. Fluorescently labeled empty LNPs were prepared by incorporating the lipophilic dye DiA (0.5 mol%) into a lipid mixture prior to microfluidic assembly and subsequent lyophilization. To initiate the assembly process, lyophilized DiA-labeled empty LNPs were reconstituted using aqueous solutions containing or without FITC-labeled mRNA, followed by brief vortexing at RT for 5–10 s. Before analysis, the assembled AllSet mRNA-LNPs were diluted to the optimal working concentration with 1×PBS (10⁻⁶ mol%). 7 -10 8 Particles mL -1 To avoid overlap events and ensure rigorous single-particle detection, fusion-mediated mRNA encapsulation efficiency was determined using the FlowNanoAnalyzer (NanoFCM Inc.) by quantifying the percentage of DiA / FITC double-positive events. For comparative analyses, baseline ALC-0315 mRNA-LNP was evaluated in parallel via routine microfluidic mixing in sodium citrate buffer (pH 4.0).

[0432] Molecular dynamics (MD) simulations.

[0433] The 200 ns molecular dynamics (MD) trajectory of the Nip-a12Dab4-RNA complex was obtained using GROMACS 2020.3 and the AMBER99sb-ildn force field. Simulations were performed using 19-nucleotide polyA, polyG, polyC, or polyU molecules and a TIP3P (three-point transferable intermolecular potential) water model. The system was equilibrated at 300 K and 1 standard atmosphere (ATM), and trajectory snapshots were recorded and analyzed every 2 ns. Trajectory analysis was performed using GROMACS built-in tools. Specifically, hydrogen bonds between Nip-a12Dab4 and RNA were quantified using geometric criteria (donor-acceptor distance <3.5 Å and hydrogen-donor-acceptor angle <30°). π-π stacking interactions were systematically evaluated by monitoring the distance between the centroids of the aromatic rings (<4.5 Å) and the angle between their normals.

[0434] Residual moisture analysis of freeze-dried AllSet LNP.

[0435] To optimize the lyophilization matrix, combinations of lyophilization protectants and formulation parameters were screened for empty AllSet LNPs. Ten different excipients were evaluated, including monosaccharides and disaccharides (trehalose, sucrose, fructose, and glucose), sugar alcohols (mannitol), and polymeric or macrocyclic protectants (dextran, HP-β-CD, PEG1500, PVA, and PVP-K12). Briefly, the dialyzed empty LNPs were adjusted to specific working concentrations. Subsequently, each of the ten lyophilization protectants was added to the LNP to achieve 40, 80, 160, and 320 mg / mL concentrations. -1 The final excipient concentration gradients and 2, 4, 8 and 12 μg / μL -1 The final LNP concentration was determined. The prepared LNP was aliquoted into glass vials and subjected to standard lyophilization cycles. After completion, the vials were backfilled with high-purity nitrogen to prevent oxidative degradation during storage. The residual moisture content of the resulting lyophilized cake was determined quantitatively using coulombic Karl Fischer (KF) titration. The residual moisture content of each formulation was calculated and expressed as a weight percentage (%, w / w) relative to the total mass of the lyophilized cake.

[0436] Reconstruction time evaluation.

[0437] To evaluate the speed and immediate-use feasibility of the AllSet platform, the reconstitution time of lyophilized empty LNPs was assessed in the aforementioned combination formulations. Vials containing the lyophilized cake were equilibrated to RT. For post-initiation assembly, a predetermined volume of aqueous mRNA solution was rapidly introduced into each vial, followed by vortexing (5–10 s). Reconstitution time was recorded using a digital timer. Complete reconstitution was defined as the duration required to produce a visually homogeneous, opalescent suspension free of undissolved particles, aggregates, or phase separation.

[0438] Cellular uptake research.

[0439] To investigate the cellular uptake efficiency of AllSet LNPs and verify their delivery universality, confocal laser scanning microscopy (CLSM) was used. HeLa cells were seeded at a density of 15,000 cells per well into 8-well Lab-Tek chamber slides and cultured overnight to allow cell adhesion. The following day, the medium was replaced with 270 μL of fresh complete medium. To demonstrate the universal encapsulation capability of the AllSet platform, diverse Cy5-labeled nucleic acid cargoes, including mRNA, siRNA, sgRNA, pDNA, ssDNA, Cas9 / sgRNA ribonucleoprotein (RNP) complex, phosphoryldiamine morpholino oligomer (PMO), and peptide nucleic acid (PNA), were individually loaded into LNPs. Subsequently, 30 μL of freshly prepared LNPs containing 300 ng of Cy5-labeled cargo were added to the cells. The cells were then incubated at 37°C for 4 h. Afterward, the medium was discarded, the cells were washed twice with cold 1×PBS, and then fixed with 4% paraformaldehyde (PFA) at RT for 40 min. After washing twice more with cold 1×PBS, use DAPI solution (2 μg / mL) -1 Cell nuclei were stained under RT for 20 min. Before imaging, the staining solution was subsequently replaced with 300 μL of fresh 1×PBS. All CLSM images were acquired using a Leica TCS SP8 confocal laser scanning microscope equipped with an HC PL APO 63× / 1.4 objective. Fluorescence emission of DAPI (nuclei) and Cy5 (cargo) was detected at 450 nm and 670 nm, respectively.

[0440] Gal8 internal body escape study.

[0441] To visualize the endosomal membrane disruption capability of UB-PIL-based mRNA-LNPs, a galactolectin-8 (Gal8) recruitment assay was performed. HeLa-mRuby3-Gal8 reporter cells were seeded at a density of 15,000 cells per well in 8-well Lab-Tek chamber slides and cultured overnight to allow for adhesion. The following day, the medium was replaced with 225 µL of fresh medium, and 75 µL of freshly prepared AllSet LNPs was added to each well. After incubation at 37°C for 4 h, the cells were washed, fixed with 4% PFA, and stained with DAPI. CLSM images were acquired, and DAPI and mRuby3 emission were recorded at 450 nm and 590 nm, respectively. Endosomal escape efficiency was evaluated by counting Gal8 spots per cell using ImageJ software (NIH).

[0442] Animal and ethical statements.

[0443] All animal experiments were approved by the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences (IOZ). Mice were housed in a barrier facility under a standard 12-hour light / dark cycle, maintained at 20–26°C and 30–60% relative humidity. Wild-type C57BL / 6 and BALB / c mice were obtained from the Institute of Zoology's Animal Breeding Core Facility. The lead editor (PE) reporter mouse was kindly provided by Professor Yupeng Chen's laboratory (Tianjin Medical University, China) and bred to homozygosity. This reporter strain carries the membrane marker EGFP allele interrupted by the TAG stop codon, thereby preventing EGFP protein expression.

[0444] In vivo delivery of luciferase mRNA.

[0445] To evaluate in vivo delivery efficacy, conventional or UB-PIL-based AllSet LNPs containing luciferase (Luc) mRNA were prepared as described above. The formulated mRNA-LNPs were administered intravenously (iv), intraperitoneally (ip), intramuscularly (im), or subcutaneously (sc) to female C57BL / 6 mice (6–8 weeks old, 18–20 g) at doses of 0.0125, 0.025, 0.05, 0.1, or 0.2 mg / kg. -1 Luc mRNA. Six hours after administration, mice were anesthetized by isoflurane inhalation and administered 100 μL of D-luciferin substrate (30 mg / mL) intraperitoneally. -1 (In PBS). After 5 min of continuous anesthesia, whole-body bioluminescence was recorded using the IVIS Lumina imaging system (PerkinElmer). Mice were then sacrificed, and major organs (liver, lungs, spleen, heart, kidneys, pancreas, and lymph nodes) were harvested for in vitro imaging. All bioluminescence data were processed and quantified using Living Image software v.4.4 (Caliper Life Sciences).

[0446] Long-term stability evaluation of AllSet LNP.

[0447] To demonstrate the AllSet platform's ability to bypass cold chain logistics, long-term stability was evaluated. Optimized empty LNPs and mRNA were independently lyophilized and refilled with high-purity nitrogen as previously described. The dried cakes were then stored in the dark at RT or 4°C. At predetermined time points (0, 7, 14, 21, 30, 60, 90, 120, 150, 180, and 240 days), the lyophilized empty LNPs and mRNA were assembled into complete mRNA-LNPs by rehydration with nuclease-free water and brief vortexing. Subsequently, the hydrodynamic diameter, polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (DLS), and the mRNA encapsulation efficiency was determined using RiboGreen. Furthermore, to verify the mRNA delivery efficiency retained by the mRNA-LNPs, in vivo evaluation was performed at predetermined time points. Freshly assembled mRNA-LNPs were administered to mice via four different delivery routes: iv (0.1 mg / mL). -1 ), ip (0.1 mg mL) -1 ), IM (0.2 mg mL) -1 ) and sc (0.2 mg mL) -1 Injection. The in vivo Luc mRNA delivery efficiency via each route was quantified as described above.

[0448] In parallel, conventionally pre-formed ALC-0315 mRNA-LNP was lyophilized and its storage stability was evaluated. Following standard microfluidic assembly and dialysis, ALC-0315 mRNA-LNP was lyophilized and stored under the same conditions (RT and 4°C). At selected time points (days 0, 7, and 30), the lyophilized baseline cake was reconstructed, and its physicochemical properties (hydrodynamic diameter, PDI, zeta potential) and mRNA encapsulation efficiency were measured. Furthermore, via IV (0.1 mg / mL) -1 ) and IM (0.2 mg mL) -1 This study investigated the in vivo mRNA delivery efficacy of ALC-0315 mRNA-LNP.

[0449] In vivo efficacy and biosafety analysis of AllSet mRNA vaccine.

[0450] Freeze-dried empty LNPs and freeze-dried mRNAs encoding different viral antigens (RSV, H1N1, or RVG) were stored independently at RT or 4°C for 6 months prior to use. Female BALB / c mice were randomly assigned to six treatment groups: (1) AllSet mRNA vaccine stored at RT for 6 months, (2) AllSet mRNA vaccine stored at 4°C for 6 months, (3) fresh freeze-dried AllSet mRNA vaccine, (4) conventionally formulated Nii-lipid-based mRNA vaccine, (5) commercial SM-102 mRNA vaccine, and (6) 1×PBS. For the AllSet group, the vaccine was post-assembled immediately before injection by rehydration and vortexing. Mice were immunized according to a standard primitivism-booster regimen, receiving intramuscular (im) injections of the appropriate formulation (5 μg mRNA per mouse) on day 0 (primitivism) and day 21 (booster). The weight of all mice was monitored every 5 days throughout the experiment.

