Lipid-conjugate nanoparticles for targeted delivery of nucleic acid therapeutics to the lungs

Abstract

This disclosure relates to the field of drug delivery systems comprising nanoparticles, particularly lipid nanoparticles (LNPs) and messenger RNA (mRNA), and to methods for target delivery of nanoparticles to the lungs.

Description

[Background technology]

[0001] Cross-reference of related applications This application claims priority and interest to International Patent Application No. PCT / CN2023 / 122744, filed on 28 September 2023, and the entire content, including all drawings and arrangement, is incorporated herein by reference.

[0002] In recent years, mRNA-based therapies have shown great therapeutic potential as vaccines or treatments after decades of development. For example, the U.S. Food and Drug Administration (FDA) recently approved two mRNA vaccines (mRNA-1273 and BNT162b2) against COVID-19, and these vaccines have been very successful in preventing the spread of the COVID-19 pandemic.

[0003] However, the therapeutic potential of mRNA-based therapies extends far beyond the realm of vaccines, encompassing treatments for infectious diseases, chronic diseases, rare genetic disorders, and tumors, all of which can be treated and / or prevented by designing specific mRNAs that encode target proteins or antigens.

[0004] As is well known, mRNA can be readily degraded by RNases, which are ubiquitous in the air or in vivo. Furthermore, because mRNA molecules have a large negative charge, they cannot be internalized by negatively charged cell membranes due to strong electrostatic repulsion. In addition, endosomal enzymes can also degrade mRNA. These factors limit the direct delivery of mRNA in vitro or in vivo. Therefore, over the past few decades, considerable effort has been devoted to the development of various nanocarriers used for mRNA delivery. Among these nanocarriers, LNPs are the most widely used due to their mature preparation process, high safety, remarkable protective effect, and excellent delivery efficiency.

[0005] Generally, mRNA co-formulated with LNPs accumulates primarily in the liver and spleen after systemic administration. Therefore, much of the clinical interest associated with LNPs is closely related to liver disease.

[0006] However, targeted mRNA delivery to extrahepatic organs or tissues remains a major challenge, limiting the applicability of nucleic acid (siRNA or mRNA) therapies. Other therapies, such as small molecules and proteins, also require effective targeted delivery systems to mitigate the adverse effects of their distribution in the body. Therefore, there is an urgent need for targeted delivery systems to address the current dilemmas in precise target organ delivery.

[0007] Targeted therapies for the lungs have been studied in hereditary, refractory lung diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a progressive and fatal lung disease. Conventional chemotherapy is the primary treatment for IPF. For example, nintedanib and pirfenidone are approved by the US FDA for the treatment of IPF. However, several undesirable side effects, such as severe nausea and diarrhea, have been observed in preclinical trials in many patients.

[0008] In recent years, gene therapy has been increasingly considered as a potential alternative treatment option. However, due to impaired respiratory function, thickening of airway and lung epithelial cells, and the inevitable increase in mucosal secretions in these patients, administering therapeutic agents to IPF patients via the respiratory route remains a challenge. Therefore, there is an urgent need for delivery strategies that ensure accurate delivery of therapeutic agents to the lungs after systemic administration.

[0009] To address these challenges, several additional lipid-based targeted delivery systems have been developed, such as selective organ-targeted lipid nanoparticles (SORT LNPs). SORT LNPs are doped with a high proportion of permanent cationic lipids, typically DOTAP, which accounts for 50% of total lipids, as a fifth component of the lipid nanoparticles (LNPs). However, SORT LNPs contain a large proportion (typically over 75%) of charged lipids, including ionized lipids and permanent cationic lipids, which can cause toxicity in clinical applications. Furthermore, tissue targeting is achieved simultaneously with a loss of expression efficiency. [Overview of the Initiative]

[0010] The present invention, as described herein, provides a lipid-based targeted delivery system having excellent lung targeting capabilities. The delivery system described herein can be used to deliver any cargo molecule of interest, including but not limited to polynucleotides or their analogs / mimies, polypeptides or their mimes / mimies, lipids, polysaccharides, and small molecules. For example, polynucleotides that can be delivered using the delivery system described herein include mRNA that can be selectively / preferentially delivered to lung tissue while simultaneously maintaining high expression efficiency of the delivered mRNA.

[0011] The delivery system described herein is characterized in part by its overall low cationic lipid content, thereby reducing the potential for toxicity.

[0012] More specifically, in one of the attached drawings, the delivery system of the present invention is a target lipid complex nanoparticle (tLCNP) comprising five or six lipids, including two different types of charged lipids such as ionized lipids and permanent cationic lipids, one or two phospholipids, one or two sterols, and one stealth lipid derivative such as PEGylated lipids. Due to lung-selective or lung-targeted delivery and high efficacy of therapeutic agents, this delivery system allows for easier regulation of nucleic acid therapy delivered by the system, and thus has clear advantages compared to existing targeted delivery systems.

[0013] More detailed embodiments of the present invention are described in the following numbered paragraphs.

[0014] 1. Target lipid complex nanoparticles (tLCNPs) that preferentially deliver therapeutic agents encapsulated / formulated within tLCNPs to the lungs of mammals, wherein the tLCNPs are (1) A first lipid composition comprising ionized lipid C1 and cationic lipid C2, (2) A second lipid composition comprising a first phospholipid PI and a second phospholipid PII, (3) Steroids and, (4) containing stealth lipids, Here, (A) The above ionized lipid C1 has the formula C1-a, C1-b, C1-c, or C1-d: [ka] During the ceremony, R1 and R2 are independently -CH3 and -(CH2) 1-8 -CH3, or -(CH2) 1-8 -OH G1 and G2 are independently -(CH2) 1-10 -and, G3 and G4 are independently -(CH2) 0-5 -and, L1 is -O-(C=O)-O-, -O-(C=O)-, -(C=O)-O-, -SS-, -O-, R3 and R4 operate independently of each other. [ka] And, R5 is -H, or -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-14 Alkyl, or C 1-14 It is alkenyl, R6 is -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-20 Alkyl, or C 1-20alkenyl, and X is C or [Chemical Formula] , and n1 is an integer of 2 to 8, (B) The aforementioned cationic lipid C2 has the formula of C2-a, C2-b, C2-c, or C2-d: [Chemical Formula] In the formula, R7, R8, and R9 are each independently -CH3, -(CH2) 1-8 -CH3, or -(CH2) 1-8 -OH, R 10 and R 11 are each independently, [Chemical Formula] , R 12 is -H, -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-14 alkyl, or C 1-14 alkenyl, R 13 is -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-20 alkyl, or C 1-20 alkenyl, M is carbon or nitrogen, A is -CH2-, -G7-L2-G8-, or -(CH2) 1-5 -(C=O)-, wherein the -(C=O)- is linked to M, G5, G6, G7, and G8 are each independently -(CH2) 0-10 -, n2 is an integer of 0 to 10, L2, L3, and L4 are each independently -CH2-, -O-(C=O)-O-, -O-(C=O)-, -(C=O)-O-, -S-S-, -O-, -S-, -HN-(C=O)-, X- Cl - and Br - These are monovalent anions (e.g., halo groups), (C) Optionally, the first phospholipid PI has the formula PI: [ka]

[0015] In the formula, R 14 , R 15 , and R 16 These are independently -H, -CH3, and -(CH2) 1-6 -CH3, or -(CH2) 1-6 -OH n3 is an integer between 5 and 20 in each case, and n4 is an integer between 1 and 5, and (D) Optionally, the second phospholipid PII has the formula PII: [ka] In the formula, R 17 , R 18 , and R 19 These are independently -H, -CH3, and -(CH2) 1-6 -CH3, or -(CH2) 1-6 -OH n5 and n6 are, independently, integers between 5 and 8 in each case. n7 is an integer between 1 and 5. Targeted lipid complex nanoparticles (tLCNPs).

[0016] 2. The steroid described above is tLCNP as described in paragraph 1, which contains, is essentially composed of, or is composed of cholesterol.

[0017] 3. The above stealth lipids are DMG-PEG2000 ( [ka] tLCNPs according to paragraph 1 or 2, comprising, essentially composed of, or composed of PEGylated lipids having PEG(-O-CH2-CH2-) repeats such as ).

[0018] 4. R5 is the tLCNP described in paragraph 1, comprising H, -(CH2)3CH3, -(CH2)5CH3, -(CH2)7CH3, or -(CH2)9CH3.

[0019] 5. R5 is the tLCNP described in paragraph 1, which includes H, -(CH=CH)(CH2)3CH3, -(CH2)3(CH=CH)CH2CH3, -(CH=CH)(CH2)4CH3, or -(CH=CH)(CH2)5CH3, or -(CH=CH)(CH2)7CH3.

[0020] 6. R6 is the tLCNP described in paragraph 1, comprising -(CH2)3CH3, -(CH2)5CH3, -(CH2)7CH3, or -(CH2)9CH3.

[0021] 7. R6 is the tLCNP described in paragraph 1, which includes -(CH=CH)(CH2)3CH3, -(CH2)3(CH=CH)CH2CH3, -(CH=CH)(CH2)4CH3, -(CH=CH)(CH2)5CH3, or -(CH=CH)(CH2)7CH3.

[0022] 8. R6 is H, -(CH2)3CH3, -(CH2)5CH3, -(CH2)7CH3, -(CH2)9CH3, or -(CH2) 11 tLCNP as described in paragraph 1, including CH3.

[0023] 9. R6 is the tLCNP described in paragraph 1, which includes H, -(CH=CH)(CH2)3CH3, -(CH2)3(CH=CH)CH2CH3, -(CH=CH)(CH2)4CH3, -(CH=CH)(CH2)5CH3, -(CH=CH)(CH2)7CH3, or -(CH2)7(CH=CH)(CH2)7CH3.

[0024] 10. R7 is -(CH2)3CH3, -(CH2)5CH3, -(CH2)7CH3, -(CH2)9CH3, or -(CH2) 11 tLCNP as described in paragraph 1, including CH3.

[0025] 11. R7 is the tLCNP described in paragraph 1, which includes -(CH=CH)(CH2)3CH3, -(CH2)3(CH=CH)CH2CH3, -(CH=CH)(CH2)4CH3, or -(CH=CH)(CH2)5CH3, -(CH=CH)(CH2)7CH3, or -(CH2)7(CH=CH)(CH2)7CH3.

[0026] 12. The above ionized lipid C1 is, [ka] , [ka] , [ka] , [ka] , or [ka] including, tLCNP as described in any one of paragraphs 1-11.

[0027] 13. The above cationic lipid C2 is, [ka] , [ka] , [ka] , [ka] , or [ka] (BHEM-cholesterol) tLCNP as described in any one of paragraphs 1-12, including the following.

[0028] 14. The first phospholipid PI mentioned above is [ka] or [ka] including, tLCNP as described in any one of paragraphs 1-13.

[0029] 15. The second phospholipid PII mentioned above is, [ka] or [ka] including, tLCNP as described in any one of paragraphs 1-14.

[0030] 16. The tLCNP according to any one of paragraphs 1 to 15, wherein the molar ratio of the cationic lipid C2 to the ionized lipid C1 is approximately 1:3 to approximately 4:1, approximately 1:1 to approximately 4:1, approximately 2:1 to approximately 4:1, approximately 3:1 to approximately 4:1, approximately 1:1, approximately 2:1, approximately 3:1, or approximately 4:1.

