Ionizable sulfur lipids and uses thereof

Abstract

The present disclosure provides compounds of Formula (I): (I), or a pharmaceutically acceptable salt thereof, which can be used to form particles (e.g., lipid nanoparticles) for delivery of nucleic acids. The disclosure further provides particle compositions comprising the compounds of Formula I and uses thereof.

Description

Background Technology

[0001] Particles for delivering nucleic acids have been the subject of much recent work. Certain particles, such as lipid nanoparticles (LNPs), are particularly useful for delivering therapies, such as nucleic acid therapies, into cells. See Tenchov et al., ACS Nano ,2021, 15, 11 16982-17015. LNPs particularly contain cationic or ionizable lipids that form stable complexes encapsulating nucleic acids via electrostatic interactions, thereby facilitating their delivery into cells. Summary of the Invention

[0002] There remains a need for cationic or ionizable lipids that can form complexes with nucleic acids but offer improved manufacturability and properties (e.g., improved nucleic acid transfection and improved stability) compared to previous lipid formulations. This disclosure provides cationic or ionizable lipid compounds comprising one or more sulfur-based moieties that avoid the problems associated with lipid compounds while exhibiting improved properties (e.g., improved transfection) and ease of manufacture.

[0003] In some embodiments, this disclosure provides a compound represented by formula I: I Or its pharmaceutically acceptable salt, wherein: L 1 and L 2 Each of the C1-Cs can be substituted independently. 30 Aliphatic groups; L 3 C1-C, which are the bonds and can be arbitrarily substituted 10 Aliphatic groups or optionally substituted 2- to 10-membered heteroaliphatic groups comprising 1 to 4 heteroatoms selected from N, O, and S; X 1 and X 2 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-, where X 1 or X 2 One or both of them are selected from -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-; Each R 1 Independently for each case the optionally substituted C1-C 20 Aliphatic or H; T 1 and T 2 Each of the C3-Cs can be substituted independently. 30 aliphatic; G is -N(R) 2 )C(S)N(R 2 )2、-OH、-N(R 2 )2、-N + (R 3 )3、-N(R 5 )C(O)R 3 -N(R) 5 )S(O)2R 3 -N(R) 5 )C(O)N(R 3 )2、-CH(NR 2 -R 4 or -S(O)2R 3 ; Each R 2 In each case, H, arbitrarily substituted C1-C6 aliphatic and OR are independently selected. 3 The group formed; or R 2 Two instances together with the atoms to which they are attached form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O and S; Each R 3 In each case, H and the arbitrarily substituted C1-C are chosen independently. 10 A group composed of aliphatic groups; and R 4 It is a 4- to 12-membered heterocycle with 1 to 4 heteroatoms selected from N, O, and S, optionally substituted, and a 4- to 12-membered heteroaryl group with 1 to 4 heteroatoms selected from N, O, and S, substituted with -(CH2). 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C6-C in )2 are replaced 12 Aryl group, or with an oxy group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C3-C in )2 are substituted12 Cycloaliphatic; Each R 5 Independently selected from H and optionally substituted C1-C6 aliphatic compounds.

[0004] In some embodiments, this disclosure provides a particle comprising the compounds and nucleic acids described herein. In some embodiments, this disclosure provides a composition (e.g., a pharmaceutical composition) comprising the particles described herein.

[0005] In some embodiments, this disclosure provides a method of treating a disease, condition, or ailment of a subject, the method comprising administering to the subject a composition comprising the particles described herein.

[0006] In some embodiments, this disclosure provides a method for preparing compounds represented by formula IV: IV A method using a pharmaceutically acceptable salt thereof, said method comprising: Compounds represented by formula V V Compounds represented by one of formulas VIa-VIc and compounds represented by one of formulas VIIa-c Contact in the presence of a reducing agent in: L 4 and L 5 Each of them is independently an optional substitution of C1-C. 30 Aliphatic groups; L 6 C1-C, which are the bonds and can be arbitrarily substituted 10 Aliphatic groups or optionally substituted 2- to 10-membered heteroaliphatic groups comprising 1 to 4 heteroatoms selected from N, O, and S; X 3 and X 4 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 40 )-、-N(R 40 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 40 )2-、-OC(S)C(R 40 )2-、-C(R 40 )2C(S)O- or -S-, where X 3or X 4 One or both of them are selected from -S(O)2N(R) 40 )-、-N(R 40 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 40 )2-、-OC(S)C(R 40 )2-、-C(R 40 )2C(S)O- or -S-; Each R 40 Independently for each case the optionally substituted C1-C 20 Aliphatic or H; T 3 and T 4 Each of the C3-Cs can be substituted independently. 20 aliphatic; G 1 -N(R) 6 )C(S)N(R 6 )2、-OH、-N(R 6 )2、-N(R 9 )C(O)R 7 -N(R) 9 )S(O)2R 7 -N(R) 9 )C(O)N(R 7 )2、-CH(NR 7 -R 8 or -S(O)2R 6 ; Each G 2 Independently, it can be either O or N2; Each G 3 Independently halogens (e.g., Cl, Br, or I), -OTs, or OTf; Each R 6 In each case, H, arbitrarily substituted C1-C6 aliphatic and OR are independently selected. 7 The group formed; or R 6 Two instances together with the atoms to which they are attached form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O and S; Each R 7 In each case, the group consisting of free H and arbitrarily substituted C1-C6 aliphatic molecules is selected independently; R 8 It is a 4- to 12-membered heterocycle with 1 to 4 heteroatoms selected from N, O, and S, optionally substituted, and a 4- to 12-membered heteroaryl group with 1 to 4 heteroatoms selected from N, O, and S, substituted with -(CH2). 0-6-OH or -(CH2) 0-6 -N(R 9 One or more of the C6-C in )2 are replaced 12 Aryl group, or with an oxy group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 9 One or more of the C3-C in )2 are substituted 12 Cycloaliphatic; and Each R 9 Independently selected from H and optionally substituted C1-C6 aliphatic compounds. Attached Figure Description

[0007] Figure 1 Bar graphs showing particle size and PDI for LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59.

[0008] Figure 2 A bar graph showing the zeta potential values ​​of LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59.

[0009] Figure 3A and Figure 3B A bar graph showing the mRNA encapsulation efficiency of LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59 ( Figure 3A ) and the agarose gel electrophoresis image of the LNP formulation ( Figure 3B ).

[0010] Figure 4 A bar graph showing the mRNA integrity (%) of LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59.

[0011] Figures 5A-5C To demonstrate the effect of LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59 on C2C12 cells ( Figure 5A ), HepG2 cells ( Figure 5B ) and RAW cells ( Figure 5C A series of bar graphs showing the effect of cell viability.

[0012] Figures 6A-6C To demonstrate the effect of LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59 on C2C12 cells ( Figure 6A), HepG2 cells ( Figure 6B ) and RAW cells ( Figure 6C A series of bar graphs showing the effect of transfection efficiency on transfection efficiency.

[0013] Figure 7 Bar graphs showing the particle size and PDI of LNP formulations containing BNT-sulfon-01, BNT-sulfon-02, BNT-sulfon-03, or BNT-sulfon-04.

[0014] Figure 8 A bar graph showing the zeta potential values ​​of LNP formulations containing BNT-sulfon-01, BNT-sulfon-02, BNT-sulfon-03, or BNT-sulfon-04.

[0015] Figure 9A and Figure 9B A bar graph showing the mRNA encapsulation efficiency of LNP formulations containing BNT-sulfon-01, BNT-sulfon-02, BNT-sulfon-03, or BNT-sulfon-04. Figure 9A ) and the agarose gel electrophoresis image of the LNP formulation ( Figure 9B ).

[0016] Figure 10 Bar graph showing the mRNA integrity (%) of LNP formulations containing BNT-sulfon-01, BNT-sulfon-02, BNT-sulfon-03, or BNT-sulfon-04.

[0017] Figures 11A-11D To demonstrate the effects of LNP formulations containing BNT-sulfon-01, BNT-sulfon-02, BNT-sulfon-03, or BNT-sulfon-04 on C2C12 cells ( Figure 11A ), HepG2 cells ( Figure 11B ), RAW cells ( Figure 11C ) and Hek293 cells ( Figure 11D A series of bar graphs showing the effect of cell viability.