[0451] To quantify vaccine-induced antigen-specific humoral immunity, serum was collected on day 14 (two weeks after primary immunization) and day 35 (two weeks after booster immunization) for enzyme-linked immunosorbent assay (ELISA) to determine antigen-specific IgG antibody titers. To monitor systemic toxicity, blood samples were collected at 3 h, 24 h, and 48 h after each administration. Serum was separated by centrifugation. Liver function markers (ALT, alanine aminotransferase; AST, aspartate aminotransferase) and renal function markers (CREA, creatinine; UREA, blood urea nitrogen) were quantified using standard colorimetric assay kits. Furthermore, serum cytokine levels, including IFN-α, IFN-γ, IL-1β, IL-6, IL-10, TNF-α, and CXCL1, were analyzed using specific ELISA kits.

[0452] In vivo PCSK9 editing and biosafety analysis of AllSet mRNA gene editing drugs.

[0453] Lyophilized empty LNPs and a mixture of lyophilized mRNA (Cas9 or ABEmax) / sgRNA (1:1 mass ratio) were stored at RT for 6 months. Wild-type C57BL / 6 mice were randomly assigned to three treatment groups: (1) AllSet LNPs stored at RT for 6 months, (2) conventionally prepared Nii-lipid-based mRNA-LNPs, and (3) 1×PBS. Prior to administration, the environmentally stored lyophilized AllSet LNPs were rehydrated and vortexed with lyophilized mRNA / sgRNA. Mice received a total RNA dose of 1.0 mg kg. -1 A single intravenous injection.

[0454] To determine the efficiency of genome editing at the target site at the DNA level, genomic DNA was extracted from homogenized liver tissue using the FastPure Blood / Cell / Tissue / Bacteria DNA Isolation Mini Kit (Vazyme). For Cas9-treated mice, target genomic sites flanking PCSK9 cleavage sites were amplified and Sanger sequenced. Insertion / deletion (indel) mutation frequencies were quantified using the Tracking of Indels by Decomposition (TIDE) network tool. For ABEmax-treated mice, next-generation sequencing (NGS) was performed on the amplified PCSK9 target sites to quantify the precise A-to-G conversion rate. To assess PCSK9 editing at the protein level, blood and liver tissue were harvested on day 7 post-injection. Serum PCSK9 protein levels and low-density lipoprotein cholesterol (LDL-C) concentrations were quantified using a mouse PCSK9 and LDL-C ELISA kit.

[0455] To evaluate systemic safety following intravenous administration, blood samples were collected at 3 h, 24 h, and 48 h post-injection. Serum levels of hepatic (ALT and AST) and renal (CREA and UREA) toxicity biomarkers were quantitatively determined using standard colorimetric assays. Serum cytokine levels, including IFN-α, IFN-γ, IL-1β, IL-6, IL-10, TNF-α, and CXCL1, were analyzed using specific ELISA kits.

[0456] In vivo lead editing of AllSet mRNA-LNP.

[0457] Lyophilized empty LNPs and a lyophilized PEmax mRNA / epegRNA mixture (weight ratio 1:2) were stored at RT for 6 months. PE reporter mice were randomly assigned to three treatment groups: (1) AllSet LNPs stored at RT for 6 months, (2) conventionally prepared Nii-lipid-based mRNA-LNPs, and (3) 1×PBS. Prior to administration, the environmentally stored lyophilized AllSet LNPs were rehydrated and vortexed with lyophilized mRNA / epegRNA. Mice received a total RNA dose of 4.0 mg kg. -1 A single intravenous injection.

[0458] One week after administration, mice were sacrificed and liver tissue was harvested for pilot editing studies via immunofluorescence staining. The excised tissue was immediately embedded in an optimal cutting temperature (OCT) compound, rapidly frozen, and sectioned into 10-μm sections. Tissue sections were fixed with 4% paraformaldehyde (PFA) for 20 min, followed by three washes with 1×PBS. To prevent nonspecific antibody binding, sections were blocked with normal goat serum (Solarbio) at RT for 1 h. Subsequently, slides were incubated overnight at 4°C with mouse anti-EGFP primary antibody (1:100 dilution, Thermo Fisher Scientific). After three washes with 1×PBS, sections were incubated at RT for 1 h with AlexaFluor 647-conjugated goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific). Finally, slides were mounted with ProLong Gold Antifade Mountant containing DAPI (for nuclear counterstaining) and sealed with coverslips. CLSM images were acquired, and EGFP-positive cells were analyzed using Phenochart 2.0.1 and ImageJ software (NIH).

[0459] To quantify in vivo lead editing efficiency, deep targeted amplicon sequencing was performed. Genomic DNA was extracted from homogenized liver tissue using the FastPure Blood / Cell / Tissue / Bacteria DNA Isolation Mini Kit (Vazyme). The target regions were then amplified, and amplicon mutations were detected using NGS and analyzed in GNEWIZ.

[0460] Statistical analysis.

[0461] Quantitative data are expressed as mean ± standard deviation (sd). Statistical analysis was performed using GraphPad Prism 10 (GraphPad Software). The significance of the comparisons shown was determined using a two-tailed unpaired t-test or one-way ANOVA. Data are expressed as mean ± sd. P <0.05 ( ), P <0.01 ( ), P <0.001 ( )and P <0.0001 ( () is considered to be statistically significant.

[0462] Composition of AIFA building blocks.

[0463] AIFA building blocks with saturated alkyl chains were synthesized via a previously reported reductive amination strategy. 21For symmetric AIFA building blocks, Fmoc-protected amino acids (Fmoc- L -Lys-OH, Fmoc- D -Lys-OH, Fmoc- L -Orn-OH, Fmoc- L -Dab-OH or Fmoc- L -Dap-OH (10 mmol, 1.0 eq) was dissolved in 1,2-dichloroethane (DCE), followed by the addition of an aldehyde (24 mmol, 2.4 eq) and acetic acid (1% v / v). The mixture was stirred at room temperature (RT) for 1 h to promote imine formation. Subsequently, sodium triacetoxyborohydride (STAB; 24 mmol, 2.4 eq) was added, and the reaction was stirred at RT for 3 days.

[0464] For asymmetric AIFA building blocks, a sequential addition method was employed. First, the Fmoc-protected amino acid (10 mmol, 1.0 eq) was reacted with a first aldehyde (12 mmol, 1.2 eq) in the presence of acetic acid (1% v / v) for 1 h, followed by reduction with STAB (12 mmol, 1.2 eq). Then, a second aldehyde (12 mmol, 1.2 eq) with a different carbon chain length and an additional portion of STAB (12 mmol, 1.2 eq) were introduced into the same reaction, and the mixture was stirred at RT for 3 days.

[0465] For both symmetric and asymmetric AIFAs, the resulting mixtures were washed twice with an aqueous trifluoroacetic acid (TFA) solution (0.1% v / v) and concentrated under reduced pressure. The crude product was purified by column chromatography using dichloromethane (DCM) / methanol (10:1, v / v) as the eluent to obtain the final AIFA building blocks.

[0466] Synthesis of (2,4,6-trioxo-1,3,5-triazine-1-yl)acetic acid (Cya).

[0467] The universal base Cya was synthesized via controlled N-alkylation of cyanuric acid. Briefly, cyanuric acid (1.0 eq) was dissolved in an alkaline aqueous solution (potassium hydroxide) under continuous magnetic stirring. Chloroacetic acid (1.0 eq) was then slowly introduced into the alkaline mixture. To promote nucleophilic substitution, the reaction mixture was stirred and maintained at 80°C for 10 h. After completion, the mixture was cooled to RT. Subsequently, the pH of the solution was adjusted to 3–5 by dropwise addition of acidic aqueous solution (1 M HCl) to protonate the carboxylate and induce precipitation of the target compound. The resulting white precipitate was collected by vacuum filtration, washed with cold deionized water, and dried under vacuum to give the purified Cya product.

[0468] Synthesis of 2,6-bis(acetamido)-4-pyridinecarboxylic acid (Dapy).

[0469] The universal base Dapy was synthesized via palladium-catalyzed carbonylation employing a CO substitution strategy followed by base-promoted hydrolysis. N,N'-(4-bromo-2,6-pyridinidyl)bis[acetamide] (630.0 mg, 2.32 mmol, 1.0 eq), palladium(II) acetate [Pd(OAc)2, 15.0 mg, 0.07 mmol, 3.0 mol%), Xantphos (60.0 mg, 0.104 mmol, 4.5 mol%), 1,2-benzisothiazol-2(3H)-formaldehyde 3-oxo-1,1-dioxide (N-formylsaccharin, 590.0 mg, 2.8 mmol, 1.2 eq), and potassium fluoride (KF, 336.0 mg, 5.8 mmol, 2.5 eq) were added sequentially to a dry 50 mL Schlenk tube equipped with a magnetic stirrer. The reaction vessel was sealed and purged three times with high-purity nitrogen (N2). Anhydrous DMF (15 mL) was added to a Schlenk tube. The reaction mixture was then heated to 80°C in an oil bath and stirred for 18 h under a nitrogen atmosphere. The dark red slurry was then cooled to RT. To initiate hydrolysis, triethylamine (Et3N, 810.0 μL, 5.8 mmol, 2.5 eq) and deionized water (420.0 μL, 23.2 mmol, 10 eq) were added to the mixture. The resulting suspension was stirred for another 3 h at RT. After completion, the solvent was removed under reduced pressure using a rotary evaporator. The resulting crude solid was washed with deionized water. The purified solid was collected by vacuum filtration and air-dried. The resulting white solid was dissolved in approximately 10 mL of a methanol-acetonitrile mixture (1:2, v / v). Diethyl ether was then slowly added dropwise to this solution to induce precipitation. This dissolution-precipitation cycle was repeated three times to obtain the final product.

[0470] Synthesis of 2-(4-nitroimidazol-1-yl)acetic acid (Nipu).