[0031] 17. The tLCNP described in paragraph 16, wherein the cationic lipid C2 is DOTMA and the ionized lipid C1 is lipid #4.

[0032] 18. The tLCNP according to any one of paragraphs 1 to 17, wherein the molar proportion of (1) to (4) in (1) (or the first lipid composition described above) is about 30% to 70%, about 40% to 60%, about 50% to 60%, about 30%, about 40%, about 50%, about 60%, or about 70%.

[0033] 19. The tLCNP described in paragraph 18, wherein the molar ratio of the cationic lipid C2 (such as DOTMA) to the ionized lipid C1 (such as lipid #4) is approximately 2:1.

[0034] 20. (a) The first lipid composition having a molar proportion of approximately 30% to 70%, approximately 40% to 60%, or approximately 50%. (b) The second lipid composition having a molar proportion of approximately 5% to 30% (approximately 5% to 15%, or approximately 10%), wherein the molar ratio of the first phospholipid PI (such as DSPC) to the second phospholipid PII (such as DOPC) is optionally 1:0 (100% DSPC) to 1:2 (33% DSPC), for example, 2:1 (67% DSPC) or 1:1 (50% DSPC), (c) The above steroids (such as cholesterol) with a molar ratio of approximately 10% to 40% (approximately 35% to 40%, or approximately 38.5%), (d) The above stealth lipid (DMG-PEG2000, etc.) having a molar proportion of approximately 0% to 5% (approximately 2% to 3%, or approximately 1.5%), (e) The tLCNP described in any one of paragraphs 1 to 19, comprising any combination described above, wherein the total molar proportion is 100%.

[0035] 21. The therapeutic agent is a tLCNP according to any one of paragraphs 1 to 20, comprising a polynucleotide (DNA and RNA such as mRNA, siRNA, miRNA, ASO, and pDNA), a hydrophobic and / or hydrophilic low molecular weight compound (e.g., having a molecular weight of less than 1000 Da, 750 Da, 500 Da, 250 Da, or 100 Da), or a bioactive lipid (such as a fatty acid or monoglyceride).

[0036] 22. The therapeutic agent described above is a tLCNP as described in paragraph 21, comprising polynucleotides such as RNA (e.g., siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly spaced short palindromic repeat (CRISPR)-associated nucleic acids, single guide RNA (sgRNA), CRISPR-RNA (crRNA), transactivated crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA)).

[0037] 23. The above RNA is a self-replicating mRNA, a non-self-replicating mRNA, or a circular RNA, as described in paragraph 22 of the tLCNP.

[0038] 24. The tLCNP described in paragraph 22 or paragraph 23, wherein the N / P molar ratio of the above tLCNP (lipid nitrogen: polynucleotide phosphate in the first lipid composition (permanently cationic lipid + ionized lipid)) is about 4, 6, or 8, for example, 6.

[0039] 25. When the tLCNP described above is administered to the mammal, at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the therapeutic agent is delivered to the lungs, as described in any one of paragraphs 1 to 24.

[0040] 26. When the tLCNP described above is administered to the mammal, about 30%, 25%, 20%, 15%, 10%, or 5% or less of the therapeutic agent is delivered to the liver (or a combination of other non-pulmonary tissues), as described in any one of paragraphs 1 to 25.

[0041] 27. The tLCNP described in any one of paragraphs 1 to 26, wherein the tLCNP has an average size of less than 200 nm, less than 150 nm, less than 100 nm, less than 90 nm, or less than 80 nm.

[0042] 28. The above tLCNP is a tLCNP described in any one of paragraphs 1 to 27, having a multivariance index (PDI) of approximately 0.10 to 0.25, or approximately 0.15 to 0.20.

[0043] 29. When the above tLCNP is administered to the above mammal, about 1% to 15%, about 2% to 10%, or about 3% to 5% of the above therapeutic agent is delivered to the spleen, as described in any one of paragraphs 1 to 28.

[0044] 30. A tLCNP described in any one of paragraphs 1 to 29, and a pharmaceutically acceptable carrier or excipient, Pharmaceutical composition.

[0045] 31. A method for selectively or preferentially delivering a therapeutic agent to the lungs of a mammal, comprising administering the therapeutic agent to the mammal, which is contained in or formulated in a tLCNP as described in any one of paragraphs 1 to 29. method.

[0046] 32. The method described in paragraph 31, wherein the mammal is a human.

[0047] 33. The method according to paragraph 31 or 32, wherein the tLCNP is administered intravenously to the mammal described above.

[0048] 34. The therapeutic agent described above comprises a polynucleotide such as RNA, as described in any one of paragraphs 31 to 33.

[0049] 35. The method according to any one of paragraphs 31 to 34, wherein the mammal has a lung disease, and the therapeutic agent is effective in treating the lung disease.

[0050] 36. The method according to paragraph 35, wherein the lung disease is lung cancer, asthma, pneumothorax, atelectasis, bronchitis (including chronic bronchitis), bronchiolitis, bronchiectasis, COPD, emphysema, lung infection (e.g., pneumonia, COVID-19, bacterial infection, viral infection), cystic fibrosis, idiopathic pulmonary fibrosis (IPF), pulmonary edema, pulmonary embolism, lymphangioleiomyomatosis (LAM), or pulmonary hypertension.

[0051] 37. A method for preparing tLCNP as described in any one of paragraphs 1 to 29, the method comprising mixing a first organic liquid phase, comprising the first lipid composition, the second lipid composition, the steroid, and the stealth lipid as described in any one of paragraphs 1 to 29, with a second aqueous phase, comprising the therapeutic agent described in any one of paragraphs 1 to 29, in a water-miscible organic solvent (e.g., ethanol), to form the tLCNP. method.

[0052] 38. The method according to paragraph 37, further comprising stepwise purification of the tLCNP by a tangential flow filtration (TFF) system.

[0053] 39. The method according to paragraph 37 or 38, wherein the organic solvent in the first organic liquid phase contains, is essentially composed of, or is composed of ethanol.

[0054] 40. The method according to any one of paragraphs 37-39, wherein the first organic liquid phase and the second aqueous phase are mixed in a microfluidic apparatus in an optionally selective volume ratio of 1:3 and / or optionally selectively at a total flow rate of about 10-15 mL / min (e.g., about 12 mL / min).

[0055] 41. The aqueous phase described above comprises 50 mM citrate buffer (pH 5.0), according to any one of paragraphs 37 to 40.

[0056] 42. The stepwise purification described above is performed in the TFF system in the TFF system described above, wherein the stepwise purification is carried out in the TFF system described above in any one of paragraphs 38 to 41.

[0057] It should be understood that any one embodiment described herein, including embodiments described solely in the examples or claims, may be combined with one or more other embodiments of the present disclosure unless such combination is inappropriate or expressly denied. [Brief explanation of the drawing]

[0058] [Figure 1] This is a schematic diagram (not to scale) showing the composition and component ratio of an embodiment of the lung-targeted (tLCNP) method of the present invention. [Figure 2] The structural formulas of several representative molecules used to manufacture the lung-targeted pulmonary nucleotide polymorphism (tLCNP) are shown. [Figure 3] ~ [Figure 6] We demonstrate that tLCNPs prepared with different lipid #4 / DOTMA ratios enable systemic delivery of mRNA from the liver to the spleen and then to the lungs. (Figure 3) In vivo and ex vivo imaging of mice intravenously injected with luciferase mRNA@tLCNPs of different lipid #4 / DOTMA ratios 6 hours post-injection. (Figure 4) Quantitative data of organs expressing luciferase (data shown as mean ± SD, n=3 biologically independent animals). (Figure 5) Heatmap of luciferase expression in different organs of mice treated with the above samples. (Figure 6) Relative luciferase expression in each organ demonstrates that lung target expression can be easily enhanced by adjusting the lipid #4 / DOTMA ratio. [Figure 7] ~ [Figure 10]Figure 7 shows tLCNPs prepared with different ionized lipids, including MC3, lipid #4, lipid #13, and lipid #9. In vivo and ex vivo imaging of mice intravenously injected with luciferase mRNA@tLCNPs of different ionized lipids 6 hours post-injection. Figure 8 shows quantitative data of organs expressing luciferase (data shown as mean ± SD, n=3 biologically independent animals). Figure 9 shows heatmaps of luciferase expression in different organs of mice treated with the above samples. Figure 10 shows that relative luciferase expression in each organ readily achieved lung target expression with tLCNPs prepared with other XH series ionized lipids (including lipid #13 and lipid #9), but not with MC3. [Figure 11] ~ [Figure 14] Figure 11 shows tLCNPs prepared with different proportions of total charged lipids, i.e., lipid #4 + DOTMA. In vivo and ex vivo imaging of mice intravenously injected with luciferase mRNA@tLCNPs of different proportions of total lipid #4 + DOTMA at 6 hours post-injection. Figure 12 shows quantitative data of organs expressing luciferase (data shown as mean ± SD, n=3 biologically independent animals). Figure 13 shows heatmaps of luciferase expression in different organs of mice treated with the above samples. Figure 14 demonstrates that relative luciferase expression in each organ can easily improve lung targeting efficiency by increasing the proportion of total cationic lipids, but that if the ratio is too high, the expression intensity is impaired. [Figure 15] ~ [Figure 18]Figure 15 shows tLCNP prepared with different N / P ratios of 4, 6, and 8. In vivo and ex vivo imaging of mice intravenously injected with luciferase mRNA@tLCNP with different N / P ratios of 4, 6, and 8 at 6 hours post-injection. Figure 16 shows quantitative data of organs expressing luciferase (data shown as mean ± SD, n=3 biologically independent animals). Figure 17 shows heatmaps of luciferase expression in different organs of mice treated with the above samples. Figure 18 shows that relative luciferase expression in each organ demonstrates that an N / P ratio of 6 is optimal for lung targeting. [Figure 19] ~ [Figure 22] Figure 19 shows tLCNPs prepared with different DSPC / DOPE ratios. In vivo and ex vivo imaging of mice intravenously injected with luciferase mRNA@tLCNPs of different DSPC / DOPE ratios at 6 hours post-injection. Figure 20 shows quantitative data of organs expressing luciferase (data shown as mean ± SD, n=3 biologically independent animals). Figure 21 shows heatmaps of luciferase expression in different organs of mice treated with the above samples. Figure 22 demonstrates that relative luciferase expression in each organ can be easily enhanced by adjusting the phospholipid composition and ratios to target lung expression. [Figure 23] ~ [Figure 26]Figure 23 shows tLCNPs prepared with different DOPC / DSPC or DOPC / DOPE ratios. In vivo and ex vivo imaging of mice intravenously injected with luciferase mRNA@tLCNPs of different DOPC / DSPC or DOPC / DOPE ratios at 6 hours post-injection. Figure 24 shows quantitative data of organs expressing luciferase (data shown as mean ± SD, n=3 biologically independent animals). Figure 25 shows heatmaps of luciferase expression in different organs of mice treated with the above samples. Figure 26 demonstrates that relative luciferase expression in each organ can be easily enhanced by adjusting the phospholipid composition and ratios to target lung expression. [Figure 27] ~ [Figure 30] Regarding lung targeting, a comparison of SORT LNP and LNP:DOTMA with our innovative tLCNP is shown. (Figure 27) In vivo and ex vivo imaging of mice intravenously injected with luciferase mRNA-loaded SORT LNP, LNP:DOTMA, and tLCNP 6 hours post-injection. (Figure 28) Quantitative data of organs expressing luciferase (data shown as mean ± SD, n=3 biologically independent animals). (Figure 29) Heatmap of luciferase expression in various organs of mice treated with the above samples. (Figure 30) Relative luciferase expression in each organ demonstrated that tLCNP showed significantly greater targeting efficiency and expression intensity than SORT LNP or LNP(DOTMA). [Figure 31] ~ [Figure 33] This shows the evaluation of tLCNP targeting in rabbits. (Figure 31) Ex vivo imaging of rabbits intravenously injected with tLCNP loaded with luciferase mRNA, 6 hours after injection. (Figure 32) Quantitative data of organs expressing luciferase. (Figure 33) Elisa test, measuring RBD concentrations in the liver, lungs, and spleen of rabbits administered tLCNP (RBD) and PBS control, 4 hours after injection. [Figure 34]This study shows the 1-day mortality rate of mice injected with PBS (control) or IL12-CBD mRNA@tLCNP. Dissection was performed to determine tumor burden in the lungs, liver, spleen, kidneys, and GI. Statistical significance was achieved by the Log-rank (Mantel-Cox) test. [Modes for carrying out the invention]

[0059] The invention described herein provides lipid nanoparticle (e.g., tLCNP) formulations that preferentially deliver cargo molecules or compositions that are incorporated / encapsulated / or otherwise formulated in LNPs such as polynucleotides or their analogs / mimickings (e.g., DNA, RNA, morpholino, PNA (peptide nucleic acid), XNA (xenon nucleic acid), rok nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA)), proteins or polypeptides, and low molecular weight compounds (molecular weights of 1500 Da, 1000 Da, 800 Da, 500 Da, 300 Da, 250 Da, 200 Da, 150 Da or less).