[0018] Figures 12A-12D To demonstrate the effect of LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59 on C2C12 cells ( Figure 12A ), HepG2 cells ( Figure 12B ), RAW cells ( Figure 12C ) and Hek293 cells ( Figure 12D A series of bar graphs showing the effect of transfection efficiency on transfection efficiency.

[0019] Figure 13A and Figure 13BA bar graph showing the in vitro hemolytic effect of LNP formulations containing BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59 ( Figure 13A ), and a bar graph showing the effect of LNP formulations on complement activation ( Figure 13B ).

[0020] Figure 14 Bar graphs showing particle size and PDI of LNP formulations containing BNT-51 and auxiliary lipids (DSPC or DOPE).

[0021] Figure 15 A bar graph showing the zeta potential values ​​of LNP formulations containing BNT-51 and auxiliary lipids (DSPC or DOPE).

[0022] Figure 16A and Figure 16B A bar graph showing the mRNA encapsulation efficiency of LNP formulations containing BNT-51 and helper lipids ( Figure 16A ) and the agarose gel electrophoresis image of the LNP formulation ( Figure 16B ).

[0023] Figure 17 Bar graph showing the mRNA integrity (%) of LNP formulations containing BNT-51 and helper lipids (DSPC or DOPE).

[0024] Figures 18A-18D To demonstrate the effect of LNP formulations containing BNT-51 and helper lipids on C2C12 cells ( Figure 18A ), HepG2 cells ( Figure 18B ), RAW cells ( Figure 18C ) and Hek293 cells ( Figure 18D A bar graph showing the effect of cell viability.

[0025] Figures 19A-19D To demonstrate the effect of LNP formulations containing BNT-51 and helper lipids on C2C12 cells ( Figure 19A ), HepG2 cells ( Figure 19B ), RAW cells ( Figure 19C ) and Hek293 cells ( Figure 19D A bar graph showing the effect of transfection efficiency on transfection efficiency.

[0026] Figure 20 Bar graphs showing the particle size and PDI of LNP formulations containing BNT-51 and occult lipids.

[0027] Figure 21 A bar graph showing the zeta potential values ​​of LNP formulations containing BNT-51 and occult lipids.

[0028] Figures 22A-22B A set of bar graphs showing the mRNA encapsulation efficiency of LNP formulations containing BNT-51 and stealth lipids ( Figure 22A ) and the agarose gel electrophoresis image of the LNP formulation ( Figure 22B ).

[0029] Figure 23 A bar graph showing the mRNA integrity (%) of LNP formulations containing BNT-51 and occult lipids.

[0030] Figures 24A-24D To demonstrate the effect of LNP formulations containing BNT-51 and stealth lipids on C2C12 cells ( Figure 24A ), HepG2 cells ( Figure 24B ), RAW cells ( Figure 24C ) and Hek293 cells ( Figure 24D A series of bar graphs showing the effect of cell viability.

[0031] Figures 25A-25D To demonstrate the effect of LNP formulations containing BNT-51 and stealth lipids on C2C12 cells ( Figure 25A ), HepG2 cells ( Figure 25B ), RAW cells ( Figure 25C ) and Hek293 cells ( Figure 25D A series of bar graphs showing the effect of transfection efficiency on transfection efficiency.

[0032] Figure 26 Bar graphs showing particle size and PDI of preformed LNPs containing BNT51 and Ac-AEEA14-DMA after undergoing at least three freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature.

[0033] Figures 27A-27C A series of bar graphs illustrating the characterization of BNT51 functionalized lipid nanoparticles prepared using Ac-AEEA14-DMA as the stealth motif and DSPE-PEG2k-α lipid; Figure 27A ) Particle size and PDI of functionalized LNP1 and LNP2; Figure 27B Agarose gel electrophoresis of control, untreated functionalized LNP1 and LNP2 (ascending), and functionalized LNP1 and LNP2 treated with release solution (descending); and ( Figure 27C The particle size and PDI of functionalized LNP1 and LNP2 were determined after at least two freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature, and after two weeks at 2°C-8°C and 25°C.

[0034] Figures 28A-28B To illustrate RNA delivered using LNP1 or LNP2 (Thy1.1)Figure 28A ) and DNA (Venus) Figure 28B A series of bars showing the percentage of transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) in all transfected PBMCs.

[0035] Figure 29 Bar graphs showing particle size and PDI of preformed LNPs containing BNT51 and Ac-AEEA14-VitE after undergoing at least three freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature.

[0036] Figures 30A-30C A series of bar graphs illustrating the characterization of BNT51 functionalized lipid nanoparticles prepared using Ac-AEEA14-VitE as the stealth motif and DSPE-PEG2k-α lipid; Figure 30A ) Particle size and PDI of functionalized LNP1 and LNP2; Figure 30B Agarose gel electrophoresis of control, untreated functionalized LNP1 and LNP2 (ascending), and functionalized LNP1 and LNP2 treated with release solution (descending); and ( Figure 30C The particle size and PDI of functionalized LNP1 and LNP2 were determined after at least two freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature, and after two weeks at 2°C-8°C and 25°C.

[0037] Figures 31A-31B To illustrate RNA delivered using LNP1 or LNP2 (Thy1.1) Figure 31A ) and DNA (Venus) Figure 31B A series of bars showing the percentage of transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) in all transfected PBMCs.

[0038] Figures 32A-32B A set of bar graphs illustrating the characterization of BNT51 functionalized lipid nanoparticles prepared using Ac-AEEA14-DMA as the stealth motif and DSPE-pAEEA14-α lipid, including ( Figure 32A ): Particle size and PDI of preformed LNPs subjected to at least two freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature; and ( Figure 32B Particle size and PDI of functionalized RNA / DNA-LNP2 under t0 and after at least two freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature.

[0039] Figures 33A-33BA set of bar graphs illustrating the characterization of BNT52 functionalized lipid nanoparticles prepared using Ac-AEEA14-DMA as the stealth motif and DSPE-pAEEA14-α lipid, including ( Figure 33A ): Particle size and PDI of preformed LNPs subjected to at least two freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature; and ( Figure 33B Particle size and PDI of functionalized RNA / DNA-LNP2 under t0 and after at least two freeze-thaw cycles of -20°C to room temperature and -80°C to room temperature.

[0040] Figure 34 A set of bar graphs illustrating the targeted transfection of T cells using the thiolipids described herein. In vitro evaluation in hPBMCs. Comparison of three different LNPs containing three different ionizable lipids shows the superior RNA and DNA transfection efficiency of the thiolipids described herein formulated in LNP2 and LNP3. The top graph depicts the percentage (y-axis) of Thy1.1-expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD19+ B cells) in all single cells and live cells. The bottom graph depicts the percentage of CD4+ T cells and CD8+ T cells expressing the Venus-nanoplasm.

[0041] Figure 35A and Figure 35B The in vitro evaluation in hPBMCs is shown using the reported thiolipin for targeted transfection of T cells. A comparison of two different LNPs containing two different ionizable lipids demonstrates the high RNA and DNA transfection efficiency of the reported thiolipin formulated in LNP1 and LNP2. Figure 35A Plot the percentage (y-axis) of Thy1.1-expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD14+ monocytes) in all single cells and live cells. Figure 35B Describe the percentage of cells expressing the Venus-nanoplasm for CD4+ T cells and CD8+ T cells.

[0042] Figure 36A and Figure 36B A set of bar graphs illustrating the characterization of LNP formulations containing BNT-72, including particle size and PDI (partial density index). Figure 36A ) and ζ potential ( Figure 36B ).

[0043] Figure 37A and Figure 37B To demonstrate the mRNA encapsulation efficiency of LNP formulations containing BNT-72 ( Figure 37A ) and mRNA integrity ( Figure 37B A set of bar charts.

[0044] Figures 38A-38C To demonstrate the effect of LNP formulations containing BNT-72 on C2C12 cells ( Figure 38A ), HepG2 cells ( Figure 38B ) and RAW cells ( Figure 38C A series of bar graphs showing the effect of cell viability.

[0045] Figures 39A-39C To demonstrate the effect of LNP formulations containing BNT-72 on C2C12 cells ( Figure 39A ), HepG2 cells ( Figure 39B ) and RAW cells ( Figure 39C A series of bar graphs showing the effect of transfection efficiency on transfection efficiency.