[0471] The general-purpose base Nipu is synthesized via a two-step reaction involving the N-alkylation of 5-nitroimidazole and the hydrolysis of an intermediate ester. Briefly, 5-nitroimidazole (1.0 eq) and anhydrous potassium carbonate (K₂CO₃, 2.0 eq) are suspended in anhydrous acetonitrile in a round-bottom flask equipped with a magnetic stirrer. The mixture is stirred at RT for 30 min under a nitrogen atmosphere to promote deprotonation. Subsequently, ethyl bromoacetate (1.2 eq) is added dropwise to the suspension via a syringe. The reaction mixture is heated to 60°C and stirred for 12 h. The mixture is then cooled to RT and quenched with distilled water. The aqueous phase is extracted three times with ethyl acetate (EtOAc). The combined organic layers are washed with water and saturated brine, dried over anhydrous sodium sulfate (Na₂SO₄), filtered, and concentrated under reduced pressure. The resulting crude residue is purified by rapid column chromatography (eluting with a hexane / EtOAc gradient) to give the intermediate ethyl 2-(5-nitro-1H-imidazol-1-yl)ethyl acetate. Subsequently, purified ethyl 2-(5-nitro-1H-imidazol-1-yl)acetate (1.0 eq) was dissolved in methanol. At 0°C, an aqueous solution of sodium hydroxide (NaOH, 2.0 M, 3.0 eq) was added dropwise to the stirred solution. The reaction mixture was then heated to RT and stirred for 4 h until ester hydrolysis was complete. The organic solvent was evaporated under reduced pressure. The remaining aqueous solution was washed with diethyl ether, cooled in an ice bath, and acidified with 1.0 M HCl to a pH of approximately 2–3. Acidification induced precipitation of the target compound. The solid was collected by vacuum filtration, washed with ice-cold deionized water, and dried under vacuum to obtain the pure product.

[0472] Synthesis of 2-(2,4,5,7-tetraoxo-8H-pyrimidino[4,5-d]pyrimidin-1-yl)acetic acid (Ppt).

[0473] The universal base Ppt was synthesized via TEMPO-mediated oxidation of an alcohol precursor. To a magnetically stirred suspension of 1-(2-hydroxyethyl)pyrimidino[4,5-d]pyrimidin-2,4,5,7(1H,3H,6H,8H)-tetraone (1.0 g, 4.2 mmol, 1.0 eq) in deionized water (70 mL), 2,2,6,6-tetramethyl-1-piperidinoxy (TEMPO, 0.70 g, 4.2 mmol, 1.0 eq) and sodium bromide (NaBr, 0.92 g, 8.4 mmol, 2.0 eq) were added. The pH of the resulting suspension was adjusted to 10 by dropwise addition of 0.4 M NaOH aqueous solution. To initiate oxidation, 5.4 mL of 11% sodium hypochlorite (NaOCl) aqueous solution was added to the reaction. The pH of the mixture was readjusted and maintained at 11 using 0.4 M NaOH aqueous solution. The resulting mixture was stirred at RT for 1.5 h. After stirring, 400 mL of ethanol was added to induce precipitation of the intermediate sodium carboxylate. The precipitated solid was collected by vacuum filtration. Subsequently, the collected solid was redissolved in deionized water (30 mL). The target compound was precipitated by adding 2 M HCl dropwise to acidify the aqueous solution. The resulting solid was collected by vacuum filtration and dried under reduced pressure to give the pure product.

[0474] Synthesis of N-[[(1,4-dihydro-6-methyl-4-oxo-2-pyrimidinyl)amino]carbonyl]-glycine (Upy).

[0475] The universal base Upy is synthesized via a two-step procedure involving the formation of an intermediate urea bond and subsequent ester hydrolysis. Briefly, 2-amino-4-hydroxy-6-methylpyrimidine (200 mg, 1.6 mmol, 1.0 eq) is suspended in anhydrous pyridine (10 mL) in a round-bottom flask equipped with a reflux condenser under a nitrogen atmosphere. Ethyl isocyanate (412.8 mg, 3.2 mmol, 2.0 eq) is then added to the stirred suspension. The reaction mixture is heated to reflux and maintained for 2 h. After completion, the mixture is cooled to RT. Acetone is added to the mixture to promote precipitation and reduce the solubility of the product. The resulting solid is collected by vacuum filtration, washed with cold acetone, and vacuum dried to give a white powdery intermediate ethyl ester product. Subsequently, intermediate ethyl ester (464 mg, 1.18 mmol, 1.0 eq) and NaOH (144.9 mg, 3.62 mmol, 3.0 eq) were suspended in a mixed solvent consisting of methanol (20 mL) and deionized water (10 mL). The reaction mixture was heated to reflux and stirred. Ester hydrolysis was completed after 2 h. The organic solvent was removed by rotary evaporation under reduced pressure. The solid residue was redissolved in a minimal amount of deionized water to give a clear aqueous solution. The solution was then cooled in an ice bath and acidified with concentrated HCl to a pH of approximately 3 to precipitate the target compound. The acidic solution was allowed to stand overnight at RT to maximize crystallization. The resulting white precipitate was collected by vacuum filtration, washed with cold deionized water and acetone, and dried under reduced pressure to give the pure product.

[0476] Synthesis of Nii-SM-102 and Nii-ALC-0315.

[0477] Nii-modified ionizable lipids (Nii-SM-102 and Nii-ALC-0315) are synthesized via standard Steglich esterification, which involves coupling the terminal hydroxyl group of the baseline lipid (SM-102 or ALC-0315) to the carboxylic acid moiety of 1-carboxymethyl-5-nitroindole (Nii). 51In short, the corresponding ionizable lipids (1.0 eq) and Nii (1.2 eq) were dissolved in anhydrous dichloromethane (DCM) under an inert nitrogen atmosphere. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 1.5 eq) and 4-dimethylaminopyridine (DMAP, 0.2 equivalences) were added to this solution to promote coupling. The reaction mixture was stirred in the dark at RT for 24 h. After completion, the reaction was quenched by adding deionized water and extracted three times with DCM. The combined organic layers were washed with saturated sodium chloride aqueous solution (saline), dried over anhydrous sodium sulfate (Na₂SO₄), and concentrated under reduced pressure using a rotary evaporator. The resulting crude products were purified by rapid silica gel column chromatography, eluting with a DCM and methanol gradient to obtain purified Nii-SM-102 and Nii-ALC-0315 lipids.

[0478] Synthesis of Nii-cKK-E12.

[0479] Nii-modified cKK-E12 (Nii-cKK-E12) was synthesized via controlled Steglich esterification between the carboxylic acid moiety of Nii and the polyhydroxy backbone of the cKK-E12 lipid. Briefly, cKK-E12 (1.0 eq) and Nii (0.8 eq) were dissolved in anhydrous DCM under an inert nitrogen atmosphere. For initiating coupling, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 1.2 eq) and a catalytic amount of 4-dimethylaminopyridine (DMAP, 0.1 eq) were added. The reaction mixture was stirred in the dark at RT for 12 h. The mixture was then quenched with deionized water and extracted with DCM. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. A crude mixture containing the desired monoesterified Nii-cKK-E12, unreacted starting material, and multi-substituted byproducts was purified by rapid silica gel column chromatography. The target monosubstituted Nii-cKK-E12 compound was separated by optimizing the elution gradient (DCM / methanol).

[0480] 1 H nuclear magnetic resonance (H nuclear magnetic resonance) 1 H NMR spectrum.

[0481] AIFA and UB-PIL 1 1H NMR spectra were recorded on a Bruker Avance III HD spectrometer (500 MHz) using deuterated chloroform (CDCl3) as solvent. Chemical shifts relative to residual solvent signals are reported in parts per million (ppm). Data processing and manual integration were performed using MestReNova software (Mestrelab Research).

[0482] Electrospray ionization mass spectrometry (ESI-MS).

[0483] Electrospray ionization mass spectrometry (ESI-MS) of AIFA and UB-PIL was recorded on a ThermoScientific LTQ FT Ultra Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an Ion Max source. Samples were dissolved in chloroform or methanol prior to analysis.

[0484] Reversed-phase high-performance liquid chromatography (HPLC) analysis.

[0485] The chemical purity of the synthesized UB-PIL was evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC). UB-PIL was dissolved in methanol to a final concentration of 1.0 mg / mL. Chromatographic analysis was performed on a VWR Hitachi Chromaster HPLC system. Ultraviolet (UV) absorbance was monitored at 214, 254, and 280 nm to verify the purity of the final product and confirm the successful conjugation of the universal base.

[0486] Conventional mRNA-LNP was prepared by microfluidic mixing.

[0487] mRNA-LNP was formulated using standard microfluidic mixing techniques. 41 The aqueous phase was prepared by diluting the mRNA in one of three different solvents: sodium citrate buffer (10 mM, pH 4.0), HEPES buffer (20 mM, pH 7.4), or nuclease-free water. The organic phase consisted of a lipid mixture dissolved in ethanol (ionizable lipids / DSPC / cholesterol / DMG-PEG2000, molar ratio 46.3:9.4:42.7:1.6). The aqueous and organic phases were rapidly mixed using a microfluidic device at a flow rate of 3:1 (aqueous phase:ethanol, v / v) to achieve a final total lipid:mRNA weight ratio of 40:1. The resulting mixture was incubated at RT for 15 min. Finally, prior to subsequent application, the prepared mRNA-LNP was diluted with 1×phosphate-buffered saline (PBS) or dialyzed against 1×PBS for 4 h to remove residual ethanol.

[0488] Characterization of mRNA-LNP.

[0489] The hydrodynamic diameter, polydispersity index (PDI), and zeta potential of mRNA-LNP were measured using a Zsizer Nano ZS instrument (Malvern Instruments) equipped with a He-Ne laser (λ = 632 nm). Samples were loaded into a DTS1070 folded capillary cell and measurements were performed at a fixed scattering angle of 173° and a constant temperature of 25°C. Each sample was analyzed in triplicate, with each measurement comprising 12 to 15 subruns. mRNA encapsulation efficiency was quantified using the Quant-iT RiboGreen RNA assay kit as previously described. The apparent pKa of mRNA-LNP was determined by a modified TNS fluorescence binding assay. Briefly, mRNA-LNP (final total lipid concentration of 60 μM) and the TNS probe (4 μM) were incubated for 5 min in a buffer containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, at pH 2.5 to 11.0. The mixture was then transferred to black 96-well plates, and fluorescence intensity was measured using a Tecan microplate reader at an excitation wavelength of 321 nm and an emission wavelength of 445 nm. The fluorescence data were normalized to the maximum observed at pH 2.5, and the apparent pKa was calculated as the pH corresponding to the half-maximum fluorescence.

[0490] Colloidal stability study.

[0491] To evaluate colloidal stability, UB-PIL-based mRNA-LNPs were lysed using Triton X-100 assays. Freshly prepared mRNA-LNPs were incubated with different concentrations of Triton X-100 (0%, 0.05%, and 1%, v / v) at RT for 5 min. The amount of approximate mRNA was then quantified using the Quant-iT RiboGreen RNA assay kit described above. The relative mRNA release was calculated using the following equation: [(A0.05-Ab) / (A1-Ab)] × 100%. Ab: amount of mRNA detected without incubation with Triton X-100; A1: amount of mRNA detected after incubation with 1% Triton X-100; A0.05: amount of mRNA detected after incubation with 0.05% Triton X-100.

[0492] Serum stability study.