[0060] The present invention as described herein is partly based on the remarkable discovery that when certain ionized lipids (such as CI ionized lipids as described herein) are mixed with certain permanently charged lipids (such as CII permanently charged lipids as described herein), the tissue distribution of tLCNPs prepared with such lipid mixtures can be dramatically shifted from primarily the liver to partly the liver and partly the spleen, and primarily the lungs. This enables tissue-specific delivery of cargo molecules carried by LNP particles, such as lung-preferential or lung-specific delivery of cargo molecules, even if not lung-exclusive delivery.

[0061] The inventions described herein are also based in part on the remarkable discovery that specific phospholipid components of tLCNPs can be fine-tuned to improve the targeting and / or expression efficiency of polynucleotide cargoes carried by tLCNPs.

[0062] Therefore, one aspect of the present invention as described herein provides a target lipid complex nanoparticle cargo molecule (tLCNP) that preferentially delivers a cargo molecule (e.g., a therapeutic agent) contained / formulated within the tLCNP to the lungs of a mammal, wherein the tLCNP comprises (1) a first lipid composition comprising an ionized lipid C1 and a cationic lipid C2, (2) a second lipid composition comprising a first phospholipid PI and a second phospholipid PII, (3) a steroid, and (4) a stealth lipid, where, (A) The above ionized lipid C1 has the formula C1-a, C1-b, C1-c, or C1-d: [ka] During the ceremony, R1 and R2 are independently -CH3 and -(CH2) 1-8 -CH3, or -(CH2) 1-8 -OH G1 and G2 are independently -(CH2) 1-10 -and, G3 and G4 are independently -(CH2) 0-5 -and, L1 is -O-(C=O)-O-, -O-(C=O)-, -(C=O)-O-, -SS-, -O-, R3 and R4 operate independently of each other. [ka] And, R5 is -H, -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-14 Alkyl, or C 1-14 It is alkenyl, R6 is -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-20 Alkyl, or C 1-20 It is alkenyl, X is C or [ka] And, n1 is an integer between 2 and 8. (B) The cationic lipid C2 described above has the formula C2-a, C2-b, C2-c, or C2-d: [ka] During the ceremony, R7, R8, and R9 are independently -CH3, -(CH2) 1-8 -CH3, or -(CH2) 1-8 -OH R 10 and R 11 Each of them operates independently. [ka] And, R 12 These are -H, -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-14 Alkyl, or C 1-14 It is alkenyl, R 13 These are -(CH2)7CH3, -(CH=CH)(CH2)5CH3, C 1-20 Alkyl, or C 1-20 It is alkenyl, M is either carbon or nitrogen. A is -CH2-, -G7-L2-G8-, or -(CH2) 1-5 -(C=O)-, where -(C=O)- is connected to M, G5, G6, G7, and G8 are independently -(CH2) 0-10 -and, n2 is an integer between 0 and 10. L2, L3, and L4 are independently -CH2-, -O-(C=O)-O-, -O-(C=O)-, -(C=O)-O-, -SS-, -O-, -S-, and -HN-(C=O)-, respectively. X - Cl - and Br - These are monovalent anions (e.g., halo groups), (C) Optionally, the first phospholipid PI has the formula PI: [ka]

[0063] In the formula, R 14 , R 15 , and R 16 These are independently -H, -CH3, and -(CH2) 1-6 -CH3, or -(CH2) 1-6 -OH n3 is an integer between 5 and 20 in each case, and n4 is an integer between 1 and 5, and (D) Optionally, the second phospholipid PII has the formula PII: [ka] In the formula, R 17 , R 18 , and R 19 These are independently -H, -CH3, and -(CH2) 1-6 -CH3, or -(CH2) 1-6 -OH n5 and n6 are, independently, integers between 5 and 8 in each case. n7 is an integer between 1 and 5.

[0064] While we do not wish to be bound by any particular theory, the ionized lipids described herein avoid the undesirable cytotoxicity associated with cationic lipids. The ionized lipids described herein have transient cationic lipids obtained at low pH (e.g., pH 5-6, such as about pH 5.0 or 5.5) and form reverse micelles that encapsulate cargo molecules (such as polynucleotides) in LNP cores. These lipids have a nearly neutral charge at physiological pH and deliver cargo molecules with significantly reduced toxicity or without cytotoxicity.

[0065] Ionized lipids useful for the present invention as described herein can be prepared, for example, based on WO2022166213A1 and WO2023104114A2 (incorporated herein by reference).

[0066] In a particular embodiment, C 1-14 Alkyl or C 1-20 Alkyl compounds include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, or their branched variants (such as isopropyl, 3-pentyl, and sec-pentyl).

[0067] In a particular embodiment, C 1-14 Alkenyl or C 1-20 Alkenyls include ethylene, propylene, 1-butene, 2-butene, isobutylene, 1-pentene, 2-pentene, 2-methyl-1-butene, 3-methyl-1-butene, 2-methyl-2-butene, 1-hexene, 2-hexene, 3-hexene, 2-methyl-1-pentene, 3-methyl-1-pentene, 4-methyl-1-pentene, 2-methyl-2-pentene, 3-methyl-2-pentene, 4-methyl-2-pentene, 2,3-dimethyl-1-butene, 3,3-dimethyl-1-butene, 2,3-dimethyl-2-butene, and 2-ethyl-1-butene. In certain embodiments, the alkenyl group contains 1, 2, 3, 4, or 5 double bonds.

[0068] In certain embodiments, the phospholipids include phosphatidylcholine such as distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and / or phosphoethanolamine, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0069] In certain embodiments, the first phospholipid PI in tLCNP has the formula PI as defined above. In certain embodiments, the second phospholipid PII in tLCNP has the formula PII as defined above. In certain embodiments, the first phospholipid PI in tLCNP has the formula PI as defined above, and the second phospholipid PII in tLCNP has the formula PII as defined above.

[0070] In certain embodiments, the steroid contains, is essentially composed of, or is composed of sterol lipids, such as cholesterol.

[0071] While we do not wish to be bound by any particular theory, sterol lipids such as cholesterol fill in gaps in the lipid membrane packing, providing structural integrity to the tLCNPs in question. Furthermore, these may facilitate membrane fusion between tLCNPs and target cells.

[0072] In certain embodiments, sterol lipids include steroids and / or secosteroids, such as animal sterols, plant sterols, and mycosterols, as well as combinations and mixtures thereof. In some embodiments, sterol lipids include cholesterol and / or plant sterols, and / or at least one secosterol, preferably at least one calciferol (vitamin D), and in particular, the steroids and / or secosteroids, especially sterols, have a Griffin HLB value of up to 1.5, and / or in particular, the steroids and / or secosteroids, especially sterols, have a log P value of at least 5.

[0073] In certain embodiments, the sterol lipid is cholesterol, cholesterol sulfate, or derivatives thereof, such as 7α-hydroxycholesterol (which may improve the delivery of oligonucleotide cargoes).

[0074] [ka] Again, while we do not wish to be bound by any particular theory, the PEGylated lipids in tLCNPs described herein prevent serum protein adsorption and inhibit uptake by mononuclear phagocytic systems (MPS), which is a major obstacle in LNP delivery. In certain embodiments, the PEGylated lipids also contain terminal functional groups such as amines or maleimides, which can be used to conjugate other molecules that improve cell targeting and uptake.

[0075] In certain embodiments, the PEGylated lipid / stealth lipid is DMG-PEG2000( [ka] It contains, is essentially composed of, or is composed of PEGylated lipids having PEG(-O-CH2-CH2-) repeats, such as ).

[0076] In certain embodiments, the PEGylated lipid / stealth lipid comprises, essentially constitutes, or is composed of C-DMG-PEG(2000) / PEG(2000)-C-DMG or ALC-0159.

[0077] In a particular embodiment, ionized lipid C1 is [ka] , [ka] , or [ka] , including.

[0078] In certain embodiments, ionized lipid C1 may also be replaced by lipids such as ALC-0315 (shown below) or SM-102 (shown below): [ka] .

[0079] In certain embodiments, cationic lipid C2 is [ka] , [ka] , [ka] , [ka] , or [ka] , including.

[0080] Glycerophospholipids are a class of phospholipids that consist of a hydrophilic head group and two hydrophobic fatty acid acyl terminals bonded to a glycerol backbone. The hydrophilic head group determines the charge of the tLCNP particle, which can theoretically be neutral, anionic (negative), or cationic (positive). In certain embodiments, the phospholipids in tLCNPs are anionic lipids such as phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA).

[0081] In a particular embodiment, the first phospholipid PI is [ka] or [ka] Includes.

[0082] In certain embodiments, the second phospholipid PII is [ka] or [ka] Includes.

[0083] In certain embodiments, the molar ratio of cationic lipid C2 to ionized lipid C1 is approximately 1:3 to approximately 4:1, approximately 1:1 to approximately 4:1, approximately 2:1 to approximately 4:1, approximately 3:1 to approximately 4:1, approximately 1:1, approximately 2:1, approximately 3:1, or approximately 4:1.

[0084] In certain embodiments, the cationic lipid C2 is DOTMA, and the ionized lipid C1 is lipid #4.

[0085] In certain embodiments, the molar proportion of (1) (or the first lipid composition) comprising (1) to (4) is approximately 30% to 70%, approximately 40% to 60%, approximately 50% to 60%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, or approximately 70%.

[0086] In certain embodiments, the molar ratio of cationic lipid C2 (such as DOTMA) to ionized lipid C1 (such as lipid #4) is approximately 2:1.