[0046] Figure 40 Bar graphs showing the size (nm) and polydispersity index (PDI) of complexes containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0047] Figure 41 A bar graph showing the Z-potential (mV) of a complex containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0048] Figure 42 Bar graph showing RNA integrity (%) of complexes containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0049] Figure 43 Bar graphs showing the permeability and pH of complexes containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0050] Figures 44A-44D To demonstrate C2C12 cells ( Figure 44A ), HepG2 cells ( Figure 44B ), RAW cells ( Figure 44C ) and Hek293 cells ( Figure 44D A series of bar graphs showing the transfection efficiency of ).

[0051] Figures 45A-45D To demonstrate C2C12 cells ( Figure 45A ), HepG2 cells ( Figure 45B ), RAW cells ( Figure 45C ) and Hek293 cells ( Figure 45D A series of bar graphs showing cell viability.

[0052] Figure 46The size and PDI of aCD3-functionalized LNPs (freshly prepared and after a freeze / thaw cycle at -80°C) prepared using different ionizable lipids according to embodiments of the present invention are depicted. The results of three measurements are shown, along with their average and error bars indicating 1× standard deviation.

[0053] Figure 47 The percentage of all transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) in transfected PBMCs prepared with different ionizable lipids according to embodiments of the present invention (freshly prepared and after one freeze / thaw cycle at -80°C).

[0054] Figure 48 The cell counts of all transfected live cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells or CD8+ T cells) treated with aCD3-functionalized LNPs prepared using different ionizable lipids according to embodiments of the present invention are depicted, wherein the aCD3-functionalized LNPs are freshly prepared or applied after a freeze / thaw cycle at -80°C.

[0055] Figure 49 To provide a graph of the size and distribution (PDI) of the provided LNPs containing the lipids reported in this article.

[0056] Figure 50 To provide a plot of the Z-potential of the provided LNP containing the lipids described herein.

[0057] Figure 51 A graph illustrating the encapsulation efficiency of the provided LNP.

[0058] Figure 52 Agarose gel electrophoresis was performed to show that the provided LNPs were free of RNA.

[0059] Figures 53A-53D A series of graphs are provided to illustrate cytotoxicity in the offered cell lines C2C12, HepG2, Raw, and Hek, as measured by viability percentage.

[0060] Figures 54A-54D A series of graphs are provided to illustrate the transfection efficiency of the provided LNPs as measured by RLU.

[0061] Figure 55 A graph showing the size and distribution (PDI) of the provided LNPs containing the lipid compounds described herein.

[0062] Figure 56 A graph illustrating the Z-potential of the provided LNP containing the lipids described herein.

[0063] Figure 57 A graph illustrating the encapsulation efficiency of the provided LNPs containing the lipids described herein.

[0064] Figure 58 To display agarose gel electrophoresis images of all provided LNPs without free RNA.

[0065] Figures 59A-59D A series of graphs are provided to illustrate cytotoxicity in the offered cell lines C2C12, HepG2, Raw, and Hek, as measured by viability percentage.

[0066] Figures 60A-60D A series of graphs are provided to illustrate the transfection efficiency of the provided LNPs as measured by RLU.

[0067] Figure 61 A graph illustrating the size and distribution (PDI) of LNPs containing the lipid compounds described herein.

[0068] Figure 62 A graph illustrating the Z-potential of the provided LNP containing the lipids described herein.

[0069] Figure 63 A graph illustrating the encapsulation efficiency of the provided LNPs containing the lipids described herein.

[0070] Figure 64 To show the image of the provided LNP without free RNA by agarose gel electrophoresis.

[0071] Figures 65A-65D A series of graphs are provided to illustrate cytotoxicity in the offered cell lines C2C12, HepG2, Raw, and Hek, as measured by viability percentage.

[0072] Figures 66A-66D A series of graphs are provided to illustrate the transfection efficiency of the provided LNPs as measured by RLU.

[0073] Figure 67 A graph illustrating the size and distribution (PDI) of LNPs containing the lipid compounds described herein.

[0074] Figure 68 A graph illustrating the Z-potential of the provided LNP containing the lipids described herein.

[0075] Figure 69 A graph illustrating the encapsulation efficiency of the provided LNPs containing the lipids described herein.

[0076] Figure 70 To show the image of the provided LNP without free RNA by agarose gel electrophoresis.

[0077] Figures 71A-71D A series of graphs are provided to illustrate cytotoxicity in the offered cell lines C2C12, HepG2, Raw, and Hek, as measured by viability percentage.

[0078] Figures 72A-72D A series of graphs are provided to illustrate the transfection efficiency of the provided LNPs as measured by RLU.

[0079] Figure 73 A graph illustrating the size and distribution (PDI) of LNPs containing the lipid compounds described herein.

[0080] Figure 74 A graph illustrating the Z-potential of the provided LNP containing the lipids described herein.

[0081] Figure 75 A graph illustrating the encapsulation efficiency of the provided LNPs containing the lipids described herein.

[0082] Figure 76 To show the image of the provided LNP without free RNA by agarose gel electrophoresis.

[0083] Figures 77A-77B A series of graphs are provided to illustrate cytotoxicity in the offered cell lines HepG2 and Hek, as measured by viability percentage.

[0084] Figures 78A-78D A series of graphs are provided to illustrate the transfection efficiency of the provided LNPs as measured by RLU. Detailed Implementation

[0085] This disclosure particularly provides the remarkable discovery of specific cationic or ionizable lipids comprising at least one sulfur-containing moiety. Such cationic or ionizable lipids exhibit improved properties (e.g., improved cell transfection) compared to previous lipids. For example, as illustrated in the examples provided herein, the sulfur lipids of this disclosure exhibit improvements over previous lipids lacking a sulfur-containing moiety, including, for example, improved transfection of cells with RNA. Particles prepared using the sulfur lipid compounds described herein exhibit a narrow size distribution (i.e., substantially uniform size), high encapsulation efficiency, low cytotoxicity, and improved biodistribution and efficacy compared to particles comprising previous lipid compounds.

[0086] Compounds and Definitions The compounds disclosed herein include those summarized above and further described by the categories, subclasses and species disclosed herein. Unless otherwise indicated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements are identified according to the periodic table, CAS edition, Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are set forth in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999; and “March's Advanced Organic Chemistry”, 5th edition, edited by Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0087] Unless otherwise stated, the structures described herein are intended to include all stereoisomers (e.g., enantiomers or diastereomers) and all geometric or conformational isomers of the structures. For example, the R and S configurations of each stereocenter are contemplated as part of this disclosure. Therefore, single stereochemical isomers of the provided compounds, as well as enantiomers, diastereomers, and geometric (or conformational) mixtures, are within the scope of this disclosure. For example, in some cases, Table 1 shows one or more stereoisomers of the compound, and unless otherwise indicated, represents each stereoisomer individually and / or in mixtures. Unless otherwise stated, all tautomers of the provided compounds are within the scope of this disclosure.

[0088] Unless otherwise indicated, the structures described herein are intended to include compounds differing only in the presence of one or more isotopically enriched atoms. For example, structures having the structures of this invention (including replacing hydrogen with deuterium or tritium, or using enriched atoms) 13 C or 14 Compounds of C (carbon substitution for carbon) are within the scope of this disclosure.

[0089] About or approximately:As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the reference value. Generally, those skilled in the art will understand the extent of variation covered by "approximately" or "about" in that context. For example, in some embodiments, the term "approximately" or "about" may cover a range of values ​​within (i.e., ±) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the mentioned value.

[0090] Administering: As used herein, the terms "administering" or "administration" generally refer to the administration of a composition to a subject to achieve delivery of the composition or an agent included in the composition to a target site or site of treatment. Those skilled in the art will recognize that a variety of routes of administration to a subject (e.g., a person) are appropriate where applicable. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., via bronchial instillation), buccal, dermal (which may be or include one or more of, for example, topical, intradermal, interdermal, transdermal, etc.), enteric, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrasheath, intravenous, intravenous, intracardiac, intra-organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., via intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may be intravenous. In some embodiments, administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve administering a fixed number of doses. In some embodiments, administration may involve intermittent (e.g., multiple doses separated by time) and / or periodic (e.g., individual doses separated by a common time period) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion) for at least a selected time period. In some embodiments, administration may include a prime-and-boost protocol. A prime-and-boost protocol may include administering a first dose of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine), followed by a second or subsequent dose of the pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) after a time interval. In the case of an immunogenic composition, a prime-and-boost protocol may result in an increased immune response in the patient.