[0493] To evaluate the protective efficacy of UB-PIL-based LNPs against physiological nucleases, serum stability was determined. Freshly prepared AllSet mRNA-LNP (mRNA concentration 1 mg / mL) was used. -1The LNP was mixed with an equal volume of mouse serum. The mixture was incubated at 37°C for 30 min to simulate physiological conditions. Afterward, the sample was diluted, and the mRNA encapsulation efficiency (EE) of each LNP formulation was quantified using the Quant-iTRiboGreen RNA assay kit as described above. The LNP group incubated with 1×PBS served as a control. Serum stability is expressed as a relative percentage of released mRNA, calculated using the following equation: EEserum / EEPBS × 100%. EEserum: mRNA encapsulation efficiency detected in LNPs treated with mouse serum; EEPBS: mRNA encapsulation efficiency detected in LNPs treated with 1×PBS.

[0494] LNP dissociation determination.

[0495] To investigate the endosomal escape ability of UB-PIL-based LNPs, FRET-based measurements were performed. 37 FRET-labeled UB-PIL-based mRNA-LNPs were formulated by incorporating the dye pair NBD-PE (donor) and Rho-PE (acceptor) into a lipid mixture prior to microfluidic mixing. Endosome-mimicking anionic liposomes (total lipid 10 mM) composed of DOPS, DOPC, and DOPE in a 25:25:50 molar ratio were prepared using standard membrane hydration methods. To simulate the acidic environment of late endosomes, the assay was performed in black 96-well plates. Briefly, 100 μL of acidic 1×PBS (pH 5.5) was added to each well, followed by 1 μL of the endosome-mimicking liposome and 10 μL of freshly prepared FRET-labeled mRNA-LNPs. The mixture was incubated at 37°C for 5 min to promote lipid interactions. The fluorescence intensity (F) of the NBD donor was measured using a Tecan microplate reader at an excitation wavelength of 465 nm and an emission wavelength of 520 nm. FRET-labeled LNPs incubated in acidic PBS without endosome-mimicking liposomes served as a negative control (Fneg), while LNPs completely destroyed with 1% (v / v) Triton X-100 were defined as a positive control (Fpos) representing 100% lipid dissociation. The percentage of lipid dissociation was quantified using the following equation: [(F-Fneg) / (Fpos-Fneg)] × 100%.

[0496] Hemolysis assay.

[0497] To evaluate membrane destabilization capacity, a red blood cell (RBC) hemolysis assay was performed. Mouse RBCs were isolated from whole blood by centrifugation (10,000 × g, 5 min). The collected RBC pellet was washed with 1×PBS (pH 7.4) and resuspended until the supernatant became colorless. To simulate physiological and endosomal environments, the purified RBCs were resuspended in 1×PBS adjusted to pH 7.4 or pH 5.5, respectively, and seeded into 96-well plates. Subsequently, freshly prepared UB-PIL-based mRNA-LNP was introduced into the RBCs to achieve a final total lipid concentration of 1.2 mM. The mixture was incubated at 37°C for 2 h to promote lipid-membrane interactions. Afterward, intact RBCs were precipitated by centrifugation (10,000 × g, 5 min), and the hemoglobin-rich supernatant was collected. The absorbance (A) of the released hemoglobin was quantified at 540 nm using a Tecan microplate reader. RBCs incubated with 1×PBS (pH 7.4) and 1% (v / v) Triton X-100 served as negative control (Aneg) and positive control (Apos, representing 100% hemolysis), respectively. The percentage of hemolysis was calculated using the following equation: [(A-Aneg) / (Apos-Aneg)] × 100%.

[0498] Cell culture.

[0499] MDA-MB-231, HeLa, and HeLa-mRuby3-Gal8 cells were supplemented with 10% (v / v) fetal bovine serum (FBS) and 100 U / mL. -1 Penicillin and 100 μg mL -1 Cells were cultured in Dulbecco modified Eagle medium (DMEM) containing streptomycin. Cells were maintained in aerated culture flasks in a humidified incubator at 37°C with a 5% CO2 atmosphere. Cells were passaged at approximately 80% confluence. A HeLa reporter cell line stably expressing the mRuby3-Gal8 fusion protein (HeLa-mRuby3-Gal8) was constructed using the Super piggyBac Transposase expression vector (System Biosciences) and the PB-CAG-mRuby3-Gal8-P2A-Zeo plasmid.

[0500] In vitro transfection efficiency and cytotoxicity assays.

[0501] In vitro mRNA delivery efficiency and cytotoxicity were evaluated using MDA-MB-231 cells. Briefly, cells were seeded at a density of 10,000 cells per well in 96-well plates and cultured overnight to allow adhesion. Before transfection, the medium was replaced with 90 μL of fresh complete medium. Subsequently, 10 μL of AllSet mRNA-LNP containing 80 ng of luciferase (Luc) mRNA was added to each well. Cells were then incubated at 37°C in a 5% CO2 atmosphere for 24 h. Afterward, luciferase expression and cell viability were quantified using the Luciferase Assay System (Promega) and Calcein-AM assays, according to the manufacturer's protocol.

[0502] In vivo delivery of luciferase mRNA.

[0503] To evaluate in vivo delivery efficiency, conventional LNPs containing luciferase (Luc) mRNA or UB-PIL-based AllSet LNPs were prepared as described above. The formulated mRNA-LNPs were administered intravenously (iv), intraperitoneally (ip), intramuscularly (im), or subcutaneously (sc) to female C57BL / 6 mice (6–8 weeks old, 18–20 g) at doses of 0.0125, 0.025, 0.05, 0.1, or 0.2 mg / kg. -1 Six hours after administration, mice were anesthetized by isoflurane inhalation and administered 100 μL of D-fluorescein substrate (30 mg / mL) intraperitoneally. -1 (in PBS). After 5 min of continuous anesthesia, whole-body bioluminescence was recorded using the IVIS Lumina imaging system (PerkinElmer). Mice were then sacrificed, and major organs (liver, lungs, spleen, heart, kidneys, pancreas, and lymph nodes) were collected for ex vivo imaging. All bioluminescence data were processed and quantified using Living Image software v.4.4 (CaliperLife Sciences).

[0504] mRNA integrity assessment.

[0505] mRNA integrity was evaluated at defined time points (0, 7, 14, 21, 30, 60, 90, 120, 150, 180, and 240 days). Independently lyophilized mRNAs stored at RT and 4°C for up to 240 days were reconstituted with nuclease-free water. As a comparison, encapsulated mRNA was extracted from the lyophilized pre-formed baseline ALC-0315 mRNA-LNP using an isopropanol precipitation protocol. Briefly, the reconstituted baseline LNP was adjusted to a final mRNA concentration of 100 μg / mL. -1Subsequently, 100 μL of LNP was mixed with 900 μL of ice-cold ammonium acetate solution (60 mM, isopropanol) and incubated at -80°C for 10 min to promote complete RNA precipitation. The mixture was then centrifuged at 15,000 × g for 15 min at 4°C. The supernatant was discarded, and the resulting RNA precipitate was washed twice with isopropanol. After drying at RT for 5 min, the purified mRNA was dissolved in 100 μL of nuclease-free water, and mRNA integrity was analyzed by capillary electrophoresis using an Agilent 5200 fragmentanalyzer.

[0506] Stability of AllSet mRNA-LNP after assembly during use.

[0507] Lyophilized empty AllSet LNPs and mRNA stored at RT for 6 months were rehydrated with nuclease-free water and vortexed for post-assembly. The resulting assembled mRNA-LNPs were then stored at 4°C. Aliquots were taken at predetermined time points (days 0, 5, 10, and 15) to monitor the storage stability of the aqueous mRNA-LNPs. Hydrodynamic diameter, polydispersity index (PDI), and zeta potential were measured using dynamic light scattering (DLS), and quantitative mRNA encapsulation efficiency (EE) was determined using Quant-iT RiboGreen as described above. In vivo mRNA delivery efficiency was evaluated at the above time points. The assembled mRNA-LNPs were delivered via intravenous (0.1 mg / kg) -1 Luc mRNA) and im (0.2 mg kg) -1 Luc mRNA was injected into C57BL / 6 mice. In vivo Luc expression levels for each injection route were quantified as described above.

[0508] AllSet siRNA-LNP in vivo TTR gene silencing.

[0509] Lyophilized empty LNPs and lyophilized TTR-targeting siRNAs were stored at RT and 4°C for 6 months. Wild-type C57BL / 6 mice were randomly assigned to six experimental groups: (1) AllSet LNPs stored at RT for 6 months, (2) AllSet LNPs stored at 4°C for 6 months, (3) Freshly lyophilized AllSet LNPs, (4) Conventionally formulated Nii-lipid-based LNPs, (5) Commercial MC3 siRNA-LNPs, and (6) 1×PBS. Prior to administration, environmentally stored AllSet LNPs were post-assembled by rehydration and vortexing. To evaluate dose-dependent knockdown efficiency, mice received a single intravenous injection of siRNA at doses of 0.1, 0.3, or 1.0 mg / kg. -1 .

[0510] The efficacy of therapeutic gene silencing in vivo was evaluated at both mRNA and protein levels. Serum samples were collected at 6 h, 24 h, and 72 h post-drug administration, and TTR protein levels were quantified using a mouse TTR-specific ELISA kit. Mice were sacrificed at the 72 h endpoint, and liver tissue was collected. Total RNA was extracted from homogenized liver tissue using TRIzol reagent. Relative hepatic TTR mRNA expression levels were subsequently determined by quantitative reverse transcription PCR (RT-qPCR).

[0511] To monitor systemic toxicity, blood samples were collected at 6 h, 24 h, and 72 h post-administration. Serum was separated by centrifugation. Liver function markers (ALT and AST) and kidney function markers (CREA and UREA) were quantified using standard colorimetric assay kits. Serum cytokine levels, including IFN-α, IFN-γ, IL-1β, IL-6, IL-10, TNF-α, and CXCL1, were analyzed using specific ELISA kits.

[0512] Example

[0513] Example 1. Design and assembly mechanism of universally modified AllSet LNPs.