[0087] In certain embodiments, the tLCNP described herein is (a) a first lipid composition having a molar proportion of about 30% to 70%, about 40% to 60%, or about 50%, and (b) a second lipid composition having a molar proportion of about 5% to 30% (e.g., about 5% to 15%, or about 10%), wherein the molar ratio of the first phospholipid PI (e.g., DSPC) to the second phospholipid PII (e.g., DOPC) is 1:0 (100% DSPC) to 1:2 (33% DSPC). (c) a second lipid composition in a ratio of 2:1 (67% DSPC) or 1:1 (50% DSPC), (d) a steroid (such as cholesterol) in a molar proportion of approximately 10% to 40% (approximately 35% to 40%, or approximately 38.5%), (d) a stealth lipid (such as DMG-PEG2000) in a molar proportion of approximately 0% to 5% (approximately 2% to 3%, or approximately 1.5%), or any combination of the above in a total molar proportion of 100%.

[0088] In certain embodiments, the tLCNP includes an internal aqueous phase that solubilizes hydrophilic cargo molecules, such as oligonucleotides, within the tLCNP.

[0089] In certain embodiments, the therapeutic agent cargo comprises polynucleotides (including DNA and RNA, such as mRNA, siRNA, miRNA, ASO, and pDNA), hydrophobic and / or hydrophilic low molecular weight compounds (e.g., having molecular weights of 1000 Da, 750 Da, 500 Da, 250 Da, or less than 100 Da), or bioactive lipids (such as fatty acids or monoglycerides).

[0090] In certain embodiments, the therapeutic agent comprises polynucleotides such as RNA (e.g., siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly spaced short palindromic repeat (CRISPR)-associated nucleic acids, single guide RNA (sgRNA), CRISPR-RNA (crRNA), transactivating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA)).

[0091] In certain embodiments, the RNA is self-replicating mRNA, non-self-replicating mRNA, or circular RNA.

[0092] In certain embodiments, the cargo is a low molecular weight. In certain embodiments, the low molecular weight is a hydrophilic agent solubilized in the aqueous compartment of tLCNP. In certain embodiments, the low molecular weight is doxorubicin, a hydrophilic agent that is an antitumor antibiotic.

[0093] In certain embodiments, the cargo is a bioactive lipid. Fatty acids, monoglycerides, and other bioactive lipids may also be incorporated into the tLCNP system of the present invention. In certain embodiments, the bioactive lipid includes lauric acid, linoleic acid, oleic acid, C6 ceramide, or sphingolipids.

[0094] In certain embodiments, the N / P molar ratio of tLCNP (lipid nitrogen:polynucleotide phosphate of the first lipid composition (permanently cationic lipid + ionized lipid)) is about 4, 6, or 8, for example, 6.

[0095] As used herein, the "N / P molar ratio" refers to the ratio of the molar amount of charged nitrogen (N) in the first lipid composition (permanent cationic lipid + ionized lipid) to the molar amount of phosphorus (P) in the polynucleotide in the LNP preparation.

[0096] In certain embodiments, when tLCNP is administered to mammals, at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the therapeutic agent is delivered to the lungs.

[0097] In certain embodiments, when tLCNP is administered to mammals, approximately 30%, 25%, 20%, 15%, 10%, or less than 5% of the therapeutic agent is delivered to the liver (or a combination of other non-pulmonary tissues).

[0098] In certain embodiments, the tLCNPs described herein have an average size of less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 90 nm, or less than about 80 nm.

[0099] In certain embodiments, the tLCNP described herein has a polyvariance index (PDI) of about 0.10 to 0.25, or about 0.15 to 0.20.

[0100] In certain embodiments, when tLCNP is administered to mammals, approximately 1% to 15%, 2% to 10%, or 3% to 5% of the therapeutic agent is delivered to the spleen.

[0101] Another aspect of the present invention provides a pharmaceutical composition / formulation comprising the tLCNP described herein and a pharmaceutically acceptable carrier or excipient.

[0102] In certain embodiments, the pharmaceutical composition / formulation is formulated for systemic administration, for example, intravenous injection / administration.

[0103] As used herein, the term “systemic administration” has the meaning commonly accepted in the art. Generally, the term refers to a method or technique of administering a molecule, drug, agent, or compound in a manner that results in the systemic absorption or accumulation of the drug in vivo in the bloodstream, followed by systemic distribution. Systemic administration includes intraocular administration.

[0104] In certain embodiments, systemic administration includes, as is commonly known in the art, pulmonary administration (such as inhalation or spray administration), intravenous administration, subcutaneous administration, catheterization, nasopharyngeal administration, or oral / gastrointestinal administration.

[0105] Further non-limiting examples of the systemic administration methods of the present invention include oral administration, sublingual administration, parenteral administration (i.e., intravenous, intraperitoneal, subcutaneous, or intramuscular administration), topical rectal administration, or other topical administration, which result in the absorption or accumulation of the administered composition in the bloodstream, followed by systemic distribution.

[0106] In one embodiment, systemic administration includes blowing and inhalation.

[0107] In one embodiment, systemic administration includes intravenous or intraperitoneal administration by bolus injection (see, for example, U.S. Patent No. 5,286,634). In another embodiment, tLCNP is administered subcutaneously. In yet another embodiment, tLCNP and its pharmaceutical composition are administered intravenously (IV).

[0108] The intravenously administered tLCNPs described herein preferentially direct / deliver cargo molecules to the lungs, if not exclusively. However, certain tLCNP formulations described herein may also deliver large amounts of cargo molecules to the spleen and may be used for spleen-targeted delivery.

[0109] Pharmaceutical compositions / formulations can be prepared by any method known or to be developed in the pharmaceutical field. Generally, such preparation methods include the steps of combining an active ingredient (e.g., the tLCNP formulation described herein) with one or more excipients and / or one or more other auxiliary components, and then, if necessary and / or desirable, shaping and / or packaging the product into desired single or multiple dose units.

[0110] In certain embodiments, the pharmaceutical composition may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple single unit doses.

[0111] As used herein, “unit dose” refers to a specific amount of a pharmaceutical composition containing a predetermined amount of the therapeutic agent (e.g., mRNA, ASO, siRNA, low-chemical drug molecule, etc.) contained in / formulated in the tLCNP formulation described herein. The amount of therapeutic agent (or an integer multiple such as 2X, 3X, 4X) is generally equal to the dose of therapeutic agent administered to the subject.

[0112] In certain embodiments, the unit dose of the pharmaceutical composition may be further divided into fractions such as 1 / 2, 1 / 3, 1 / 4, and 1 / 5 of the unit dose. Such fractions of the unit dose may be useful for specific patient populations, such as pediatric patients.

[0113] The relative or exact amounts of therapeutic agents, pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical compositions of the present invention that are incorporated / formulated within the tLCNP formulations described herein vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route through which the composition is administered.

[0114] In certain embodiments, the pharmaceutical formulation may further contain pharmaceutically acceptable excipients. These excipients include any and all solvents, dispersions, diluents, or other liquid vehicles, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for the desired specific dosage form as used herein. Remington's *The Science and Practice of Pharmacy*, 21st Edition, Argennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006) discloses various excipients used to formulate pharmaceutical compositions and known techniques for their preparations. Any use of any conventional excipient is considered within the scope of the present invention unless it is incompatible with the substance or its derivatives, for example, if it produces any undesirable biological effect or interacts adversely with other components of the pharmaceutical composition.

[0115] In some embodiments, the pharmaceutically acceptable excipients are of at least 95%, 96%, 97%, 98%, 99%, or 100% purity. In some embodiments, the excipients are approved for use in human and veterinary medicine. In some embodiments, the excipients are approved by the U.S. Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and / or International Pharmacopoeia.

[0116] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Such excipients may optionally be included in pharmaceutical formulations containing immunogenic nanoparticles as described herein. Excipients such as cocoa butter, suppository waxes, colorants, coatings, sweeteners, flavorings, and fragrances may be present in the composition at the discretion of the formulationer.

[0117] Examples of diluents include, but are not limited to, sodium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and combinations thereof.

[0118] Examples of granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium glycolate starch, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium glycolate starch), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (bee gum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0119] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate], beegum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymer, carboxyvinyl polymer), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN20 (登録商標) ], polyoxyethylene sorbitan [TWEEN60 (登録商標)], polyoxyethylene sorbitan monooleate [TWEEN80 (登録商標) ], Sorbitan monopalmitate [SPAN40 (登録商標) ], sorbitan monostearate [SPAN (登録商標) 60], sorbitan tristearate [SPAN (登録商標) 65], Glyceryl monooleate, Sorbitan monooleate [SPAN (登録商標) 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ (登録商標) 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, sorbitol), sucrose fatty acid ester, polyethylene glycol fatty acid ester (e.g., CREMOPHOR (登録商標) ), polyoxyethylene ether (for example, polyoxyethylene lauryl ether [BRIJ (登録商標) 30) (polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic acid (登録商標) Examples include, but are not limited to, F 68, poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0120] Examples of disintegrants include, but are not limited to, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate.

[0121] Examples of binders include, but are not limited to, starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwal gum, gatti gum, isapole shell mucus, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), aluminum magnesium silicate (bee gum), and larch alabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, waxes, water, alcohols, and combinations thereof.

[0122] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, α-tocopherol, ascorbic acid, acorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium pyrosulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Examples of alcohol-based preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium pyrosulfite, potassium sulfite, potassium pyrosulfite, and GLYDANT PLUS. (登録商標) PHENONIP (登録商標) Methylparaben, Germall 115, Germaben II, Neolone™, Kathon™, and EUXYL (登録商標) Examples of preservatives may include, but are not limited to, those listed above. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0123] Examples of buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dicalcium phosphate, phosphate, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dicalocotium phosphate, monopotassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dicalocotium phosphate, monosodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and combinations thereof.

[0124] Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0125] Examples of oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cadet, chamomile, canola, caraway, carnauba, castor oil, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, and litsea buckthorn. Examples of oils include, but are not limited to, ba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, susquana, savory, sea buckthorn, sesame oil, shea butter, silicone, soybean, sunflower, tea tree oil, thistle oil, camellia, vetiver, walnut, and wheat germ oil. Exemplary specific oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0126] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, and mixtures thereof). In addition to inert diluents, the oral composition may contain auxiliary agents such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavoring agents, and fragrance agents. In a particular embodiment for parenteral administration, the therapeutic agent of the present invention is CREMOPHOR (登録商標) It is mixed with solubilizers such as alcohols, oils and fats, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

[0127] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using appropriate dispersants or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension, or emulsion in a non-toxic, parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution. Acceptable carriers and solvents include water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile fixative oils have conventionally been used as solvents or suspension media. For this purpose, any mild fixative oil, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid are used in injectable preparations.

[0128] Injectable formulations can be sterilized, for example, by filtration through a bacterial capture filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0129] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate, and / or a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) dissolution retarders such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) adsorbents such as kaolin and bentonite clay; i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, or pills, the dosage form may include a buffering agent.

[0130] Similar solid compositions can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in pharmaceutical formulation technology. These may optionally contain opacifiers and may be compositions that selectively release the active ingredient only at specific sites in the intestinal tract, or preferentially, in a delayed manner. Examples of usable embedding compositions include polymers and waxes. Similar solid compositions can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0131] In certain embodiments, the therapeutic agent encapsulated in tLCNP is in microencapsulation form with one or more excipients, as described above. Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled-release coatings, and other coatings well known in pharmaceutical formulation technology. In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may also contain additional substances other than the inert diluent, such as tableting aids, e.g., tableting lubricants, magnesium stearate, or microcrystalline cellulose, as is common practice. In the case of capsules, tablets, and pills, the dosage form may contain buffers. These may optionally contain opacifiers and may be compositions that selectively release the therapeutic agent only, or preferentially, in a delayed manner at specific sites in the intestinal tract.