[0091] Aliphatic:The term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched hydrocarbon chain, substituted or unsubstituted, that is fully saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon (also referred to herein as "cycloaliphatic") that is fully saturated or contains one or more unsaturated units but is not aromatic, and has a single or more connection point with the rest of the molecule. Unless otherwise specified, the aliphatic group contains 1-12 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms (e.g., C64 ... 1-6 In some embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms (e.g., C15, C25, C35, C45, C55, C65, C75, C65, C75, C65, C75, C65, C75, C65, C75, C65, C75, C65, C75, C65, C65, C65, C7 ... 1-5 In other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms (e.g., C14, C24, C34, C44, C54, C64, C7 ... 1-4 In other embodiments, the aliphatic group contains 1-3 aliphatic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-3 In other embodiments, the aliphatic group contains 1-2 aliphatic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-2 Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl or ynyl groups, and their hybrids. Preferred aliphatic groups are C16 and C26. 1-6 alkyl.

[0092] Alkyl: The term "alkyl" as used alone or as part of a larger part refers to having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 1-12 C 1-10 C 1-8 C 1-6 C 1-4 C 1-3 Or C 1-2 The saturated, optionally substituted straight-chain or branched hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0093] Alkylene: The term "alkylene" refers to a divalent alkyl group. In some embodiments, "alkylene" is a divalent straight-chain or branched alkyl group. In some embodiments, the "alkylene chain" is polymethylene, i.e., -(CH2). n- where n is a positive integer, for example, 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. The optionally substituted alkylene chain is a polymethylene in which one or more methylene hydrogen atoms are optionally replaced by substituents. Suitable substituents include those described below for substituted aliphatic groups and also include those described in this specification. It should be understood that two substituents of an alkylene group can together form a ring system. In some embodiments, two substituents can together form a 3- to 7-membered ring. Substituents can be on the same or different atoms. The suffix "-ene" or "-enyl" when attached to certain groups herein is intended to refer to the bifunctional portion of the group. For example, "-ene" or "-enyl" when attached to "cyclopropyl" becomes "cyclopropylene" or "cyclopropylenyl" and is intended to refer to a bifunctional cyclopropyl group, such as... .

[0094] Alkenyl: The term "alkenyl" used alone or as part of a larger part refers to having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-12 C 2-10 C 2-8 C 2-6 C 2-4 Or C 2-3 The optional substituted linear, branched, or cyclic hydrocarbon group of the alkenyl group. Exemplary alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0095] Alkynyl: The term "alkynyl" used alone or as part of a larger part refers to a group having at least one triple bond and having (unless otherwise specified) 2–12, 2–10, 2–8, 2–6, 2–4, or 2–3 carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-12 C 2-10 C 2-8 C 2-6 C 2-4 Or C 2-3 The alkynyl group may be a straight-chain or branched hydrocarbon group that has been optionally substituted. Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0096] Aryl:The term "aryl" refers to an aryl group having a total of 6 to 14 ring members (e.g., C6-C). 14 The system comprises single-ring and double-ring systems, wherein at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. In some embodiments, the "aryl" contains 6 to 12 total ring members (e.g., C6-C). 12 The term "aryl" is used interchangeably with the term "aryl ring." In some embodiments, "aryl" refers to an aromatic ring system including, but not limited to, phenyl, biphenyl, naphthyl, anthracene, etc., which may have one or more substituents. Unless otherwise specified, "aryl" is a hydrocarbon. In some embodiments, the "aryl" ring system is an aromatic ring (e.g., phenyl) fused to a non-aromatic ring (e.g., cycloalkyl). Examples of fused aryl rings include... , and .

[0097] Related: When used herein, the term means that two events or entities are “related” to each other if the presence, level, and / or form of one event or entity is associated with the presence, level, and / or form of another event or entity. For example, a particular entity (e.g., peptide, genetic trait, metabolite, microorganism, etc.) is considered related to a particular disease, condition, or susceptibility (e.g., in a relevant population) if the presence, level, and / or form of that entity is associated with the incidence and / or susceptibility to that disease, condition, or ailment. In some embodiments, two or more entities are “related” to each other if they interact directly or indirectly to bring them physically close to each other and / or keep them close. In some embodiments, two or more physically related entities are covalently connected; in some embodiments, two or more physically related entities are not covalently connected but are non-covalently related, for example, by means of hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0098] BicyclicThe term "bicyclic" or "bicyclic system" refers to any bicyclic system having one or more common atoms between the two rings, i.e., a carbocyclic or heterocyclic system, saturated or having one or more unsaturated units. Therefore, the term includes any permitted ring fusion, such as ortho-fused or spirocyclic. The term "heterobicyclic" as used herein is a subset of "bicyclic" requiring the presence of one or more heteroatoms in one or both rings. Such heteroatoms may be present at the ring junctions and optionally substituted, and may be selected from nitrogen (including N-oxides), oxygen, sulfur (including oxidized forms such as sulfones and sulfonates), phosphorus (including oxidized forms such as phosphates), boron, etc. In some embodiments, the bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. The term "bridging bicyclic" as used herein refers to any bicyclic system having at least one bridge, i.e., a carbocyclic or heterocyclic system, saturated or partially unsaturated. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or valence bonds connecting two bridgeheads, wherein a “bridgehead” is any skeletal atom of a ring system bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, the bridging bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridging bicyclic groups are well known in the art and include those groups described below, wherein each group is attached to the remainder of the molecule at any substituted carbon or nitrogen atom. Unless otherwise specified, the bridging bicyclic group is optionally substituted with one or more substituents as described for aliphatic groups. Additionally or alternatively, any substituted nitrogen atom in the bridging bicyclic group is optionally substituted. Exemplary bicycles include: An exemplary bridging dual ring includes: .

[0099] Biological sample:As used herein, the term "biological sample" generally refers to a sample obtained from or derived from a biological source of interest (e.g., tissue or organism or cell culture), as described herein. In some embodiments, the source of interest includes organisms, such as animals or humans. In some embodiments, the biological sample is or contains biological tissue or fluid. In some embodiments, the biological sample may be or contain bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; cell-containing body fluids; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washing or lavage solutions, such as catheter lavage or bronchoalveolar lavage fluid; aspirates; scrapings; bone marrow samples; tissue biopsy samples; surgical samples; feces, other body fluids, secretions and / or excretions; and / or cells derived therefrom, etc. In some embodiments, the biological sample is or contains cells obtained from an individual. In some embodiments, the obtained cells are or include cells from the individual from which the sample was obtained. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as the context will be clear, the term “sample” refers to a formulation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, semi-permeable membrane filtration is used. Such a “processed sample” may contain nucleic acids or proteins, for example, extracted from the sample or obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components.

[0100] Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or medium applied together with the composition. In some exemplary embodiments, the carrier may include sterile liquids, such as, for example, water and oils, including petroleum, animal, vegetable, or synthetically derived oils, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or comprises one or more solid components.

[0101] Combination therapy:As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents or modalities). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "dose" of the first regimen are administered before any dose of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administration" of combination therapy may involve administering another one or more agents or modalities to a subject receiving one or more agents or modalities from the combination. For the sake of brevity, combination therapy does not require the administration of individual agents together in a single composition (or even necessarily simultaneous administration), but in some embodiments, two or more agents or their active portions may be administered together in the combination composition or even in the combination compound (e.g., as part of a single chemical complex or covalent entity).

[0102] Corresponding As used herein, the term "equivalent" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to each other but are similar enough to allow for comparison between them, so that those skilled in the art will recognize that reasonable conclusions can be drawn based on observed differences or similarities. In some embodiments, equivalent sets of conditions, situations, individuals, or groups are characterized by a number of substantially identical features and one or a few different features. Those skilled in the art will understand, in the context, what degree of consistency is required in any given situation of two or more such agents, entities, situations, sets of conditions, etc., considered equivalent. For example, those skilled in the art will recognize that when the features are substantially identical in a sufficient number and type, the sets of situations, individuals, or groups are equivalent to each other to guarantee reasonable conclusions that differences in results or observed phenomena obtained under different sets of situations, individuals, or groups, or using different sets of situations, individuals, or groups, are caused by or indicate changes in those different features.