[0514] A universally modified peptide-ionizable lipid (UB-PIL) designed for encapsulating mRNA in neutral aquatic environments was synthesized via solid-phase support. 21-24 (SPSS) Synthesis. Initially, a carboxylated derivative of 3-nitropyrrole (Nip) was chosen (Nip is a universal base known to stabilize DNA double strands through base stacking rather than hydrogen bonding²). 5 And conjugated it to the N-terminus of the previously reported liver-targeting a12Dab4 PIL to obtain a universally modified Nip-a12Dab4 ( Figure 3 , Figures 103-104 , Figure 192 Unmodified counterpart a12Dab4, nonionized control Nip-c12Dab4, and FDA-approved baseline ALC-0315 were used for comparison. Figures 3-4 , Figures 105-106 Subsequently, twelve mRNA-LNPs were prepared by microfluidic mixing under three different aqueous conditions: acidic sodium citrate (10 mM, pH 4.0), neutral HEPES (20 mM, pH 7.4), and pure water (pH 6.8). Figure 5Physicochemical characterization showed that both Nip-modified lipids (Nip-a12Dab4 and Nip-c12Dab4) achieved approximately 80% mRNA encapsulation efficiency (EE) under all test conditions. In contrast, formulations lacking universal bases (a12Dab4 and ALC-0315) achieved mRNA loading only in acidic buffer and failed completely in neutral solution. Figure 6 , Figure 107 Subsequent in vivo evaluations using firefly luciferase (Fluc) mRNA were consistent with these encapsulation profiles. Nip-a12Dab4 mediated strong expression regardless of the preparation buffer, while a12Dab4 and ALC-0315 only functioned when prepared in citrate buffer. Figure 7 Notably, despite its high EE, nonionized Nip-c12Dab4 completely failed in vivo. Mechanistic studies indicated that the loss of transfection efficiency was due to the lack of a protonatable tertiary amine, which eliminated the pH buffering and endosome escape capabilities of the Nip-c12Dab4 LNP. 26,27 ( Figure 108 After validating the microfluidic preparation of mRNA-LNPs in neutral solution, the inventors further investigated whether mRNA could be loaded into pre-assembled empty LNPs after being immersed in water. Encapsulation strategies were evaluated using empty LNPs in their native aqueous state and lyophilized form. Figure 8 , Figure 109 Specifically, empty Nip-a12Dab4 and a12Dab4 LNPs were prepared, and a portion of these formulations were subjected to a lyophilization cycle including a primary drying stage and a secondary drying stage to produce a stable dry cake. Figure 110 Consistent encapsulation rates were observed in both states of LNP when the aqueous suspension was mixed with a neutral mRNA solution or when the dry cake was reconstructed with a neutral mRNA solution. The unmodified a12Dab4 control required both acidic conditions and external heating (>60°C) to achieve a maximum EE of approximately 80%, with single treatments achieving only moderate mRNA encapsulation (10%–60%). Figure 9 Conversely, both aqueous and rehydrated lyophilized Nip-a12Dab4 LNPs rapidly encapsulated mRNA in pure water at room temperature, achieving >90% EE (excision efficiency) within seconds. Figure 9 , Figure 111 ).

[0515] Encapsulation of mRNA in pure water suggests an electrostatically independent loading mechanism. The inventors propose that mRNA acts as a molecular bridge, driving empty LNP fusion and spontaneous mRNA internalization. Figure 10To verify this mechanism, the inventors first performed nanoparticle tracking analysis (NTA). After the addition of mRNA, the LNP concentration decreased by approximately 52.0%, while the average particle size increased by approximately 32 nm, strongly indicating particle fusion. Figure 11 , Figure 112 The inventors further validated this process using a fluorescence resonance energy transfer (FRET) elimination assay. In this assay, membrane fusion drives lipid mixing and increases the spatial distance between FRET pairs, thereby reducing FRET efficiency¹ 7 ( Figure 113 At various total lipid to mRNA mass ratios, the addition of mRNA solution to the lyophilized Nip-a12Dab4 formulation triggered significant FRET elimination, confirming substantial membrane fusion. Figure 12 , Figure 113 In contrast, the FRET effect was maintained in the a12Dab4 and ALC-0315 controls, which lack a universal base. Figure 113 Furthermore, dual-color nanoflow cytometry (DiA-labeled LNPs and FITC-labeled mRNA) demonstrated that the addition of mRNA drastically reduced the proportion of empty LNPs from 99.9% to 5.8%, suggesting a highly efficient encapsulation process. Figure 13 Notably, this post-assembled Nip-a12Dab4 formulation outperformed the conventional microfluidic ALC-0315 formulation, which, despite being prepared in acidic buffer, retained 31.1% of the empty LNP population. Figure 114 Next, the inventors sought to explore the intermolecular forces driving non-electrostatic bridging post-assembly. Molecular dynamics (MD) simulations were performed to model the interactions between Nip-a12Dab4 and 19-nucleotide RNA homooligomers (polyA, polyG, polyC, and polyU). Figure 14 Simulations revealed a persistent π-π stacking between Nip bases and all four natural nucleobases. Figure 14 Simultaneously, multiple hydrogen bond interactions were observed between the UB-PIL amide backbone and RNA molecules, primarily involving the phosphate moiety of RNA. In summary, these findings indicate that AllSet post-assembly is thermodynamically driven by concerted π-π stacking and hydrogen bonding, bypassing the traditional dependence on electrostatic interactions. 28,29 ( Figure 15 , Figure 64 ).

[0516] To confirm the functional integrity of this strategy, the inventors further compared the in vivo delivery efficiency of post-assembled Nip-a12Dab4 mRNA-LNP from lyophilized cakes with that of freshly prepared Nip-a12Dab4 mRNA-LNP in standard citrate buffer. Specifically, the lyophilized LNPs were constructed by hydration with a neutral mRNA solution and then diluted with a pre-prepared buffer to achieve isotonicity prior to administration. Figure 115 Table 1). Six hours after intravenous administration, no significant difference in bioluminescent signal in mouse liver was observed between the two preparation methods, confirming that the integration of the lyophilized empty LNP formulation with the post-assembly strategy fully preserves the bioavailability of the universally modified mRNA-LNP. Figure 16 ).

[0517] Example 2. Continuous chemical evolution of UB-PIL for enhanced in vivo delivery.

[0518] Despite achieving efficient mRNA encapsulation in neutral solution, the in vivo delivery efficacy of Nip-a12Dab4 remains lower than that of conventional a12Dab4 and ALC-0315 formulations. Figure 7 Inspired by biological evolution, the inventors employed an iterative "mutation-selection" method to conduct six rounds of in vivo guided chemical evolution to optimize the delivery efficiency of UB-PIL.³ 0 ( Figure 17 , Figure 116 In each round, specific structural motifs are systematically mutated, and the lipids that perform best serve as the basis for the next round.

[0519] First, the motif of alkylated ionized Fmoc-protected amino acids (AIFAs) was optimized²¹. Figures 117-120 , Figures 184-191 , Figures 218-223 In the initial round, the AIFA side chain length was systematically altered. Lysine (K)-based Nip-a12K4 mediated the highest mRNA expression in the liver, while Dap-derived UB-PIL failed to produce efficient delivery in vivo. Figure 18 Mechanistic studies revealed a positive correlation between side chain length and the apparent pKa of LNPs. Figures 65-66 , Figures 121-122 The best-performing Nip-a12K4 achieved a pKa of 6.13, which falls within the optimal range (6.0–6.8)³¹ for liver-targeted mRNA delivery. In the second round, the number of AIFAs was adjusted from 1 to 5. Figures 123-124 The formulation containing two AIFAs (Nip-a12K2) was superior to other formulations, achieving a 3.1-fold increase in bioluminescence compared to Nip-a12K4. Figure 19This optimization reflects the potential balance of intermolecular forces governing intracellular release³². Specifically, Nip-a12K1, with only two hydrogen bond donors, exhibits weak hydrogen bond binding to mRNA, while lipids with four to six donors (a12K3, K4, and K5) induce excessive mRNA complexation, thereby hindering efficient cytoplasmic mRNA release. 33-35 ( Figure 67 , Figure 125 Therefore, Nip-a12K2 with three hydrogen bond donors achieved optimal binding affinity for maximum transfection. Subsequently, the effect of base type was evaluated ( Figures 126-133 Nineteen Nip-a12K2 analogs were synthesized using five natural nucleobases and fourteen universal bases, and subsequently formulated for in vivo screening (0.1 mg / kg). - ¹ FlucmRNA). Bioluminescent imaging showed that universal base modification achieved a significant 60% hit rate for valid candidates, superior to native nucleobases (0% hit rate). Figure 68 Structure-activity relationship (SAR) analysis showed that specific universal bases affect the colloidal stability, serum resistance, and endosome escape ability of LNPs, which together determine their transfection efficacy²¹. Figure 68 , Figure 134 Notably, the 5-nitroindole (Nii)-modified lipid Nii-a12K2 exhibited the highest efficiency in achieving saturated bioluminescent signaling in the liver, and was 2.2 times superior to Nip-a12K2 (…). Figure 20 ).

[0520] To prevent signal saturation, the mRNA dose was reduced to 0.05 mg / kg. - ¹, for the fourth round of evolution focusing on universal base count and AIFA stereochemistry ( Figures 135-137 The inclusion of multiple Nii bases can impair delivery efficiency because excessive π-π stacking may limit intracellular mRNA release.³ 6 ( Figure 21 Regarding stereochemistry, UB-PILs with heterochiral AIFA (LD and DL configurations) were shown to be significantly superior to those composed of isochiral (LL and DD configurations) variants. The Nii-LD-a12K2 lipids maintained a saturation signal even at this reduced dose and outperformed the initial LL configuration analog by 4.5 times. It is speculated that the heterochiral configuration reduces the spatial distance between the two ionized amine head groups. This structural compression increases the critical packing parameter (CPP) of UB-PILs, favoring transformation to a fusion-promoting antihexagonal (HII) phase in an acidic endosome environment, thereby facilitating endosome escape. 37,38 ( Figure 69 , Figure 138In the fifth round, the dose was further reduced to 0.025 mg / kg. - ¹ Optimization of universal bases and spacers between the PIL backbone at different dosage levels ( Figures 139-140 After in vivo evaluation of various saturated carbon chains and oligoethylene glycols, Nii-C4-LD-a12K2 with a four-carbon (C4) spacer group was identified as the optimal structure, which exhibited a saturated bioluminescent signal in the liver, with an intensity twice that of Nii-LD-a12K2. Figure 22 , Figure 70 , Figure 141 In the final round of evolution, at an ultra-low mRNA dose of 0.0125 mg / kg... - ¹(250 ng) to study tail chain asymmetry ( Figures 142-145 Introducing the asymmetric a10a12 tail yielded the final lead candidate Nii-C4-LD-a10a12K2, which maintained strong mRNA expression even at nanograms of mRNA and outperformed its symmetric counterpart by 3.5 times. Figure 23 Mechanistically, the asymmetric lipid tail disrupts uniform hydrophobic stacking, thereby reducing LNP stiffness and enhancing membrane fluidity. 39,40 Dynamic fluidity promotes lipid fusion with the endosome membrane, thereby facilitating endosome escape and cytoplasmic mRNA translocation. This asymmetric architecture, highlighting tail chain flexibility, is a key determinant of LNP fusion-promoting and intracellular mRNA delivery. Figure 71 , Figure 146 ).