[0132] In certain embodiments, dosage forms for topical and / or transdermal administration of therapeutic agents include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is mixed with pharmaceutically acceptable excipients and / or any necessary preservatives and / or buffers, as needed, under sterile conditions. Furthermore, the use of transdermal patches is also considered. Such patches often have the additional advantage of providing controlled delivery of the active ingredient to the body. Such dosage forms may be prepared, for example, by dissolving and / or dispersing the active ingredient in a suitable medium. Alternatively, the rate may be controlled by either providing a rate-controlled membrane and / or dispersing the active ingredient in a polymer matrix and / or gel.

[0133] Suitable devices for delivering the intradermal pharmaceutical compositions described herein include, for example, soluble microneedle array patches as described in U.S. Patent Publication Nos. US 2019 / 0358441 Al, US 2019 / 0240469 Al, US 2019 / 0151638 Al, US 2019 / 0151638 Al, US 2018 / 0001070 Al, US 2017 / 0209553 Al, US 2016 / 0263362 Al, US 2017 / 0196966 Al, US 2016 / 0213908 Al, US 2016 / 0015952 Al, US 2015 / 0112250 Al, US 2012 / 0150023, etc. Furthermore, devices with short needles, such as those described in U.S. Patents 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662, should also be considered. The intradermal composition may be administered by a device that limits the effective penetration length of the needle into the skin, such as those described in PCT Publication WO99 / 34850 and its functional equivalents. Jet injectors are suitable for delivering the vaccine to the dermis via a liquid jet syringe and / or via a needle that produces a jet that penetrates the stratum corneum and reaches the dermis. Jet injection devices include, for example, U.S. Patent Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, and 5,339,1 This is described in publications such as No. 63, No. 5,312,335, No. 5,503,627, No. 5,064,413, No. 5,520,639, No. 4,596,556, No. 4,790,824, No. 4,941,880, No. 4,940,460, and PCT publications WO97 / 037705 and WO97 / 013537. Ballistic powder / particle delivery devices that use compressed gas to accelerate the powder form of the vaccine through the outer layers of the skin to the dermis are suitable. Alternatively, conventional syringes may be used for the classic Mantou method of intradermal administration.

[0134] In certain embodiments, formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid formulations such as liniments, lotions, oil-in-water and / or water-in-oil emulsions (creams, ointments and / or pastes, etc.), and / or solutions and / or suspensions. In certain embodiments, a topically administered formulation may contain, for example, about 1% to about 10% (w / w) of the therapeutic agent (immunogenic nanoparticles as described herein), but the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. In certain embodiments, a topically administered formulation may further contain one or more of the additional ingredients described herein.

[0135] In certain embodiments, a pharmaceutical composition comprising the tLCNP formulation described herein may be prepared, packaged, and / or marketed as a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may, for example, comprise particles containing or comprising a therapeutic agent encapsulated in the tLCNP described herein. Such a composition is, conveniently, in the form of a dry powder for administration using a self-propelled solvent / powder dispersion container, such as a device comprising a dry powder reservoir to which a stream of propellant may be delivered to disperse the powder, and / or a device containing an active ingredient dissolved and / or suspended in a low-boiling point propellant in a sealed container.

[0136] Low-boiling-point propellants generally include liquid propellants having a boiling point of less than 65°F at atmospheric pressure. In certain embodiments, the propellant may constitute 50% to 99.9% (w / w) of the composition, and the therapeutic agent may constitute 0.1% to 20% (w / w) of the composition. The propellant may further contain additional components such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size similar to that of the particles containing the active ingredient).

[0137] In certain embodiments, the pharmaceutical compositions comprising the therapeutic agents described herein and formulated for pulmonary delivery may provide nanoparticles in the form of droplets of solution and / or suspension. In certain embodiments, such formulations may be prepared, packaged, and / or sold as aqueous and / or diluted alcoholic solutions and / or suspensions (optionally sterile) containing the active ingredient, and may be conveniently administered using any spray and / or atomizing device. In certain embodiments, such formulations may further contain one or more additional components, including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methyl hydroxybenzoate. In certain embodiments, the average diameter of the droplets provided by this administration route may range from about 0.1 pm to about 200 pm.

[0138] In certain embodiments, formulations described herein that are useful for pulmonary delivery are useful for intranasal delivery of the pharmaceutical compositions of the present invention. Another exemplary formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to about 500 pm. Such formulations can be administered in the manner in which snuff is ingested, i.e., by rapid inhalation through the nasal passages from a container of powder held near the nostrils.

[0139] In certain embodiments, formulations suitable for intranasal administration may, for example, contain about 0.1% (w / w) to 100% (w / w) of the tLCNP formulation described herein, and may contain one or more of the additional components described herein. In certain embodiments, the pharmaceutical composition of the tLCNP formulation described herein may be prepared, packaged, and / or sold in formulations suitable for oral administration. Such formulations may, for example, be in the form of tablets and / or lozenges manufactured using conventional methods, and may contain, for example, 0.1% to 20% (w / w) of the tLCNP formulation described herein, with the remainder being an orally soluble and / or biodegradable composition and optionally one or more of the additional components described herein. Alternatively, in certain embodiments, formulations suitable for oral administration may contain a powder containing immunogenic nanoparticles, and / or aerosolized and / or aerosolized solutions and / or suspensions. In certain embodiments, such powders, aerosols, and / or aerosolized formulations, when dispersed, have an average particle size and / or droplet size in the range of about 0.1 pm to about 200 pm, and may further contain one or more of the additional components described herein.

[0140] General considerations regarding the formulation and / or manufacture of pharmaceutical preparations may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.

[0141] In some embodiments, therapeutically effective doses of the tLCNP formulations described herein are delivered to patients and / or animals before, concurrently with, and / or after the diagnosis of the disease. In some embodiments, therapeutic doses of the compositions of the present invention are delivered to patients and / or animals before, concurrently with, and / or after the onset of symptoms of the disease. In some embodiments, the amount of the tLCNP formulations described herein is sufficient to treat, alleviate, improve, reduce, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of the disease. In some embodiments, the amount of the tLCNP formulations described herein is sufficient to induce a detectable immune response in a subject. In some embodiments, the amount of the tLCNP formulations described herein is sufficient to induce a detectable T-cell response in a subject. In some embodiments, the amount of the tLCNP formulations described herein is sufficient to induce a detectable antibody and T-cell response in a subject. In some embodiments, the advantage of the nanoparticles provided is that the nanoparticles can induce a potent response at much lower antigen concentrations than required by conventional controls.

[0142] Compositions described herein may be administered using any therapeutically effective dose and route of administration. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the infection, the specific composition, its mode of administration, its mode of activity, etc. Compositions containing tLCNP formulations described herein are typically formulated in dose-unit forms to facilitate administration and ensure uniformity of dose. However, it should be understood that the total daily dose of the composition will be determined by the attending physician within the bounds of sound medical judgment. The specific therapeutically effective dose (or prophylactically effective dose) for any particular subject or organism will vary depending on various factors, including the disease and its severity, the activity of the specific active ingredient used, the specific composition used, the subject's age, weight, general health, sex, and diet, timing of administration, route of administration, and the excretion rate of the specific active ingredient used, the duration of treatment, drugs used in combination with or concurrently with the specific active ingredient used, as well as similar factors well known in the medical field.

[0143] In some embodiments, the described pharmaceutical composition is administered by various routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, percutaneous, intradermal, rectal, vaginal, intraperitoneal, topical (by powder, ointment, cream, and / or infusion, etc.), mucous membrane, nasal, buccal, enteral, sublingual, intratracheal infusion, bronchial infusion, and / or inhalation, and / or oral spray, nasal spray, and / or aerosol. Specifically, oral administration, intravenous injection, intramuscular injection, and / or subcutaneous injection are considered. In some embodiments, nanoparticles are administered parenterally. In some embodiments, nanoparticles are administered intravenously. In some embodiments, nanoparticles are administered orally.

[0144] Generally, the most appropriate route of administration depends on various factors, including the properties of the tLCNP formulation described herein (e.g., stability in the gastrointestinal environment) and the condition of the subject (e.g., whether the subject can tolerate oral administration).

[0145] In certain embodiments, the therapeutic agents described herein may be administered once or more times daily in amounts ranging from about 0.1 μg / kg to about 100 μg / kg, about 0.2 μg / kg to about 50 μg / kg, about 0.3 μg / kg to about 25 μg / kg, about 0.4 μg / kg to about 10 μg / kg, or about 0.5 μg / kg to about 2.5 μg / kg per day of body weight of the subject, in order to obtain the desired therapeutic effect. In certain embodiments, the desired dose may be delivered three times a day, twice a day, once a day, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose may be delivered using multiple doses (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more doses). In certain embodiments, the dose of Mi may be delivered every two weeks, every one month, every two months, every three months, every four months, every five months, or every six months.

[0146] In some embodiments, the present invention encompasses a “therapeutic cocktail” comprising a group of tLCNP formulations described herein. In some embodiments, all of the tLCNP formulations described herein within a nanoparticle population contain a single type of cargo. In some embodiments, different nanoparticles within a nanoparticle population contain different cargoes.

[0147] Another aspect of the present invention provides a method for selectively or preferentially delivering a therapeutic agent to the lungs of a mammal, comprising administering the therapeutic agent, which is incorporated into or formulated in the tLCNP described herein, to the mammal.

[0148] In certain embodiments, mammals are humans. In certain embodiments, mammals are non-human animals, non-human mammals, or non-human primates (e.g., livestock such as cattle, bulls, sheep, goats, horses, pigs, and camels; pets such as cats or dogs; or rodents such as rabbits, mice, rats, hamsters, and guinea pigs).

[0149] In certain embodiments, tLCNP is administered intravenously to mammals.

[0150] In certain embodiments, the therapeutic agent comprises a polynucleotide such as RNA (e.g., mRNA).

[0151] In certain embodiments, the mammal has a pulmonary disease, and the therapeutic agent is effective for treating the pulmonary disease.

[0152] In certain embodiments, the pulmonary disease is lung cancer, asthma, pneumothorax, atelectasis, bronchitis (including chronic bronchitis), bronchiolitis, bronchiectasis, COPD, emphysema, pulmonary infection (e.g., pneumonia, COVID-19, bacterial infection, viral infection), cystic fibrosis, idiopathic pulmonary fibrosis (IPF), pulmonary edema, pulmonary embolism, lymphangioleiomyomatosis (LAM), or pulmonary hypertension.

[0153] Another aspect of the present invention provides a method of preparing the tLCNP of the present invention, the method comprising mixing a first organic liquid phase comprising the first lipid composition, the second lipid composition, a steroid, and a stealth lipid of the present disclosure described herein with a water-miscible organic solvent (e.g., ethanol), and the second aqueous phase comprises the therapeutic agent of the present disclosure to form the tLCNP.

[0154] In the above embodiments, the above method is performed in a microfluidic mixing apparatus / chip. An automated microfluidic device or microfluidic chip is a fast and efficient method for preparing LNPs. These apparatuses enable rapid mixing in a highly controllable and reproducible manner, achieving homogeneous LNPs and high encapsulation efficiency. In these apparatuses, separate streams of an ethanolic lipid mixture and an aqueous oligonucleotide solution are rapidly combined. The present tLCNP is formed when the two streams are mixed and collected into a single collection tube. Parameters such as flow rate ratio and total flow rate can be modified to fine-tune the tLCNP.