[0103] Composition: Those skilled in the art will recognize that the term "composition" can be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise specified, a composition may be in any form, such as a gas, gel, liquid, solid, etc.

[0104] Cycloaliphatic As used herein, the term "cycloaliphatic" refers to a monocyclic C14 molecule that is fully saturated or contains one or more unsaturated units, but is not aromatic, and has a single or more connection points with the rest of the molecule. 3-8 Hydrocarbons or bicyclic C 6-10 hydrocarbon.

[0105] CycloalkylAs used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic system with optional substitution of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0106] Deoxyribonucleic acid (DNA) As used herein, the term "DNA" refers to a polymer molecule that is typically double-stranded and contains nucleotides of adenine, cytosine, guanine, and thymine, and a deoxyribose backbone structure as specified in the definition of "nucleic acid / polynucleotide". In some embodiments, the DNA is linear DNA, plasmid DNA, microcircular DNA, nanoparticle DNA, dog bone DNA, or a transposon.

[0107] Deoxyribonucleotide: As used herein, the term "deoxyribonucleic acid" refers to both unmodified and modified deoxyribonucleic acid. For example, unmodified deoxyribonucleic acid includes purine bases adenine (A) and guanine (G), and pyrimidine bases cytosine (C) and thymine (T). Modified deoxyribonucleic acid may include one or more modifications, including but not limited to, for example, (a) terminal modifications, such as 5' terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse bonding, etc.), 3' terminal modifications (e.g., conjugation, reverse bonding, etc.), (b) base modifications, such as substitution with a modified base, a stable base, a destabilized base, or a base or conjugated base paired with an extended partner lineage base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution, and (d) internucleotide linking modifications, including modifications or substitutions of phosphodiester links.

[0108] Dosage form or Unit dosage form: Those skilled in the art will recognize that the term "dosage form" can be used to refer to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) administered to a subject. Typically, each such unit contains a predetermined amount of the active agent. In some embodiments, such an amount is a unit dose (or a whole portion thereof) suitable for administration according to a dosing regimen determined to be associated with desired or beneficial outcomes (i.e., the use of a therapeutic dosing regimen) when administered to the relevant population.

[0109] Dosing regimen or Treatment regimen:Those skilled in the art will recognize that the terms "dosing regimen" and "treatment regimen" can be used to refer to a set of unit doses (usually more than one) administered individually to a subject, typically separated by time periods. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen includes multiple doses, each separated from the other doses in time. In some embodiments, individual doses are separated from each other by time periods of equal length; in some embodiments, the dosing regimen includes multiple doses and at least two different time periods separating the individual doses. In some embodiments, all doses within the dosing regimen have the same unit dose amount. In some embodiments, the different doses within the dosing regimen have different amounts. In some embodiments, the dosing regimen includes a first dose of a first dose amount, followed by one or more additional doses of a second dose amount different from the first dose amount. In some embodiments, the dosing regimen includes a first dose of a first dose amount, followed by one or more additional doses of a second dose amount identical to the first dose amount. In some embodiments, when administered in a relevant population, the dosing regimen is associated with a desired or beneficial outcome (i.e., a therapeutic dosing regimen).

[0110] Effective amount The term "effective amount" refers to an amount of compound sufficient to achieve a beneficial or desired outcome (e.g., therapeutic, ameliorative, inhibitory, or preventative outcome). An effective amount may be administered in a single or multiple applications, by application, or in a dose, and is not intended to be limited to a particular formulation or route of administration.

[0111] Excipient: As used herein, the term "excipient" refers to a non-therapeutic agent that can be incorporated into a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc.

[0112] Halogen: The term "halogen" or "halogen group" refers to F, Cl, Br, or I.

[0113] HeteroaliphaticAs used herein, the term "heteroaliphatic" or "heteroaliphatic group" refers to an optionally substituted hydrocarbon moiety having 1 to 5 heteroatoms in addition to carbon atoms, which may be straight-chain (i.e., unbranched), branched, or cyclic ("heterocyclic") and may be fully saturated or contain one or more unsaturated units but is not aromatic. The term "heteroatom" means nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise specified, a heteroaliphatic group contains 1 to 10 carbon atoms, wherein 1 to 3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroaliphatic group contains 1 to 4 carbon atoms, wherein 1 to 2 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen, and sulfur. In other embodiments, the heteroaliphatic group contains 1 to 3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced by a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, straight-chain or branched heteroalkyl, heteroalkenyl, and heteroynyl groups. For example, heteroaliphatic groups of 1 to 10 atoms include the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2-CH2-O-CH2-CH2-O-CH3, etc.

[0114] Heteroaryl The terms “heteroaryl” and “heteroaryl-”, used alone or as part of a larger portion (e.g., “heteroarylalkyl” or “heteroarylalkoxy”), refer to monocyclic or bicyclic groups having 5 to 10 ring atoms (e.g., 5 to 6-membered monocyclic heteroaryl or 9 to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π-electrons shared in a cyclic array; and having 1 to 5 heteroatoms in addition to carbon atoms. Heteroaryl groups include, but are not limited to, thiophene, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiazolyl, thiazolyl, pyridinyl, pyrazinyl, indazinyl, purine, naphthinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, pyrrolopyridinyl, pyrrolopyrazinyl, thiophenolopyrimidinyl, triazolopyridinyl, and benzoisoxazolyl. As used herein, the terms "heteroaryl" and "heteroary-" also include groups in which a heteroaryl ring is fused to one or more aryl, cycloaliphatic, or heterocyclic rings, wherein the linking group or linking point is located on the heteroaryl ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothiopheneyl, benzofuranyl, dibenzofuranyl, indazoleyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzooxazolyl, quinolinyl, isoquinolinyl, terolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4- H-Quinazinyl, carbazolyl, acridinel, phenazinyl, phenothiazinyl, phenotoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl,” or “heteroaryl group,” any of which includes optionally substituted rings.

[0115] Heteroatom: As used herein, the term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of basic nitrogen.

[0116] Heterocycle: As used herein, the terms “heterocycle,” “heterocyclic group,” “heterocyclic group,” and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, 6- to 10-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic portion that is saturated or partially unsaturated and has one or more, such as 1 to 4, heteroatoms as defined above, in addition to a carbon atom. When used to refer to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen may be N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolealkyl), or NR. + (e.g., in N-substituted pyrrolidinyl groups). The heterocyclic ring may be attached to its side group at any heteroatom or carbon atom that produces a stable structure, and any ring atom may optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, azirrobutyl, oxacyclobutyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolyl, piperazine, dioxalyl, dioxopentyl, diazaphenyl, oxazolyl, thioazolyl, morpholinyl, and thiomorpholinyl. The heterocyclic group may be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, more preferably monocyclic or bicyclic. Bicyclic heterocyclic rings also include groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indololinyl, isoindolinyl, benzodioxanepentenyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. The bicyclic heterocyclic ring can also be a spirocyclic system (e.g., a 7- to 11-membered spirocyclic fused heterocyclic ring having one or more heteroatoms as defined above (e.g., one, two, three, or four heteroatoms) in addition to a carbon atom). The bicyclic heterocyclic ring can also be a bridging ring system (e.g., a 7- to 11-membered bridging heterocyclic ring having one, two, or three bridging atoms).

[0117] Nanoparticle:As used herein, the term "nanoparticle" refers to a small, discrete entity, typically shorter than about 1000 nanometers (nm) and typically shorter than 500 nm, or even 100 nm or less. In many embodiments, nanoparticles are characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 µm and about 500 nm, or between about 1 nm and 1000 nm. In many embodiments, a population of microparticles is characterized by an average size (e.g., the longest dimension) below about 1000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and typically above about 1 nm. In many embodiments, microparticles may be substantially spherical (e.g., such that their longest dimension is their diameter). In some embodiments, the diameter of the nanoparticles is less than 100 nm, as defined by the National Institutes of Health. In some embodiments, the nanoparticles are micelles, wherein they comprise closed compartments separated from the bulk solution by a micelle membrane, and typically comprise amphiphilic entities that surround and enclose spaces or compartments (e.g., to define an interior cavity). In some embodiments, the micelle membrane comprises at least one polymer, such as, for example, a biocompatible and / or biodegradable polymer.