[0521] Through continuous chemical evolution, Ni-C4-LD-a10a12K2, possessing a single Ni base, a C4 spacer, an anchiral LD-AIFA backbone, and an asymmetric a10a12 tail, was identified as the leader UB-PIL. Figure 24 When compared to an ultra-low 250 ng mRNA dose as a baseline, this lead UB-PIL was 3.5-fold and 16.9-fold superior to ALC-0315 and the initial Nip-a12Dab4, respectively. Figures 25A-26 , Figure 72 More importantly, no significant difference in in vivo efficacy was observed between post-assembled lyophilized AllSet LNPs and fresh LNPs prepared in standard acidic buffer. This consistency further demonstrates that the Nii-C4-LD-a10a12K2 AllSet LNP achieves efficient and ultra-low dose mRNA delivery without compromising structural or biological integrity after lyophilization cycles. Summarizing the entire evolutionary process, comprehensive structural criteria for creating effective UB-PILs and related AllSet LNPs are provided. Figure 73 ).

[0522] At the same time, the inventors also tried to integrate Nii-C4- LD The -a10a12K2 mRNA-LNP was lyophilized as a whole and then reconstituted with aqueous solution for in vivo testing. The results showed that the in vivo mRNA delivery efficiency mediated by the whole lyophilized LNP was not significantly different from that of the standard aqueous solution preparation group and the AllSet lyophilized group. Figure 25B ).

[0523] Example 3. The wide applicability of AllSet LNP in load and LNP systems.

[0524] After identifying Nii-C4-LD-a10a12K2 (abbreviated as Nii-lipid) as a lead lipid, the inventors then sought to explore the versatility of the corresponding AllSet platform in various payload and LNP systems. Using lyophilized AllSet LNPs or standard aqueous formulations, Nii-lipid achieved approximately 90% encapsulation efficiency (EE) for various RNAs in pure water, including linear double-stranded siRNAs, single-stranded sgRNAs / pegRNAs, and mRNAs ranging in length from 1,000 to 7,400 nt. Figures 27-28 , Figures 74-75 , Figures 147-148 Furthermore, this platform can efficiently accommodate larger and more complex payloads, achieving approximately 89% EE for Cas9 / sgRNA ribonucleoproteins (RNPs) and approximately 75% EE for circular double-stranded pDNA. In contrast, the benchmark ALC-0315 can only encapsulate these payloads in acidic buffer using standard microfluidic mixing methods. Figure 28 , Figures 149-150 Considering that conventional ionizable lipids relying on electrostatic recombination typically cannot stably encapsulate ultrashort oligonucleotides due to insufficient charge density, the inventors hypothesized that the universal base moiety in Nii-lipid could overcome this limitation by providing additional molecular anchoring via π-π stacking and hydrogen bonding. Consistent with this rationale, AllSet LNPs achieved approximately 52% EE for very small 2-nt cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), significantly higher than ALC-0315 LNPs (approximately 25% EE). To further validate the superiority of this non-electrostatic assembly mechanism, the inventors challenged AllSet LNPs with uncharged nucleic acids that completely circumvent conventional electrostatic interactions. Notably, Nii-lipid achieved 60-74% EE for neutral phosphoryldiamine morpholino oligonucleotides (PMO) and peptide nucleic acids (PNA), while ALC-0315 failed to encapsulate these cargoes at all under all test conditions. Subsequent cellular uptake assays demonstrated that HeLa cells readily internalized multiple AllSet LNPs, highlighting the broad application potential of the AllSet platform for delivering various classes of therapeutic cargoes. Figure 29, Figure 151 ).

[0525] Based on its multi-functional payload delivery capabilities, the inventors subsequently focused on expanding the applicability of AllSet technology in various established LNP systems. Figures 30-31 , Figures 76-77 First, AllSet LNPs are integrated into the organ-targeting platform, specifically PILOT (peptide-ionizable lipid-driven organ targeting) and SORT (selective organ targeting) LNPs. 21,41 By conjugating the universal bases of Nii to the N-terminus of the established lung-targeting (Am-K2a12K4) and spleen-targeting (a12K4E-Ca) PIL, Am-K2K(Nii)a12K4 and a12K4K(Nii)E-Ca UB-PIL are generated. Figure 32 , Figures 152-153 After simple hydration and vortexing, lyophilized empty PILOT LNPs prepared with these modified UB-PILs efficiently encapsulated mRNA, achieving approximately 99% (Am-K2K(Nii)a12K4) and approximately 91% (a12K4K(Nii)E-Ca) EE (Excision Emissions). Figure 154 Crucially, its inherent organotropy was maintained after in vivo administration, directing 87.9% and 91.7% of its mRNA expression to the lungs and spleen, respectively. Figure 33 , Figure 154 Furthermore, AllSet technology is highly compatible with the SORT strategy. By incorporating SORT molecules (25% DOTAP or 15% DOPS) into a lyophilized Nii-lipid formulation, functional mRNA was delivered to the lungs and spleen upon rehydration, thus validating the adaptability of AllSet to lipid doping methods. Figures 78-80 , Figures 155-156 ).

[0526] Subsequent research expanded to widely used commercially available ionizable lipids. For lipids with modifiable sites, such as the head hydroxyl groups of ALC-0315 and SM-102, Nii conjugated derivatives (Nii-ALC-0315 and Nii-SM-102) were synthesized. Figure 34 , Figures 157-158 , Figures 216-217 , Figures 260-263 When prepared as lyophilized empty LNPs, these modified lipids mediated highly efficient mRNA encapsulation (85%-93% EE) and drove strong hepatic mRNA expression upon hydration, while their unmodified counterparts failed to encapsulate mRNA or mediate intracellular expression under the same conditions. Figures 35-36 , Figure 159Furthermore, it was confirmed that Nii modification is not limited to the lipid head group. Conjugating Nii to the hydrophobic tail of cKK-E12 enables strong post-assembly mRNA encapsulation and in vivo efficacy from lyophilized LNP cakes. Figures 81-82 , Figures 160-161 For commercial lipids lacking reactive sites, such as MC3 and LP-01, an alternative strategy is employed, in which the lead Nii-lipid is introduced as the fifth lipid component. Figure 37 Titration studies revealed that a minimum 5% molar addition of Nii-lipid was sufficient to achieve >90% mRNA EE in lyophilized AllSet LNPs. Figure 38 , Figure 162 Notably, these AllSet LNPs produced saturated bioluminescence in the liver, significantly outperforming the standard MC3 and LP-01 formulations prepared in citrate buffer. Figure 39 In summary, these findings highlight the dual versatility of the AllSet platform (payload and LNP vector), demonstrating its great potential for post-assembly of a variety of nucleic acid-LNP therapeutic agents.

[0527] Example 4. Long-term environmental stability of AllSet LNP.

[0528] After verifying the broad versatility of the AllSet platform, the inventors attempted to systematically screen lyophilization protectants to ensure optimal physicochemical properties and long-term stability. An ideal lyophilized AllSet formulation must exhibit an elegant appearance, low residual moisture, rapid remodeling kinetics, and the ability to maintain standard LNP physicochemical characteristics and in vivo efficacy during extended storage. Figure 40 Due to trehalose (80 mg / mL) - ¹) It has a high glass transition temperature, allowing for aggressive freeze-drying cycles and increased storage temperatures, and was therefore initially chosen as a freeze-drying protectant. 4 ². To determine the optimal excipients, a diverse combination of ten candidates was evaluated, covering monosaccharides and disaccharides (trehalose, sucrose, fructose, and glucose), sugar alcohols (mannitol), polysaccharides (glucan), cyclic oligosaccharides (hydroxypropyl-β-cyclodextrin, HP-β-CD), and synthetic polymers (polyethylene glycol 1500, PEG1500; polyvinyl alcohol, PVA; and polyvinylpyrrolidone K12, PVP-K12). The macroscopic morphology of the lyophilized empty LNP cakes was first evaluated. Formulations using disaccharides, HP-β-CD, and PVA produced white and elegant cakes characterized by a uniform, porous, spongy structure. Figure 41In contrast, other excipients failed to maintain structural integrity. PEG1500 and PVP-K12 matrices exhibited extensive cracking and fissures; the dextran formulation showed significant shrinkage, while glucose and mannitol both experienced macroscopic collapse. Residual moisture content was subsequently quantified by Karl Fischer titration. The moisture content ranged from 40 to 320 mg / mL. - ¹Within the range of excipient concentrations, monosaccharides / disaccharides and mannitol maintain a moisture level below 2% ( Figure 42 The key point is that sucrose ranges from 80 to 320 mg / mL. - ¹Minimum residual moisture (<1%) was achieved between concentrations. When the sucrose concentration was fixed at 160 mg / mL... - ¹And the LNP concentration changes (2-12 μg μL) - ¹) This excellent drying efficiency was maintained (<1% moisture). Furthermore, lyophilization of individual mRNA components using sucrose also yielded an elegant appearance and minimal residual moisture in the tested lyophilization protectants (¹). Figures 163-164 ).

[0529] Since rapid reconstitution is necessary for immediate practical use, hydration kinetics were subsequently evaluated. Four sugars (trehalose, sucrose, fructose, and glucose) were present in concentrations ranging from 40 to 160 mg / mL. - ¹This enables instant reconstruction within 7-10 seconds, while other excipients require >20 seconds ( Figure 43 It is worth noting that using 160 mg / mL - ¹The sucrose formulation maintained a rapid remodeling time of 6–10 seconds, independent of LNP concentration. Following this rapid remodeling, the physicochemical properties of the post-assembled LNPs were evaluated. 80 or 160 mg / mL was used. - ¹Sucrose freeze-dried formulations exhibit the most ideal profile, characterized by the smallest particle size (165-175 nm), narrow polydispersity index (PDI), and maximum mRNA encapsulation efficiency (>95%). Figure 44 , Figure 165 Subsequent in vitro assays in MDA-MB-231 cells revealed negligible cytotoxicity in all test groups, while the cytotoxicity was significantly lower with 160 mg / mL. - ¹Sucrose freeze-dried formulations achieve the highest transfection efficiency ( Figure 45 , Figure 166 Finally, the mass ratio of LNP to sucrose (160 mg / mL) was compared. - The evaluation of ¹sucrose determined the optimal in vivo performance window to be between 1% and 5%. Figure 46 Based on these results, 160 mg / mL was selected in subsequent studies. - ¹Sucrose was used as a lyophilization protectant for both empty LNPs and unprepared mRNA.