[0155] In certain embodiments, the above method is performed with a T- or Y-junction mixer. These mixers can be assembled from common and inexpensive laboratory materials. The T- or Y-connector can be fitted with two inlets connected to separate syringes containing the lipid mixture or oligonucleotide solution, and one outlet for directing LNPs to a collection tube. Optionally, the inlet flow rate can be controlled with a syringe pump.

[0156] In certain embodiments, the above method is performed using ethanol injection therapy. Mixing of the ethanolic lipid mixture and the aqueous oligonucleotide solution is carried out on a magnetic stir plate. The ethanolic lipid mixture is injected into the acidic aqueous oligonucleotide solution with constant stirring, and mixing is continued for 30 minutes.

[0157] A more convenient alternative method for ethanol injection therapy is manual mixing. The ethanolic lipid mixture is transferred to the acidic aqueous oligonucleotide solution and mixed for 15 seconds by rapid pipetting. The mixture is then allowed to stand for 10 minutes.

[0158] In certain embodiments, the above method further comprises extrusion to reduce particle size and generate a uniform particle size distribution.

[0159] In certain embodiments, the above method further comprises dialyzing the tLCNP in a storage buffer using a suitable molecular weight cutoff (MWCO) tube. This step removes unencapsulated cargo, excess lipid components, and ethanol from the final preparation. Dialysis also adjusts the pH of the LNPs from the acidic preparation buffer to a neutral storage solution. This step is generally performed with ethanol injection therapy or manual mixing methods.

[0160] In certain embodiments, the above method further comprises filtration for sterilizing the LNPs. In certain embodiments, the filtration step is performed using a 0.22 μm filter to remove bacteria or other contaminants.

[0161] In certain embodiments, the method further includes stepwise purification of tLCNPs using a tangential flow filtration (TFF) system.

[0162] In certain embodiments, the organic solvent in the first organic liquid phase contains, essentially consists of, or is composed of ethanol.

[0163] In certain embodiments, the first organic liquid phase and the second aqueous phase are mixed in a microfluidic device in an optional volume ratio of 1:3 and / or optionally at a total flow rate of about 10 mL to 15 mL / min (e.g., about 12 mL / min).

[0164] In certain embodiments, the aqueous phase includes 50 mM citrate buffer (pH 5.0).

[0165] In certain embodiments, stepwise purification is performed in a TFF system in citrate buffer (pH 5.0) and Tris-HCl buffer (pH 7.2) containing 7.5% sucrose.

[0166] In certain embodiments, the method further includes measuring the particle size of the tLCNPs (e.g., average size and / or size distribution). In certain embodiments, the size is determined by dynamic light scattering (DLS) measurement, for example, using a Zetasizer Nano ZS from Malvern Instruments Ltd.

[0167] In certain embodiments, the method further includes measuring the encapsulation efficiency (EE%) of cargo such as mRNA. In certain embodiments, the EE% is, for example, RIBOGREEN (登録商標) Determined using an assay. RIBOGREEN (登録商標) This dye exhibits fluorescence when it binds to single-stranded mRNA, but it cannot penetrate LNPs.

[0168] In certain embodiments, the method further includes imaging a sample of tLCNPs by cryo-electron microscopy (Cryo-EM) to investigate their nano-precipitated core structure. The cryo-EM images show that mRNA is encapsulated within what appears to be tLCNPs, forming a multilayer core structure.

[0169] In accordance with the general aspects of the present invention described herein, the following embodiments further illustrate specific aspects of the present invention and do not limit the present invention in any way. Furthermore, the specific embodiments described in the embodiments are all specific embodiments of the present invention and can be combined with or are intended to be combined with other aspects of the present invention or other embodiments described herein above.

[0170] Examples Example 1: Production of self-replicating mRNA and conventional mRNA in vitro transcription of srRNA Self-replicating mRNA (srRNA) was produced from a backbone plasmid vector based on the Trinidad Roba Venezuelan Encephalitis (VEEV) alphavirus genome, according to a reported method (Blakney et al., ACS Nano 13:5920-5930, 2019, incorporated herein by reference).

[0171] Next, post-transcription capped srRNA was synthesized as described above (McKay et al., Nat.Commun. 11, 3523, 2020, incorporated herein by reference). Specifically, uncapped RNA was pre-prepared using 1 μg of linearized DNA template in a MEGAScript reaction (Ambion, UK) according to the manufacturer's protocol described. The transcript was then purified overnight at -20°C by LiCl precipitation, pelletized by centrifugation at 14,000 rpm at 4°C for 20 minutes, washed once with 70% ethanol, and resuspended in UltraPure H2O after centrifugation at 14,000 rpm at 4°C for 5 minutes. Subsequently, the purified transcript was capped simultaneously via the ScriptCap m7G capping system (CellScript, Madison, WI, USA) and the ScriptCap 2'-O-methyltransferase kit (CellScript) according to the manufacturer's protocol described. The capped transcript was purified by LiCl precipitation using the same procedure as described in detail above, resuspended in UltraPure H2O, and stored at -80°C for future use.

[0172] mRNA in vitro transcription Conventional luciferase mRNA production consists of three steps. First, the plasmid DNA encoding luciferase is amplified, extracted, and purified. Next, the plasmid is linearized and purified by chromatography and / or LiCl / ethanol precipitation. Finally, mRNA is enzymatically synthesized in vitro using the linearized plasmid DNA as a template, followed by purification and storage at -80°C for use.

[0173] Example 2 Preparation of tLCNP tLCNP was synthesized by rapidly mixing the ethanol phase and the aqueous phase at a volume ratio of 1:3 at a total flow rate of 12 mL / min using a conventional microfluidic device. The aqueous phase is 50 mM citrate buffer (pH 5.0) containing mRNA or other cargo molecules. The ethanol phase comprises ionizable lipid (Lipid #4, Immorna), DOTMA (SHOCHEM), cholesterol (Jiangsu Southeast Nanomaterials) / Sito 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Jiangsu Southeast Nanomaterials) / DOPE (AVT) / DOPC (AVT), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (SINOPEG).

[0174] These lipid components were mixed at a molar ratio of 16.7:33.4:38.5:6.6:3.3:1.5 (ionizable lipid: cationic lipid: cholesterol: DSPC: DOPC: DMG-PEG2000). The N / P ratio was fixed at 6. The prepared "mRNA@tLCNP" composition was purified stepwise by a TFF system in citrate buffer (pH 5.0) containing 7.5% sucrose and Tris-HCl buffer (pH 7.2). Thereafter, mRNA@tLCNP was diluted to the target concentration, and stored at 2°C to 8°C for short-term storage (less than 3 months) and at -80°C for long-term storage (more than 3 months).

[0175] Therefore, as shown in Figure 2, the present mRNA@tLCNP was synthesized by a microfluidic mixing device using ionizable lipid (C I), permanent cationic lipid (C II), two types of phospholipids (P I and P II), stealth lipid DMG-PEG, and one type of sterol (cholesterol, β-sitosterol, or other cholesterol derivatives).

[0176] Example 3 Characterization of tLCNP The size and polydispersity index (PDI) of the mRNA@tLCNP nanoparticles prepared in Example 2 described above were determined by dynamic light scattering (DLS, Brookhaven 90 Plus PALS), and the mRNA concentration was measured using Ribogreen (登録商標) assay.

[0177] Thereafter, a TFF purification process was used to remove ethanol, and the mRNA@tLCNP was transferred to a Tris-HCl buffer containing sucrose suitable for intravenous administration. Finally, the prepared product was subjected to DLS and Ribogreen (登録商標) assay for characterization to determine its size and encapsulation efficiency (EE) / concentration.

[0178] Example 4 In Vivo Evaluation of tLCNP The series of in vivo experiments described herein demonstrate the flexibility and optimal conditions for lung targeting and mRNA expression using the present tLCNP delivery system.

[0179] All animal experiments were approved by the Animal Experiment Committee of Hangzhou Normal University and comply with the Chinese government regulations on the management and use of animals. Briefly, BALB / c mice (female, 6-8 weeks old) were divided into groups of n=3. Mice were intravenously injected with luciferase mRNA@tLCNP (mRNA dose: 5 μg) dispersed in Tris-HCl buffer (pH 7.2) containing 7.5% sucrose. Mice were injected with D-luciferin potassium (150 mg kg-1, intraperitoneal injection) and imaged 6 hours after injection using an IVIS Lumina system (Perkin Elmer). Thereafter, organs were isolated and imaged in the same manner.

[0180] New Zealand White rabbits (female, 15 weeks old) were also used to evaluate lung targeting. Specifically, a dose of 100 μg of mRNA was intravenously injected into the marginal ear vein, IVIS Lumina imaging was performed 6 hours after injection, and RBD measurement was performed 4 hours after injection.

[0181] DOTMA doping of lipid #4 Luciferase mRNA@tLCNP samples with lipid #4 / DOTMA ratios of 1:0, 3:1, 1:1, 1:2, 1:3, and 1:4 (i.e., 100%, 75%, 50%, 33%, 25%, and 20% of total charged lipids (lipid #4 + DOTMA), respectively) were prepared using the protocol described in Example 2. This series of samples covered a range of DOTMA percentages (relative to total lipid #4 / DOTMA) from approximately 0% to 80% (i.e., 0%, 25%, 50%, 67%, 75%, and 80% of total charged lipids (lipid #4 + DOTMA), respectively).

[0182] [Table 1]

[0183] As shown in Table 1, all mRNA@tLCNP samples had a small size (less than 200 nm) suitable for their intended therapeutic use, a PDI of less than 0.25, and suggested good uniformity in size distribution. Furthermore, they exhibited excellent inclusion efficiency (EE%) exceeding 97% (over 99.5% in the presence of DOTMA). In particular, all LNPs containing different proportions of lipid #4 / DOTMA mixture had particle sizes less than 100 nm based on DLS measurements, with most being less than 90 nm and one less than 80 nm. See Table 1.

[0184] [Table 2]

[0185] As shown in Figures 3-6 and Table 2, as the doping rate of DOTMA in lipid #4LNP increased from 0% to 80%, organ-targeted mRNA expression gradually moved from the liver to the spleen and then to the lungs with a targeting efficiency of 81.0%.

[0186] Considering that lung targeting efficiency is nearly equivalent between a lipid #4 / DOTMA ratio of 1:2 (i.e., 33% lipid #4 / 67% DOTMA) at 78.8% and a lipid #4 / DOTMA ratio of 4:1 (i.e., 80% lipid #4 / 20% DOTMA) at 81.0%, a lipid #4 / DOTMA ratio of 1:2 (i.e., 33% lipid #4 / 67% DOTMA) is a potential optimal choice for balancing safety and efficiency.

[0187] Doping other different ionized lipid-based LNPs with DOTMA To demonstrate that lung targeting can be achieved with other ionized lipid-based tLCNP systems, tLCNPs were formulated using three other ionized lipids, including MC3, lipid #9, and lipid #13, instead of lipid #4.

[0188] As shown in Figures 7-10 and Tables 3-4, the sizes of lipid #9 tLCNPs and lipid #13 tLCNPs exceed 200 nm, but the larger size clearly did not have a significant impact on lung targeting, as both of these tLCNPs achieved high lung targeting efficiencies of 83.8% and 90.8%, respectively (Table 4).