[0118] Nucleic acid / polynucleotideAs used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises a mixture of DNA and RNA. In some embodiments, the nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, the nucleic acid is or comprises a single-stranded nucleic acid. In some embodiments, the nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, the nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, the nucleic acid comprises a backbone comprising one or more phosphodiester bonds. In some embodiments, the nucleic acid comprises a backbone comprising phosphodiester bonds and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may comprise a backbone comprising one or more thiophosphates, dithiophosphates, phosphoramides, phosphite-borane complexes, or 5'-N-phosphite bonds and / or one or more peptide bonds (e.g., as in "peptide nucleic acid"). In some embodiments, the nucleic acid contains one or more or all of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid contains one or more or all of the non-natural residues. In some embodiments, the non-natural residues comprise nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residues comprise one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars in the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (such as RNA or a polypeptide). In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by isolating from a natural source, enzymatically synthesizing (e.g., by polymerase chain reaction based on a complementary template, e.g., in vivo or in vitro), propagating in a recombinant cell or system, or by chemical synthesis.In some implementation schemes, the length of the nucleic acids is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, or 14. 0, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 25 00, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500 0, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides.

[0119] Nucleic acid particle: Nucleic acid particles can be used to deliver nucleic acids to target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from at least one cation or cation-ionizable lipid or lipid-like material, at least one cationic polymer (such as protamine), or mixtures thereof, and nucleic acids. Nucleic acid particles include formulations based on lipid nanoparticles (LNPs) and formulations based on liposome complexes (LPX).

[0120] Nucleotide: As used herein, the term "nucleotide" refers to its accepted meaning. When the number of nucleotides is used as an indicator of, for example, the size of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand of, for example, a polynucleotide.

[0121] Parenterally: As used herein, the phrases “parenteral administration” and “extraterrestrial administration” have their meanings as understood in the art, referring to administration methods other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-occipital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0122] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond between ring atoms. The term “partially unsaturated” is intended to cover rings having multiple unsaturated sites, but not to include aromatic (e.g., aryl or heteroaryl) moiety as defined herein.

[0123] Patient or Subject: As used herein, the terms "patient" or "subject" refer to any organism to which the provided composition is applied or can be applied, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient or subject has or is susceptible to one or more conditions or disorders. In some embodiments, the patient or subject exhibits one or more symptoms of a condition or disorder. In some embodiments, the patient or subject has been diagnosed with one or more conditions or disorders. In some embodiments, the patient or subject is receiving or has received a therapy for the diagnosis and / or treatment of a disease, condition, or disorder.

[0124] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount suitable for administration in a treatment or dosing regimen that demonstrates a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those suitable for: oral administration, such as oral enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., tablets targeted for absorption via the buccal, sublingual, and systemic routes), pills, powders, granules, or pastes applied to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection as, for example, a sterile solution or suspension or a sustained-release formulation; topical administration, such as as creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or mouth; intravaginal or rectal administration, such as as pessaries, creams, or foams; sublingual; ocular; transdermal; or via the nose, lungs, and other mucosal surfaces.

[0125] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” means, to the extent of reasonable medical judgment, compounds, materials, compositions, and / or dosage forms that are suitable for contact with tissues in humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio.

[0126] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to a salt suitable for use in a pharmaceutical context, meaning a salt that, to a reasonable medical judgment, is suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and in proportion to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. in... J. Pharmaceutical Sciences Pharmaceutically acceptable salts are described in detail in , 66: 1-19 (1977). Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed using inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by other methods used in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-p-ethylhexanoate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0127] Furthermore, acids generally considered suitable for forming pharmaceutically useful salts from basic pharmaceutical compounds are discussed in, for example, the following literature: P. Stahl et al., Camille G. (edited) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences 1977, 66 (1), 1-19; P. Gould, International Journal of Pharmaceutics 1986, 33 , 201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; and The Orange Book(From the Food & Drug Administration, Washington, DC website). This publicly available information is incorporated herein by reference.

[0128] Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N salts. + (C 1-4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using balancing ions (such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate).

[0129] Physiological conditions: As used herein, with the meaning understood in the art, physiological conditions refer to the conditions under which cells or organisms live and / or reproduce. In some embodiments, the term refers to the conditions of the external or internal environment in which an organism or cellular system may exist in nature. In some embodiments, physiological conditions are those present in the body of a human or non-human animal, particularly those present at and / or within a surgical site. Physiological conditions typically include, for example, a temperature range of 20°C–40°C, a pressure of 1 atmosphere, a pH of 6–8, a glucose concentration of 1–20 mM, an oxygen concentration at atmospheric levels, and the gravity encountered on Earth. In some embodiments, laboratory conditions are manipulated and / or maintained under physiological conditions. In some embodiments, physiological conditions are encountered in an organism.

[0130] Multi-ring As used herein, the term "polycyclic" refers to a saturated or unsaturated ring system having two or more rings (e.g., heterocyclic, heteroaryl, cycloalkyl, or aryl rings) having 7 to 20 atoms, wherein one or more carbon atoms are shared by two adjacent rings. For example, in some embodiments, a polycyclic system refers to a saturated or unsaturated ring system having three or more rings (e.g., heterocyclic, heteroaryl, cycloalkyl, or aryl rings) having 14 to 20 atoms, wherein one or more carbon atoms are shared by two adjacent rings. The rings in a polycyclic system can be fused (i.e., bicyclic or tricyclic), spirocyclic, or combinations thereof. An example polycyclic compound is a steroid.

[0131] polypeptideAs used herein, the term "polypeptide" or "peptide" generally has the accepted meaning of a polymer having at least three or more amino acids. Those skilled in the art will appreciate that the term "polypeptide" is intended to be broad enough to encompass not only polypeptides having the complete sequences listed herein, but also polypeptides representing functional, biologically active, or characteristic fragments, portions, or domains (e.g., retaining at least one active fragment, portion, or domain) of such complete polypeptides. In some embodiments, the polypeptide may contain L-amino acids, D-amino acids, or both, and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the polypeptide may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptide-like substances).

[0132] refer to: As used herein, standards or controls are described for comparison with respect to their implementation. For example, in some embodiments, the agent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is substantially tested and / or measured simultaneously with the test or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, as those skilled in the art will understand, the reference or control is measured or characterized under conditions or circumstances equivalent to those under which the evaluation is conducted. Those skilled in the art will understand when sufficient similarity exists to justify reliance on and / or comparison with a particular possible reference or control.

[0133] Ribonucleotides: As used herein, the term "ribonucleotide" encompasses both unmodified and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications, including but not limited to, for example, (a) terminal modifications, such as 5' terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse bonding, etc.), 3' terminal modifications (e.g., conjugation, reverse bonding, etc.), (b) base modifications, such as substitution with a modified base, a stable base, a destabilized base, or a base or conjugated base paired with an extended coupler lineage base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleotide linking modifications, including modified or substituted phosphodiester links. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including both modified and unmodified ribonucleotide triphosphates.

[0134] Ribonucleic acid (RNA):As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, RNA is single-stranded. In some embodiments, RNA is double-stranded. In some embodiments, RNA comprises both single-stranded and double-stranded portions. In some embodiments, RNA may contain as described above... Nucleic acid / polynucleotide The main chain structure is as defined in the definition. RNA can be regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA). In some embodiments, the RNA is mRNA. In some embodiments where the RNA is mRNA, the RNA typically contains a poly(A) region at its 3' end. In some embodiments where the RNA is mRNA, the RNA typically contains a recognized cap structure at its 5' end, for example, for recognizing the mRNA and linking it to a ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic synthesis and / or by chemical synthesis).