[0530] After determining the optimal lyophilization conditions, the long-term storage stability of AllSet LNPs was evaluated. Individually lyophilized empty LNPs and mRNAs were filled with nitrogen and maintained at 4°C or room temperature (RT, 25°C) for up to 8 months, using conventional co-lyophilized ALC-0315 mRNA-LNPs as a baseline. Capillary electrophoresis (CE) revealed that the lyophilized mRNAs maintained >90% structural integrity within 8 months at both 4°C and RT. Figure 167 In contrast, severe mRNA degradation was observed in the co-lyophilized ALC-0315 formulation, exhibiting a loss of >10% integrity within the first month, and significantly reduced by approximately 70% and 80% at 4°C and RT, respectively, by the eighth month. These findings are consistent with previous studies indicating that mRNA stored alone has better stability than mRNA in mRNA-LNP. 4 ³. Notably, after 8 months of storage, the post-assembled AllSet mRNA-LNP maintained homogeneous physicochemical properties at both 4°C and RT, exhibiting a stable particle size of 150–160 nm, a PDI between 0.1 and 0.15, and an EE exceeding 90%. Figures 47-48 , Figures 168-169 Crucially, this physicochemical stability directly translates into sustained in vivo potency throughout the entire 8-month storage period at ambient temperature. Following assembly, strong mRNA expression of AllSet mRNA-LNP was maintained without detectable attenuation across multiple routes of administration. Specifically, strong bioluminescent signals in the liver were preserved after intravenous (iv) or intraperitoneal (ip) administration. Figure 49 , Figures 83-84 Simultaneously, continuous Fluc expression within the lymphatic system was achieved through intramuscular (im) injection to the right inguinal lymph node and subcutaneous (sc) delivery to both inguinal lymph nodes. Figure 49 , Figures 83-84 Formulations maintained at 4°C showed the same long-term stability profile. Figure 170 In contrast, the co-lyophilized ALC-0315 baseline showed a significant loss of in vivo efficacy immediately upon reconstructing after lyophilization, and the bioluminescent signal further decreased within a week, becoming undetectable by day 30. Figures 85-86 In summary, these findings demonstrate that the AllSet platform not only avoids the storage-induced degradation inherent in traditional co-lyophilized LNP systems, but also provides excellent long-term stability, thus ensuring conversion potential even after extended storage at ambient temperatures.

[0531] The conversion of nanoformulations requires manufacturing reproducibility and operational robustness. 44The batch-to-batch consistency of the platform was evaluated across four independently prepared AllSet batches. No significant differences were observed in physicochemical characteristics or in vivo transfection efficiency, confirming a reliable and reproducible manufacturing process. Figures 87-89 Furthermore, to simulate the inherent variable conditions of a real-world, immediate-use environment, the effect of ambient hydration temperature was investigated. Significantly, the lyophilized components reliably self-assembled into functional mRNA-LNPs across a wide temperature gradient from 4°C to 35°C, maintaining homogeneous physicochemical properties and in vivo efficacy. Figures 90-92 Finally, the stability of the assembled formulation in use was evaluated. After preparation, the aqueous AllSet mRNA-LNP maintained its structural integrity and delivery potency for at least 15 days at 4°C, without showing significant physicochemical degradation. Figures 93-95 In summary, these results establish the AllSet platform as a highly robust, cold-chain-independent technology that provides a transformable strategy for addressing the bottleneck of sustained storage of nucleic acid therapeutics.

[0532] Example 5. AllSet LNP is used for field assembly in a variety of applications.

[0533] Inspired by its excellent long-term stability, the inventors envision the AllSet platform as a universal "ready-to-use" toolkit for the rapid, immediate, and on-site preparation of precision genetic drugs. Figure 50 Therefore, the on-site preparation of various nucleic acid therapeutic agents was evaluated using AllSet LNPs that had been stored at RT for 6 months. Figure 51 The capability of AllSet for rapid mRNA vaccine production was evaluated. Lyophilized mRNA cakes (encoding RSV preF, H1N1, or RVG antigens) were reconstituted in pure water and injected into lyophilized empty AllSet LNPs. The resulting vials were briefly vortexed for several seconds, and the post-assembled mRNA vaccine was diluted to an isotonic solution for intramuscular injection. Figure 52 , Figure 171 In BALB / c mice, a primary-boost immunization strategy induced strong antigen-specific IgG titers after primary immunization, which were significantly amplified after the booster dose. Figure 53 For all three tested vaccines, the antibody response induced by AllSet LNP stored at 4°C and RT for 6 months was found to be equivalent to that induced by freshly prepared lyophilized LNP, standard aqueous LNP, and the commercial SM-102 control. Furthermore, a favorable safety and tolerability profile was confirmed. Normal weight gain was observed throughout the immunization period, and serological markers (ALT, AST, CREA, and UREA) and systemic inflammatory cytokines remained at baseline levels at all evaluation time points (3, 24, and 48 hours post-injection). Figures 54-55 , Figures 172-174 ).

[0534] Next, we investigated the AllSet platform's ability to deliver complex, multi-component gene-editing therapeutics. The PCSK9 gene was chosen as the relevant model because it encodes a key regulator of cholesterol homeostasis and represents a clinically validated therapeutic target for treating familial hypercholesterolemia and reducing cardiovascular disease risk. 45 For CRISPR-Cas9 gene knockout and adenine base editing, the PCSK9-targeting sgRNA was co-lyophilized with the corresponding mRNA (Cas9 or ABEmax) at a 1:1 mass ratio. After rehydration and on-site assembly, a single intravenous dose of AllSet mRNA-LNP (1 mg / kg) was administered. - ¹Total RNA) Figures 56-57 , Figures 175-176 Genomic DNA was extracted from the liver for sequencing, and mouse serum was collected to measure PCSK9 protein, low-density lipoprotein cholesterol, and triglyceride concentrations. Sanger sequencing revealed that AllSet Cas9 mRNA-LNP achieved an insertion / deletion frequency of 63.4% in the liver, reaching a saturation editing level of approximately 64%. 45 ( Figure 57 This highly efficient genome disruption induces a >90% decrease in serum PCSK9 protein, accompanied by a >65% decrease in low-density lipoprotein cholesterol (LDL-C) and a >25% decrease in triglyceride levels by day 7. Figure 58 When evaluated using the AllSet ABEmax formulation, next-generation sequencing (NGS) validated a near-saturated A-to-G base conversion efficiency of 62.7%. Figure 59 Therefore, precise nucleotide substitution mediates 92.6% inhibition of the PCSK9 protein (). Figure 60 This blockade further led to a significant systemic lipid-lowering effect, with LDL-C and triglyceride levels decreasing by 65.2% and 26.2%, respectively. Figure 60 Most importantly, after being stored at ambient temperature for six months, the in vivo efficacy of these formulations was fully retained, achieving the same level of efficacy as freshly prepared standard LNPs. Figures 57-60 The safety profile indicated that only a slight, transient increase in liver enzymes and IL-6 was induced 3 hours after injection, which completely returned to baseline levels within 24 hours. Figures 177-178 ).

[0535] To push the payload limits, the platform was challenged with a significantly larger leader editor (PE) mRNA. Using a transgenic reporter mouse model carrying a membrane-tagged EGFP sequence interrupted by a TAG stop codon, successful PE-mediated TAG-to-CAG conversion restored EGFP fluorescence²¹. Figure 61Engineered lead-editing guide RNA (epegRNA) and PEmax mRNA were co-lyophilized at a 2:1 mass ratio and then assembled with stored AllSet LNPs. Figure 179 ). Single dose 4 mg kg - One week after intravenous administration, a large number of EGFP-positive cells were observed throughout the liver by immunofluorescence imaging in both the AllSet and fresh-prepared control. Figure 62 , Figures 180-181 Crucially, NGS quantitatively confirmed that the editing efficiency of AllSet LNPs stored for 6 months was 5.76%, demonstrating efficacy comparable to the freshly prepared standard formulation (5.23%). Figure 63 These findings highlight AllSet's ability to stably deliver highly complex gene editor mRNAs without relying on a cold chain.

[0536] Finally, AllSet's ready-to-use utility is extended from mRNA to siRNA-mediated gene silencing. 46,47 ( Figure 96 The TTR (transthyretin) gene was selected as a clinically relevant target. Silencing hepatic TTR expression is considered a disease modification strategy to reduce amyloid deposition, and is intended for the treatment of hereditary and wild-type transthyretin amyloidosis. 48 The lyophilized siRNA targeting TTR was rapidly reconstructed and injected into AllSet LNPs stored at 4°C or RT for six months. Figures 97-99 ). Administered intravenously in single doses (0.1, 0.3, and 1 mg kg). - Following this, strong and dose-dependent TTR mRNA knockdown was achieved in the liver at 72 hours, with >95% silencing detected at the highest dose. Figures 100-101 It is worth noting that at lower doses (0.1 and 0.3 mg / kg) - ¹) Compared to the FDA-approved MC3 baseline, the AllSet formulation induced significantly greater hepatic mRNA knockdown and subsequent decrease in serum TTR at 24 h. Figures 101-102 Crucially, the in vivo silencing efficacy of these 6-month stored formulations was found to be comparable to that achieved by the freshly prepared lyophilized counterpart and the standard aqueous control. Regarding systemic tolerability, only a slight and transient increase in liver function markers (ALT and AST) and IL-6 was observed 3 hours post-treatment. Figures 182-183 In summary, these results establish AllSet LNP as a versatile delivery platform for delivering broad-spectrum nucleic acid therapeutics without relying on cold chain infrastructure.

[0537] discuss

[0538] The clinical success of mRNA-LNP has established a robust platform for modern vaccinology and gene therapy. 49 However, most approved mRNA-LNP therapeutics rely on nucleoside-modified mRNAs requiring cold chain storage, limiting their global accessibility³. Achieving their full translational impact requires overcoming these logistical barriers, especially in resource-constrained environments where cold chain dependence and complex manufacturing restrict equitable distribution. To address this, the inventors rationally designed the AllSet platform, which physically separates the lyophilized lipid carrier from the mRNA to eliminate storage-induced degradation. By introducing universally modified peptide-ionizable lipids (UB-PILs), the inventors shifted the driving force of mRNA encapsulation from classical electrostatic interactions to specific supramolecular recognition, including π-π stacking and hydrogen bonding. Through continuous chemical evolution and comprehensive structure-activity relationship (SAR) analysis of UB-PILs, the inventors identified a lead UB-PIL, Nii-C4-LD-a10a12K2, superior to the FDA-approved ALC-0315 lipid, and discovered key molecular determinants governing delivery efficacy.