[0189] [Table 3]

[0190] [Table 4]

[0191] However, MC3 tLCNPs do not possess lung targeting ability. While we do not wish to be bound by any particular theory, this discrepancy may be caused by the presence of ester bonds in the branched tails of ionized lipids #4, #9, and #13. In other words, ester bonds located in the hydrophobic tails of ionized lipids are thought to have a significant effect on lung targeting. The data presented herein also demonstrate that a range of ionized lipids can be used in the lung-targeted tLCNPs of the present invention (Table 4).

[0192] Adjusts the total proportion of lipids #4 and DOTMA lipids. Partly in light of the smaller size of tLCNPs prepared based on lipid #4, which facilitates quality control for subsequent development, the following screening and optimization were performed based on tLCNPs from lipid #4.

[0193] To further optimize lung targeting and expression efficiency, the effect of the proportion of total charged lipids (lipid #4 + DOTMA) was investigated using different proportions of total charged lipids. As shown in Tables 5-6 and Figures 11-14, lung targeting efficiency showed an increasing trend dependent on the proportion of total charged lipids. The best lung targeting was achieved with 60% total charged lipids (lipid #4 + DOTMA), while further increasing the proportion of total charged lipids to 70% did not contribute to higher lung targeting.

[0194] [Table 5]

[0195] Furthermore, the expression intensity in the lungs showed a similar trend within a total charged lipid percentage of 30% to 60%, however, a total charged lipid percentage of 70% resulted in a dramatic decrease in expression (Figure 12 and Table 6).

[0196] [Table 6]

[0197] Adjust the N / P ratio As shown in Figures 15-18 and Tables 7-8, the N / P ratio was also optimized in this tLCNP delivery system. Regarding targeting efficiency and expression intensity in the lungs, N / P=6 was found to be optimal compared to the other two test values ​​(Table 8).

[0198] [Table 7]

[0199] [Table 8]

[0200] Doping with other phospholipids boosts targeting and expression efficiency. The data described above demonstrates that the tLCNP in question (e.g., specific lipid #4-based tLCNPs) can achieve lung targeting of over 90%. However, the use of a high proportion of charged lipids in some of these formulations may result in potentially high levels of side effects.

[0201] [Table 9]

[0202] The data presented herein (Figures 19–22 and Tables 9–10) provide further optimizations for improving overall lung targeting and expression based on a moderate total charged lipid ratio of approximately 50%. In this embodiment, lung targeting efficiency can be increased to approximately 83.2% by partially or completely replacing DSPC with DOPE (e.g., doping DSPC with DOPE) and altering liver-to-lung delivery (Figures 22 and Table 10), although mRNA expression is slightly reduced (Figures 20 and 21).

[0203] [Table 10]

[0204] Further combination optimization of DSPC and DOPC, or DOPE and DOPC was tested (Figure 23 to Figure 26 and Table 11 to Table 12). The data showed that the expression of the marker gene luciferase in all groups was mainly concentrated in the lungs. In particular, when the relative expression in each organ was calculated, doping DSPC with DOPC at ratios of 1:2 and 1:1 efficiently altered the delivery from the liver and spleen to the lungs, and lung targeting was improved from 69.8% to 83.3% and 88.6%. Meanwhile, the expression level also reached 1.757×10 6 p / s / cm 2 / sr to 2.627×10 6 p / s / cm 2 / sr and 2.530×10 6 p / s / cm 2 / sr (Table 12).

[0205]

Table 11

[0206]

Table 12

[0207] The above data showed that by introducing primary amine-capped phospholipids having unsaturated fatty acid chains such as DOPE, lung targeting can be enhanced without promoting endosomal escape and subsequent expression (Figure 19 to Figure 22 and Table 10). Interestingly, introducing quaternary ammonium-capped phospholipids having unsaturated fatty acid chains such as DOPC created dual beneficial effects in terms of lung targeting and expression intensity (Figure 23 to Figure 26 and Table 12).

[0208] Overall, these results demonstrate that the two approaches described herein—doping conventional LNPs with permanent cationic lipids (C2 lipids) and quaternary ammonium-capped phospholipids (PII lipids) having unsaturated fatty acid chains—synthetically enhanced both lung targeting and cargo mRNA expression efficiency.

[0209] Example 5: Comparison of different lung target delivery systems To verify the superiority of this tLCNP-based lung-targeted delivery system, a direct comparison of lung-targeted delivery efficiency between SORT LNP and this tLCNP was performed in vivo experiments (Figures 27-30 and Tables 13-14).

[0210] [Table 13]

[0211] The tLCNPs based on DOTMA and lipid #4 were prepared with optimized composition and lipid ratio formulations: lipid #4:DOTMA:DSPC:DOPC:cholesterol:DMG-PEG = 16.7:33.4:6.6:3.3:38.5:1.5. Two SORT LNP formulations containing DOTAP+MC3 and DOTAP+lipid #4, respectively, were prepared using the same procedure (Table 13).

[0212] The size and CE of all products were within the controlled range for application. Similarly, the marker gene luciferase was expressed primarily in the lungs in all groups. SORT LNP containing DOTAP and lipid #4 showed a higher lung targeting efficiency (69.3%) than SORT LNP containing DOTAP and MC3 (reported formulation) (55.4%), but both were significantly lower than the lung targeting efficiency of the tLCNP in question (95.2%) (Table 14).

[0213] Furthermore, tLCNP is SORT LNP:DOTAP+Lipid #4(7.09×10 5 ) and SORT LNP:DOTAP+MC3(6.26×105 / p / s / cm 2 The expression intensity of luciferase via both delivery formulations ( / sr) was compared, and the overwhelming expression intensity of luciferase (1.83 × 10⁻¹⁰) was determined. 7 p / s / cm 2 The result was shown in Table 14 ( / sr).

[0214] [Table 14]

[0215] The results described above demonstrate that tLCNP in question offers clear advantages over SORT LNP in terms of selective delivery and expression efficiency of nucleic acid therapeutics to the lungs, and that it has great potential for clinical use.

[0216] Finally, DOTMA-based LNPs were also prepared for comparison with tLCNPs regarding lung targeting. The data showed that DOTMA-based LNPs (without lipid #4) also had high lung targeting efficiency (79.8%), but their expression intensity (1.27 × 10⁻¹⁶) was lower. 4 p / s / cm 2 The / sr value is significantly lower than that of the tLCNP system in question.

[0217] The evidence presented above indicates that the tLCNP delivery system in question has great potential for clinical applications in treating or preventing specific lung diseases.

[0218] Example 6 Evaluation of tLCNP targeting of the lungs in large animals To demonstrate that this lung-targeted delivery system is species-independent, tests were conducted in rabbits by injecting tLCNPs loaded with mRNA encoding luciferase and mRNA expressing the receptor-binding domain (RBD) sequence of the SARS-CoV-2 virus ("COVID-19 RBD"), as shown in Table 15. Luciferase expression was determined by in vivo and ex vivo IVIS Lumina imaging. RBD expression in major organs (liver, lungs, and spleen) was determined by ELISA.

[0219] [Table 15]

[0220] The data showed a similar trend in lung targeting in rabbits, reaching approximately 82.7% (Figures 31 and 32). Furthermore, ELISA tests to measure RBD concentrations were performed on the liver, lungs, and spleen of rabbits 4 hours after injection of tLCNP(RBD) and PBS. As shown in Figure 33, the relative RBD concentration detected in the lungs (1454 pg / μg) was significantly higher than that in the liver (47 pg / μg) and spleen (53 pg / μg).

[0221] These data demonstrate that the lung-targeted tLCNP delivery system of the present invention is independent of specific animal species or specific cargo molecule / therapeutic agents in terms of its observed advantageous properties.

[0222] Example 7 Evaluation of targeted therapy with IL12-CBD mRNA@tLCNP in mice with metastatic LL2 lung tumors This embodiment demonstrates the delivery efficiency of tLCNP to loaded mRNA drugs for use in treating cancer.

[0223] Specifically, mRNA encoding a mouse interleukin-12-collagen-binding domain fusion protein (IL12-CBD) was synthesized and used for delivery (using the tLCNP in question) and treatment of the developed primary and metastatic LL2 lung cancer models.

[0224] In short, IL12-CBD mRNA@tLCNP was prepared using the method described in Example 2 and then characterized essentially as described in Example 3. The size / PDI and encapsulation efficiency were found to be 74.67 nm / 0.135 and 94.5%, respectively.

[0225] Mouse Lewis lung cancer (LLC) cells were purchased from ATCC (Manassas, Virginia, USA) and cultured in RPMI DMEM containing FBS (10%), penicillin (50 μg / mL), and streptomycin (50 μg / mL). The cells were maintained at 37°C in a humidified atmosphere with 5% CO2. Next, 100 μL of LLC cells (2 × 10⁶ per mouse) were added. 5 A suspension was injected into the tail vein of mice (6-8 week old C57BL / 6 male mice) to generate a lung tumor model. Mice were randomly divided into an IL12-CBD mRNA@tLCNP treatment group (n=10) and a PBS control group (n=10). On days 9, 13, 17, and 21 after tumor cell injection, mice were intravenously administered either IL12-CBD mRNA@tLCNP (10 μg IL12-CBD mRNA / mouse) or PBS. Mortality was measured daily.

[0226] As shown in Figure 34, lung tumor mice treated with IL12-CBD mRNA@tLCNP showed increased survival rates compared to the control group treated with PBS.

Claims

1. A targeted tLCNP that preferentially delivers a therapeutic agent encapsulated / formulated within the tLCNP to the lungs of a mammal, wherein the tLCNP is (1) A first lipid composition comprising ionized lipid C1 and cationic lipid C2, (2) A second lipid composition comprising a first phospholipid PI and a second phospholipid PII, (3) Sterol lipids and (4) Stealth lipids and, Here, (A) The ionized lipid C1 has the formula C1-a, C1-b, C1-c, or C1-d: 【Chemistry 1】 During the ceremony, R １ and R ２ Each of them is independent of -CH ３ ,-(CH ２ ) １－８ -CH ３ , or - (CH ２ ) １－８ -OH, G １ and G ２ are each independently -(CH ２ ) １－１０ - G ３ and G ４ It is independently, - (CH ２ ) ０－５ - and L １ は、-O-(C=O)-O-、-O-(C=O)-、-(C=O)-O-、---S-、-O-- R ３ and R ４ Each of them operates independently. 【Chemistry 2】 And, R ５ is -H, or -(CH ２ ) ７ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , C １－１４ Alkyl, or C １－１４ It is alkenyl, R ６ is, -(CH ２ ) ７ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , C １－２０ Alkyl, or C １－２０ It is alkenyl, X is C or 【Transformation 3】 And, n １ These are integers from 2 to 8, (B) The cationic lipid C2 has the formula C2-a, C2-b, C2-c, or C2-d: 【Chemistry 4】 During the ceremony, R ７ , R ８ , and R ９ Each of them is independent of -CH ３ ,-(CH ２ ) １－８ -CH ３ , or - (CH ２ ) １－８ -OH, R １０ and R １１ Each of them operates independently. 【Transformation 5】 And, R １２ is -H, -(CH ２ ) ７ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , C １－１４ Alkyl, or C １－１４ It is alkenyl, R １３ is, -(CH ２ ) ７ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , C １－２０ Alkyl, or C １－２０ It is alkenyl, M is carbon or nitrogen, A is -CH ２ -, -G ７ -L ２ -G ８ - or - (CH ２ ) １－５ -(C=O)-, where -(C=O)- is connected to M, G ５ G ６ G ７ , and G ８ It is independently, - (CH ２ ) ０－１０ - and n ２ is an integer between 0 and 10, L ２ , L ３ , and L ４ Each of them is independent of -CH ２ -, -O-(C=O)-O-, -O-(C=O)-, -(C=O)-O-, -S-S-, -O-, -S-, -HN-(C=O)-, X － Cl － and Br － These are monovalent anions (e.g., halo groups), (C) Optionally, the first phospholipid PI has the formula PI: 【Transformation 6】 In the formula, R １４ , R １５ , and R １６ are each independently -H, -CH ３ , -(CH ２ ) １－６ -CH ３ , or -(CH ２ ) １－６ -OH. n ３ is, in each case independently, an integer of 5 to 20, and n ４ is an integer from 1 to 5, and, (D) Optionally, the second phospholipid PII has the formula PII: 【Transformation 7】 In the formula, R １７ , R １８ , and R １９ These are, independently, -H and -CH. ３ ,-(CH ２ ) １－６ -CH ３ , or - (CH ２ ) １－６ -OH, n ５ and n ６ In each case, independently, these are integers from 5 to 8. n ７ These are integers from 1 to 5. Target tLCNP.