[0135] Replacement or Optional replacement: As described herein, the compounds of the present invention may contain an "optionally substituted" portion. Generally, the term "substituted" means, regardless of whether it is preceded by the term "optionally," that one or more hydrogens of the specified portion are replaced by suitable substituents. "Substituent" applies to one or more hydrogens explicitly or implicitly present in the structure (e.g., It means at least ;and It means at least , , or Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure may be substituted by more than one substituent selected from the specified group. The combination of substituents contemplated in this invention is preferably a combination of substituents that can form stable or chemically viable compounds. As used herein, the term "stable" means a compound that does not substantially change when subjected to conditions that allow it to be generated, detected, and in some embodiments recovered, purified, and used for one or more purposes provided herein. A group described as "substituted" preferably has 1 to 4 substituents, more preferably 1 or 2 substituents. A group described as "optionally substituted" may be unsubstituted or "substituted" as described above.

[0136] The suitable monovalent substituent on the substituted carbon atom of the "optionally substituted" group is independently a halogen; -(CH2) 0- 4R°;-(CH2) 0-4 OR°;-O(CH2)0-4 R°、-O-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 CH(OR°)2;-(CH2) 0-4 SR°;-(CH2) 0-4 Ph, which can be replaced by R°; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be substituted by R°; -CH=CHPh, which can be substituted by R°; -(CH2) 0-4 O(CH2) 0-1 -Pyridyl group, which can be substituted with R°; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R°)2;-(CH2) 0-4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH2) 0-4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2) 0-4 N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2) 0-4 C(O)R°;C(S)R°;-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 C(O)SR°;-(CH2) 0-4 C(O)oSiR°3;-(CH2) 0-4 OC(O)R°;-OC(O)(CH2) 0-4 SR°;-(CH2) 0-4 SC(O)R°;-(CH2) 0-4 C(O)NR°2; -C(S)NR°2; -C(S)SR°; -SC(S)SR°, -(CH2) 0-4 OC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2) 0- 4SSR°;-(CH2) 0-4 S(O)2R°;-(CH2) 0-4 S(O)₂OR°;-(CH₂) 0-4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0-4S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -P(O)2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; SiR°3; -(C 1-4 (linear or branched alkylene)ON(R°)2; or -(C 1-4 (straight-chain or branched alkylene)C(O)ON(R°)2, wherein each R° may be substituted as defined below and independently be hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (5 to 6-membered heteroaryl ring), or a 3 to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, although defined above, two independently occurring R° together with one or more intermediate atoms to form a 3 to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0137] Suitable monovalent substituents on R° (or a ring formed by two independently occurring R° atoms and their intermediate atom) are independently halogens or -(CH2). 0-2 R l -(halogenated R) l -(CH2) 0-2 OH, -(CH2) 0-2 OR l -(CH2) 0-2 CH(OR l )2、-O(halogenated R l -CN, -N3, -(CH2) 0-2 C(O)R l -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR l -(CH2) 0-2 SR l -(CH2) 0- 2SH、-(CH2) 0-2 NH2、-(CH2) 0-2 NHR l -(CH2) 0-2 NR l 2, -NO2, -SiR l 3. -oSiR l 3. -C(O)SR l 、 -(C 1-4(straight-chain or branched alkylene)C(O)OR l or -SSR l , where each R l It is either unsubstituted or, when preceded by "halogenated", substituted by only one or more halogens, and independently selected from C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on the saturated carbon atom of R° include =O and =S.

[0138] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include the following groups: =O ("oxo"), =S, =NNR. * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * =NR * =NOR * -O(C(R) * 2)) 2-3 O- or -S(C(R) * 2)) 2-3 S-, where each independently occurring R * Selected from hydrogen, and C that can be substituted as defined below. 1-6 Aliphatic, or unsubstituted, 5- to 6-membered saturated, partially unsaturated, or aryl rings having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents for the ortho-substituted carbon atom attached to the "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each independently occurring R * Selected from hydrogen, and C that can be substituted as defined below. 1-6 Aliphatic, or unsubstituted, having 5-6 saturated, partially unsaturated, or aryl rings with 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0139] R * Suitable substituents on the aliphatic group include halogens, -R l -(halogenated R) l -OH, -OR l -O (halogenated R) l -CN, -C(O)OH, -C(O)OR l -NH2, -NHR l -NR l 2 or -NO2, where each R lIt is either unsubstituted or, when preceded by a "halogenated" group, substituted by only one or more halogens, and is independently C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0140] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include -R † -NR † 2. -C(O)R † -C(O)OR † -C(O)C(O)R † -C(O)CH2C(O)R † -S(O)2R † -S(O)2NR † 2. -C(S)NR † 2. -C(NH)NR † 2 or -N(R) † )S(O)2R † ; where each R † Independently, hydrogen, and C that can be substituted as defined below. 1-6 Aliphatic, unsubstituted -oPh, or unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl rings having 0 to 4 independently selected heteroatoms chosen from nitrogen, oxygen, or sulfur, or, although defined above, two independently occurring R... † Together with one or more intermediate atoms, it forms an unsubstituted 3 to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring with 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur.

[0141] R † Suitable substituents on the aliphatic group are independently halogens, -R l -(halogenated R) l -OH, -OR l -O (halogenated R) l -CN, -C(O)OH, -C(O)OR l -NH2, -NHR l -NR l 2 or -NO2, where each R l It is either unsubstituted or, when preceded by a "halogenated" group, substituted by only one or more halogens, and is independently C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0142] Small molecules: As used herein, the term "small molecule" refers to a low molecular weight organic and / or inorganic compound. Generally, a "small molecule" is a molecule smaller than about 5 kilodaltons (kD). In some embodiments, a small molecule is smaller than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, a small molecule is smaller than about 800 Daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is smaller than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, the small molecule is not a polymer.

[0143] In some embodiments, the small molecule does not include a polymer portion. In some embodiments, the small molecule is not and / or does not contain proteins or polypeptides (e.g., not an oligopeptide or peptide). In some embodiments, the small molecule is not and / or does not contain polynucleotides (e.g., not an oligonucleotide). In some embodiments, the small molecule is not and / or does not contain polysaccharides; for example, in some embodiments, the small molecule is not a glycoprotein, proteoglycan, glycolipid, etc. In some embodiments, the small molecule is not a lipid.

[0144] In some embodiments, the small molecule is a modulator (e.g., an inhibitor or activator). In some embodiments, the small molecule is a bioactive agent. In some embodiments, the small molecule is detectable (e.g., containing at least one detectable moiety). In some embodiments, the small molecule is a therapeutic agent.

[0145] Those skilled in the art who read this disclosure will understand that certain small molecule compounds described herein may be provided and / or used in any of a variety of forms, such as, for example, crystalline forms (e.g., polymorphs, solvates, etc.), salt forms, protected forms, prodrug forms, ester forms, isomer forms (e.g., optical and / or structural isomers), isotopic forms, etc.

[0146] Those skilled in the art will understand that certain small molecule compounds have structures that can exist in one or more stereoisomers. In some embodiments, such small molecules may be used according to this disclosure as individual enantiomers, diastereomers, or geometric isomers, or as mixtures of stereoisomers; in some embodiments, such small molecules may be used as racemic mixtures other than those disclosed herein.

[0147] Those skilled in the art will understand that certain small molecule compounds have structures that can exist in one or more tautomer forms. In some embodiments, such small molecules can be used according to this disclosure either as individual tautomers or in a form that interconverts between tautomer forms.

[0148] Those skilled in the art will understand that some small molecule compounds allow isotope substitution (e.g., 2 H or 3 H replaces H; 11 C 13 C or 14 C replaces 12 C; 13 N or 15 N replaces 14 N; 17 O or 18 O replaced 16 O; 36 Cl replaced 35 Cl or 37 Cl; 18 F replaces 19 F; 131 I replace 127 The structure of (I, etc.). In some embodiments, such small molecules may be used according to this disclosure in one or more isotope-modified forms or mixtures thereof.

[0149] In some embodiments, reference to a specific small molecule compound may refer to a specific form of the compound. In some embodiments, the specific small molecule compound may be provided and / or used in salt form (e.g., as an acid addition salt or a base addition salt, depending on the compound); in some embodiments, the salt form may be a pharmaceutically acceptable salt form.