[0539] The non-electrostatic bridging mechanism endows the AllSet platform with formulation simplicity and therapeutic versatility. By circumventing the need for acidic buffers, external heating, and dedicated microfluidic equipment, the post-assembly process requires only pure water hydration and a brief room-temperature vortex. The mild formulation environment maintains the structural integrity of fragile payloads and significantly expands the encapsulable cargo spectrum. The inventors have demonstrated that AllSet LNPs are not only effective at encapsulating large cargoes (e.g., mRNA, pDNA, and Cas9 RNPs), but also at encapsulating ultrasmall or neutral charged nucleic acids (including cGAMP, PNA, and PMO) that completely circumvent conventional benchmark lipids such as ALC-0315. Furthermore, this universal base-mediated assembly exhibits broad applicability. The AllSet technology can be integrated into established commercial formulations (e.g., SM-102, ALC-0315, and MC3) and advanced organ-targeting platforms (e.g., PILOT and SORT) by directly conjugating universal bases to existing lipid backbones or by incorporating a lead Nii-lipid as an additional component. From a translational perspective, developing environmentally stable genetically modified drugs will greatly facilitate equitable deployment in resource-limited regions where the supply chains required for cold chain transportation are often insufficient. 44The AllSet platform allows vector / cargo integrity and in vivo potency to be maintained at room temperature for at least 8 months, eliminating reliance on cryogenic infrastructure. Crucially, this superior thermal stability robustly preserves the bioactivity of a wide range of genetic drugs, from prophylactic vaccines to CRISPR-based genome editing and siRNA-mediated gene silencing therapies. Furthermore, the on-demand pure water hydration and vortexing methods significantly simplify preparation. This allows for the cost-effective stockpiling and rapid on-site formulation of a broad range of nucleic acid therapeutics, supporting a shift towards decentralized manufacturing and regional distribution hubs, thereby reducing reliance on centralized imports. 3,44,50 .

[0540] In summary, the inventors have developed a thermostable AllSet LNP platform based on UB-PIL, which enables the rapid and on-site generation of multifunctional nucleic acid therapeutics. By eliminating the need for cold chain logistics and complex formulation conditions, the AllSet platform offers a potential solution to make diverse genetic medicines more accessible and affordable, ultimately advancing global health equity.

[0541] sheet

[0542] Table 1. Buffer composition.

[0543]

[0544] Table 2. Coding sequences of mRNAs for luciferase, RSV preF antigen, H1N1 antigen, RVG antigen, Cas9 protein, ABEmax protein, and PEmax protein.

[0545]

[0546] Table 3. LNP formulations tested in this invention.

[0547]

[0548] Table 4. sgRNAs used in this invention.

[0549]

[0550] The chemically modified sgPCSK9 was synthesized by GenScript Biotech.

[0551] Table 5. Membrane-labeled EGFP sequences containing the TAG stop codon.

[0552]

[0553] Table 6. The epegRNA used in this invention.

[0554]

[0555] Chemically modified epigRNA-mEGFP is synthesized using IDT. PBS: Primer binding site. RTT: Reverse transcription template.

[0556] Table 7. Primers used for Sanger sequencing and NGS sequencing.

[0557]

[0558] Table 8. Amplicon sequences analyzed by NGS sequencing.

[0559]

[0560] Table 9. siRNA used in this invention.

[0561]

[0562] The chemically modified siRNA was synthesized by Suzhou Biosyntech.

[0563] Table 10. TTR mouse qPCR primers used in this invention.

[0564]

[0565] References

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[0617] All references cited in this invention, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as each reference individually and expressly indicated by reference and fully listed herein. The foregoing description is intended only to illustrate the technical solutions of this invention and does not limit its scope of protection. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solutions of this invention do not depart from the essence and scope of this invention.

Claims

1. A universally base-modified ionizable lipid, characterized in that, The universally base-modified ionizable lipids include (i) Ionizable lipid backbone, and (ii) A universal base group covalently linked to the ionizable lipid backbone, The universal base group is directly attached to the ionizable lipid backbone, or attached to the ionizable lipid backbone via a spacer group.

2. The universally base-modified ionizable lipid according to claim 1, characterized in that, The universal base group is derived from one or more universal bases selected from the following or their derivatives: hypoxanthines, purines, isoquinolinones, pyrimidines, and so on. Azides, nitropyrroles, nitroindoles, nitroimidazolides, formylpyrroles, nitrobenzodioxanepentenes, benzotriazoles, cyanuric acids, diaminotriazines, acylaminopyridines, pyrimidines, pteridine analogs, urea-based pyrimidinones, and general bases or their derivatives.

3. The universally base-modified ionizable lipid according to claim 1, characterized in that, The universal base group is derived from a universal base or its derivative that has a reactive functional group.

4. The universally base-modified ionizable lipid according to claim 1, characterized in that, The number of universal base groups covalently linked to the ionizable lipid backbone is 1-10.

5. The universally base-modified ionizable lipid according to claim 1, characterized in that, The ionizable lipid backbone is derived from ionizable lipids with reactive sites.

6. The universally base-modified ionizable lipid according to claim 5, characterized in that, The ionizable lipid with reactive sites is an ionizable lipid containing a protonable amino group and one or more hydrophobic tails.

7. The universally base-modified ionizable lipid according to claim 5, characterized in that, The ionizable lipid with reactive sites is a peptide-based ionizable lipid PIL with the structure of Formula II. Formula II, in, A4 represents a hydrogen atom or an alkyl group. n is an integer from 1 to 30. X is O or S. R1 represents the N-terminus of the PIL, and R1 is a hydrogen atom or modified with an acetyl group, amino acid, and / or other functional groups. R2 represents the side group contained in the PIL, and R2 is independent each time it appears. Natural or non-natural amino acid side groups, and at least one R2 in the PIL is , R3 represents the C-terminus of the PIL, and R3 is a hydroxyl group or has an amino group, an amino acid group, and / or other functional group modification. m is an integer between 0 and 10. A1 and A2 are hydrophobic tails and are independently substituted alkyl chains of 4 to 25 carbon atoms, either saturated or unsaturated, linear or branched.

8. The universally base-modified ionizable lipid according to claim 7, characterized in that, n is 2.

9. The universally base-modified ionizable lipid according to claim 7, characterized in that, The adjacent amino acid building blocks in the PIL are in an anatomical configuration.

10. The universally base-modified ionizable lipid according to claim 7, characterized in that, m is 4.

11. The universally base-modified ionizable lipid according to claim 7, characterized in that, The universal base group is covalently linked to any covalently linked site in the PIL.

12. The universally base-modified ionizable lipid according to claim 7, characterized in that, The universal base group is covalently attached to the N-terminus, C-terminus, amino acid side group, or hydrophobic tail of the PIL.

13. The universally base-modified ionizable lipid according to claim 12, characterized in that, The universal base group is linked to the N-terminal amino group of the PIL via a spacer group.

14. The universally base-modified ionizable lipid according to claim 7, characterized in that, A1 and A2 are saturated alkyl chains.

15. The universally base-modified ionizable lipid according to claim 7, characterized in that, The universally modified ionizable lipid is Nii-C4- LD -a10a12K2: ， Nip-a12Dab4, Am-K2K(Nii)a12K4 or a12K4K(Nii)E-Ca: ， ， 。 16. Use of universal bases in modifying ionizable lipids to endow said ionizable lipids with the ability to assemble nucleic acids in a neutral aqueous phase.

17. The use according to claim 16, characterized in that, The neutral aqueous phase is an aqueous medium with a pH of 6.0 to 8.

0.

18. A lipid nanoparticle, characterized in that, The lipid nanoparticles comprise ionizable lipids with universal base modification according to any one of claims 1 to 15.

19. A method for preparing lipid nanoparticles encapsulating nucleic acids, wherein the lipid nanoparticles comprise ionizable lipids with universal base modification according to any one of claims 1 to 15, characterized in that, The method includes: (a) A lipid phase containing the universally modified ionizable lipid is mixed with an aqueous phase containing the nucleic acid via microfluidics to form lipid nanoparticles encapsulating the nucleic acid, wherein the aqueous phase is an acidic or neutral medium. (b) Empty lipid nanoparticles without nucleic acid are prepared from a lipid composition containing the universally modified ionizable lipids, and the empty lipid nanoparticles are then incubated with the nucleic acid to form lipid nanoparticles encapsulated with nucleic acid, wherein the empty lipid nanoparticles are prepared in an acidic or neutral medium. (c) Empty lipid nanoparticles without nucleic acid are prepared from a lipid composition comprising the universally modified ionizable lipids, and the empty lipid nanoparticles and the nucleic acid are lyophilized separately to obtain separated lyophilized empty lipid nanoparticles and lyophilized nucleic acid; before use, the separated lyophilized empty lipid nanoparticles and lyophilized nucleic acid are post-assembled to form lipid nanoparticles encapsulating nucleic acid, wherein the post-assembly is performed in an acidic or neutral medium; or (d) After forming nucleic acid-containing lipid nanoparticles, the nucleic acid-containing lipid nanoparticles are freeze-dried as a whole and reconstituted before use to obtain reconstituted nucleic acid-encapsulated lipid nanoparticles.

20. The method according to claim 19, characterized in that, In (c), the empty lipid nanoparticles or the nucleic acid are mixed with a freeze-drying protectant before freeze-drying.

21. The method according to claim 19, characterized in that, The neutral medium is an aqueous medium with a pH of 6.0 to 8.

0.

22. The method according to claim 19, characterized in that, The post-assembly is performed at a temperature of 4°C to 40°C.

23. A kit for preparing lipid nanoparticles encapsulating nucleic acids, characterized in that, The kit includes: (i) a first container comprising lyophilized empty lipid nanoparticles, said empty lipid nanoparticles comprising ionizable lipids with universal base modification according to any one of claims 1 to 15; and (ii) A second container containing lyophilized nucleic acids; The components in the first and second containers are adapted for post-assembly before use to form lipid nanoparticles encapsulating nucleic acids.

24. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises lipid nanoparticles according to claim 18 or lipid nanoparticles encapsulating nucleic acids as defined in any one of claims 19 to 23, and a pharmaceutically acceptable carrier.

25. The use of the lipid nanoparticles according to claim 18, the kit according to claim 23, or the pharmaceutical composition according to claim 24 in the preparation of a medicament, characterized in that, The drug is used for vaccine therapy, protein replacement therapy, gene editing therapy, gene silencing therapy, or in vivo cell engineering therapy.

26. The use according to claim 25, characterized in that, The drug is used to treat or prevent diseases including respiratory syncytial virus infection, H1N1 influenza A, rabies virus infection / rabies, familial hypercholesterolemia, cardiovascular disease, and hereditary or wild-type transthyretin amyloidosis.

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