2. The tLCNP according to claim 1, wherein the steroid contains, is essentially composed of, or is composed of cholesterol.

3. The aforementioned stealth lipid is DMG-PEG2000 ( 【Transformation 8】 PEG (-O-CH) such as ) ２ -CH ２ -) The tLCNP according to claim 1 or 2, which includes, is essentially composed of, or is composed of a repeat.

4. R ５ H, -(CH ２ ) ３ CH ３ ,-(CH ２ ) ５ CH ３ ,-(CH ２ ) ７ CH ３ , or - (CH ２ ) ９ CH ３ The tLCNP according to claim 1.

5. R ５ is H, -(CH=CH)(CH ２ ) ３ CH ３ ,-(CH ２ ) ３ (CH=CH)CH ２ CH ３ , -(CH=CH)(CH ２ ) ４ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , or -(CH=CH)(CH ２ ) ７ CH ３ The tLCNP according to claim 1, including the above.

6. R ６ is, -(CH ２ ) ３ CH ３ ,-(CH ２ ) ５ CH ３ ,-(CH ２ ) ７ CH ３ , or - (CH ２ ) ９ CH ３ The tLCNP according to claim 1, including the above.

7. R ６ is -(CH=CH)(CH ２ ) ３ CH ３ ,-(CH ２ ) ３ (CH=CH)CH ２ CH ３ , -(CH=CH)(CH ２ ) ４ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , or -(CH=CH)(CH ２ ) ７ CH ３ The tLCNP according to claim 1, including the above.

8. R ６ H, -(CH ２ ) ３ CH ３ ,-(CH ２ ) ５ CH ３ ,-(CH ２ ) ７ CH ３ ,-(CH ２ ) ９ CH ３ , or - (CH ２ ) １１ CH ３ The tLCNP according to claim 1, including the above.

9. R ６ is H, -(CH=CH)(CH ２ ) ３ CH ３ ,-(CH ２ ) ３ (CH=CH)CH ２ CH ３ , -(CH=CH)(CH ２ ) ４ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , -(CH=CH)(CH ２ ) ７ CH ３ , or - (CH ２ ) ７ (CH=CH)(CH ２ ) ７ CH ３ The tLCNP according to claim 1, including the above.

10. R ７ is, -(CH ２ ) ３ CH ３ ,-(CH ２ ) ５ CH ３ ,-(CH ２ ) ７ CH ３ ,-(CH ２ ) ９ CH ３ , or - (CH ２ ) １１ CH ３ The tLCNP according to claim 1, including the above.

11. R ７ is -(CH=CH)(CH ２ ) ３ CH ３ ,-(CH ２ ) ３ (CH=CH)CH ２ CH ３ , -(CH=CH)(CH ２ ) ４ CH ３ , -(CH=CH)(CH ２ ) ５ CH ３ , -(CH=CH)(CH ２ ) ７ CH ３ , or - (CH ２ ) ７ (CH=CH)(CH ２ ) ７ CH ３ The tLCNP according to claim 1, including the above.

12. The ionized lipid C1 is, 【Chemistry 9】 、 【Chemistry 10】 、 【Chemistry 11】 、 【Chemistry 12】 , or 【Chemistry 13】 including, The tLCNP according to any one of claims 1 to 11.

13. The cationic lipid C2 is 【Chemistry 14】 、 【Chemistry 15】 、 【Chemistry 16】 、 【Chemistry 17】 , or [Chemistry 18] including, The tLCNP according to any one of claims 1 to 12.

14. The first phospholipid PI is, 【Chemistry 19】 or 【Chemistry 20】 The tLCNP according to any one of claims 1 to 13, including the tLCNP.

15. The second phospholipid PII is, 【Chemistry 21】 or 【Chemistry 22】 including, The tLCNP according to any one of claims 1 to 14.

16. The tLCNP according to any one of claims 1 to 15, wherein the molar ratio of the cationic lipid C2 to the ionized lipid C1 is about 1:3 to about 4:1, about 1:1 to about 4:1, about 2:1 to about 4:1, about 3:1 to about 4:1, about 1:1, about 2:1, about 3:1, or about 4:

1.

17. The tLCNP according to claim 8, wherein the cationic lipid C2 is DOTMA and the ionized lipid C1 is lipid #4.

18. The tLCNP according to any one of claims 1 to 17, wherein the molar proportion of (1) to (4) in (1) (or the first lipid composition) is about 30% to 70%, about 40% to 60%, about 50% to 60%, about 30%, about 40%, about 50%, about 60%, or about 70%.

19. The tLCNP according to claim 18, wherein the molar ratio of the cationic lipid C2 (such as DOTMA) to the ionized lipid C1 (such as lipid #4) is approximately 2:

1.

20. (a) The first lipid composition having a molar proportion of approximately 30% to 70%, approximately 40% to 60%, or approximately 50%. (b) The second lipid composition having a molar proportion of about 5% to 30% (about 5% to 15%, or about 10%, etc.), wherein the molar ratio of the first phospholipid PI (such as DSPC) to the second phospholipid PII (such as DOPC) is optionally 1:0 (100% DSPC) to 1:2 (33% DSPC), for example, 2:1 (67% DSPC) or 1:1 (50% DSPC), (c) The steroid (such as cholesterol) having a molar proportion of approximately 10% to 40% (approximately 35% to 40%, or approximately 38.5%) (d) The stealth lipid (DMG-PEG2000, etc.) having a molar proportion of approximately 0% to 5% (approximately 2% to 3%, or approximately 1.5%), (e) The total mole ratio is 100%, including any one of the above combinations, The tLCNP according to any one of claims 1 to 19.

21. The therapeutic agent comprises a polynucleotide (DNA and RNA such as mRNA, siRNA, miRNA, ASO, and pDNA), a hydrophobic and / or hydrophilic low molecular weight compound (for example, having a molecular weight of 1000 Da, 750 Da, 500 Da, 250 Da, or less than 100 Da), or a bioactive lipid (such as a fatty acid or monoglyceride), according to any one of claims 1 to 20.

22. The therapeutic agent comprises a polynucleotide, such as RNA (e.g., siRNA, miRNA, prim-miRNA, messenger RNA (mRNA), clustered regularly spaced short palindromic repeat (CRISPR)-associated nucleic acids, single guide RNA (sgRNA), CRISPR-RNA (crRNA), transactivating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA)), according to claim 21, wherein the therapeutic agent comprises a polynucleotide.

23. The tLCNP according to claim 22, wherein the RNA is self-replicating mRNA, non-self-replicating mRNA, or circular RNA.

24. The tLCNP according to claim 22 or 23, wherein the molar ratio of N / P of the tLCNP (the first lipid composition (permanently cationic lipid + ionized lipid) lipid nitrogen: polynucleotide phosphate) is about 4, 6, or 8, for example, 6.

25. When the tLCNP is administered to the mammal, at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the therapeutic agent is delivered to the lungs, the tLCNP according to any one of claims 1 to 24.

26. The tLCNP according to any one of claims 1 to 25, wherein when the tLCNP is administered to the mammal, about 30%, 25%, 20%, 15%, 10%, or 5% or less of the therapeutic agent is delivered to the liver (or a combination of other non-pulmonary tissues).

27. The tLCNP according to any one of claims 1 to 26, wherein the tLCNP has an average size of less than 200 nm, less than 150 nm, less than 100 nm, less than 90 nm, or less than 80 nm.

28. The tLCNP according to any one of claims 1 to 27, wherein the tLCNP has a polyvariance index (PDI) of about 0.10 to 0.25, or about 0.15 to 0.

20.

29. The tLCNP according to any one of claims 1 to 28, wherein when the tLCNP is administered to the mammal, about 1% to 15%, about 2% to 10%, or about 3% to 5% of the therapeutic agent is delivered to the spleen.

30. A tLCNP according to any one of claims 1 to 29, comprising a pharmaceutically acceptable carrier or excipient, Pharmaceutical composition.

31. A method for selectively or preferentially delivering a therapeutic agent to the lungs of a mammal, comprising administering the therapeutic agent to the mammal, which is contained in or formulated in a tLCNP according to any one of claims 1 to 29. method.

32. The method according to claim 31, wherein the mammal is a human.

33. The method according to claim 31 or 24, wherein the tLCNP is administered intravenously to the mammal.

34. The method according to any one of claims 31 to 33, wherein the therapeutic agent comprises a polynucleotide such as RNA (e.g., mRNA).

35. The method according to any one of claims 31 to 34, wherein the mammal has a lung disease, and the therapeutic agent is effective in treating the lung disease.

36. The method according to claim 35, wherein the lung disease is lung cancer, asthma, pneumothorax, atelectasis, bronchitis (including chronic bronchitis), bronchiolitis, bronchiectasis, COPD, emphysema, lung infection (e.g., pneumonia, COVID-19, bacterial infection, viral infection), cystic fibrosis, idiopathic pulmonary fibrosis (IPF), pulmonary edema, pulmonary embolism, lymphangioleiomyomatosis (LAM), or pulmonary hypertension.

37. A method for preparing tLCNP according to any one of claims 1 to 29, the method comprising mixing a first organic liquid phase comprising the first lipid composition, the second lipid composition, the steroid, and the stealth lipid according to any one of claims 1 to 29, with a second aqueous phase comprising the therapeutic agent according to any one of claims 1 to 29 in a water-miscible organic solvent (e.g., ethanol) to form the tLCNP. method.

38. The method according to claim 37, further comprising stepwise purification of the tLCNP by a tangential flow filtration (TFF) system.

39. The method according to claim 37 or 38, wherein the organic solvent in the first organic liquid phase contains, is essentially composed of, or is composed of ethanol.

40. The method according to any one of claims 37 to 39, wherein the first organic liquid phase and the second aqueous phase are mixed in a microfluidic device in an optionally selective volume ratio of 1:3 and / or optionally selectively at a total flow rate of about 10 mL / min to 15 mL / min (e.g., about 12 mL / min).

41. The method according to any one of claims 37 to 39, wherein the aqueous phase comprises a 50 mM citrate buffer (pH 5.0).

42. The method according to any one of claims 38 to 41, wherein the stepwise purification is performed in the TFF system in citrate buffer (pH 5.0) and Tris-HCl buffer (pH 7.2) containing 7.5% sucrose.

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