[0150] In some embodiments, when the small molecule compound is a compound that is present or found in nature, the compound may be provided and / or used according to this disclosure in a form different from that which is present or found in nature. Those skilled in the art will understand that in some embodiments, a formulation of a particular small molecule compound (containing an absolute or relative amount of the compound or a particular form thereof that differs from the absolute or relative (relative to another component of the formulation, including, for example, another form of the compound) amount present in the reference formulation of interest (e.g., from a primary sample from a source of interest, such as a biological or environmental source) of the compound or form thereof) differs from the compound present in the reference formulation or source. Therefore, in some embodiments, for example, a formulation of a single stereoisomer of a small molecule compound may be considered a form of compound different from a racemic mixture of the compound; a particular salt of a small molecule compound may be considered a form different from another salt form of the compound; a formulation containing only one conformational isomer ((Z) or (E)) containing a double bond may be considered a form of compound different from another conformational isomer ((E) or (Z)) containing a double bond; a formulation in which one or more atoms are isotopes different from those present in the reference formulation may be considered a different form, etc.

[0151] Those skilled in the art should further understand that, in small molecule structures, symbols as used herein... This refers to the junction between two atoms. Alternatively, the symbol... It refers to the connection point of a ring in a spiral manner.

[0152] treat: As used herein, the terms “treat,” “treatment,” or “treating” refer to any method used to partially or completely alleviate, improve, reduce, suppress, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or features of a disease, condition, and / or disorder. Treatment may be administered to a subject who does not exhibit signs of a disease, condition, and / or disorder. In some embodiments, treatment may be administered to a subject who exhibits only early signs of a disease, condition, and / or disorder, for purposes such as reducing the risk of developing lesions associated with the disease, condition, and / or disorder.

[0153] Ionizable sulfur lipid compounds This disclosure particularly provides cationic or ionizable thiolipin compounds that can be used to form particles containing nucleic acids. As described herein, in some embodiments, this disclosure provides compounds represented by Formula I: I Or its pharmaceutically acceptable salt, wherein: L 1 and L2 Each of the C1-Cs can be substituted independently. 30 Aliphatic groups; L 3 C1-C, which are the bonds and can be arbitrarily substituted 10 Aliphatic groups or optionally substituted 2- to 10-membered heteroaliphatic groups comprising 1 to 4 heteroatoms selected from N, O, and S; X 1 and X 2 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-, where X 1 or X 2 One or both of them are selected from -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-; Each R 1 Independently for each case the optionally substituted C1-C 20 Aliphatic or H; T 1 and T 2 Each of the C3-Cs can be substituted independently. 30 aliphatic; G is -N(R) 2 )C(S)N(R 2 )2、-OH、-N(R 2 )2、-N + (R 3 )3、-N(R 5 )C(O)R 3 -N(R) 5 )S(O)2R 3 -N(R) 5 )C(O)N(R 3 )2、-CH(NR 2 -R 4 or -S(O)2R 3 ; Each R 2In each case, H, arbitrarily substituted C1-C6 aliphatic and OR are independently selected. 3 The group formed; or R 2 Two instances together with the atoms to which they are attached form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O and S; Each R 3 In each case, H and the arbitrarily substituted C1-C are chosen independently. 10 A group composed of aliphatic groups; and R 4 It is a 4- to 12-membered heterocycle with 1 to 4 heteroatoms selected from N, O, and S, optionally substituted, and a 4- to 12-membered heteroaryl group with 1 to 4 heteroatoms selected from N, O, and S, substituted with -(CH2). 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C6-C in )2 are replaced 12 Aryl group, or with an oxy group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C3-C in )2 are substituted 12 Cycloaliphatic; Each R 5 Independently selected from H and optionally substituted C1-C6 aliphatic compounds.

[0154] As described in this article, L 1 and L 2 Each of the C1-Cs can be substituted independently. 30 Aliphatic group. In some embodiments, L 1 C1-C is an optional substitute 30 Aliphatic. In some implementations, L 1 For C1-C 10 Aliphatic. In some implementations, L 1 C1-C is an optional substitute 30 Alkylene. In some embodiments, L 1 C1-C is an optional substitute 30 Alkenyl group. In some embodiments, L 1 For C1-C 10 Alkylene. In some embodiments, L 1 -(CH2) 1-10 - In some implementations, L 2 C1-C is an optional substitute 30 Aliphatic. In some implementations, L 2 For C1-C 10Aliphatic. In some implementations, L 2 C1-C is an optional substitute 30 Alkylene. In some embodiments, L 2 C1-C is an optional substitute 30 Alkenyl group. In some embodiments, L 2 For C1-C 10 Alkylene. In some embodiments, L 2 -(CH2) 1-10 - In some implementations, L 1 It is -(CH2)6-, and L 2 It is -(CH2)8-. In some implementations, L 1 and L 2 Each is C1-C 30 Alkylene. In some embodiments, L 1 and L 2 Each is -(CH2) 6-12 - In some implementations, L 1 and L 2 Each is -(CH2) 6-10 - In some implementations, L 1 and L 2 Each is -(CH2)6-. In some implementations, L 1 and L 2 Each is -(CH2)7-. In some implementations, L 1 and L 2 Each is -(CH2)8-. In some implementations, L 1 and L 2 Each is -(CH2)9-.

[0155] In some implementations, L 1 and L 2 For the same reason. In some implementations, L 1 and L 2 For different reasons.

[0156] As described in this article, X 1 and X 2 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-, where X 1 or X2 One or both of them are selected from -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-. In some implementations, X 1 and X 2 One of them is the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-, and X 1 and X 2 The other one is -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-. In some implementations, X 1 and X 2 Each is independently selected from -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-. In some implementations, X 1 and X 2 One of them is a bond, -OC(O)- or -C(O)O-, and X 1 and X 2 The other one is -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-. In some implementations, X1 For bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-. In some implementations, X 1 -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-. In some implementations, X 1 For the bond, -OC(O)- or -C(O)O-. In some implementations, X 2 For bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-. In some implementations, X 2 -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-. In some implementations, X 2 For the bond, -OC(O)- or -C(O)O-. In some implementations, X 2 As a key. In some implementations, X 2 For -OC(O)-. In some implementations, X 2 It is -C(O)O-.

[0157] In some implementation schemes, X 1 and X 2 Each is independently selected from -S(O)2N(R) 1 )-、-N(R 1-S(O)2 and -S(O)2-. In some implementations, X 1 -S(O)2N(R) 1 In some implementations, X 1 It is -S(O)₂N(H)⁻. In some implementations, X 1 -S(O)2N(R) 1 )-, where R 1 For C1-C 10 Aliphatic. In some implementations, X 1 -N(R) 1 )S(O)2. In some implementations, X 1 It is -N(H)S(O)2. In some implementations, X 1 -N(R) 1 )S(O)2, where R 1 For C1-C 10 Aliphatic. In some implementations, X 1 For -S(O)2-. In some implementations, X 2 -S(O)2N(R) 1 In some implementations, X 2 It is -S(O)₂N(H)⁻. In some implementations, X 2 -S(O)2N(R) 1 )-, where R 1 For C1-C 10 Aliphatic. In some implementations, X 2 -N(R) 1 )S(O)2. In some implementations, X 2 It is -N(H)S(O)2. In some implementations, X 2 -N(R) 1 )S(O)2, where R 1 For C1-C 10 Aliphatic. In some implementations, X 2 It is -S(O)2-.

[0158] In some implementation schemes, X 1 -S(O)2N(R) 1 )-, where R 1 For C1-C 10 Aliphatic, and X 2 For -C(O)O-. In some implementations, X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 Independently for R 1 For C1-C10 Aliphatic. In some implementations, X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 -(CH2) 3-10 -CH3. In some implementations, X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 It is -(CH2)3-CH3. In some implementations, X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 It is -(CH2)4-CH3. In some implementations, X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 It is -(CH2)5-CH3. In some implementations, X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 It is -(CH2)6-CH3. In some implementations, X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 It is -(CH2)7-CH3.

[0159] In some implementation schemes, X 1 and X 2 For the same reason. In some implementations, X 1 and X 2 For different reasons.

[0160] As described in this article, T 1 and T 2 Each of the C3-Cs can be substituted independently. 30 Aliphatic. In some implementations, T 1 For C3-C 30 Aliphatic. In some implementations, T 1 C3-C as an optional substitute 30 Alkyl group. In some embodiments, T 1 C3-C as an optional substitute 20 Alkyl group. In some embodiments, T 1 C5-C as an optional replacement 20Alkyl group. In some embodiments, T 1 For optional substitution of linear C3-C 20 Alkyl group. In some embodiments, T 1 optional substituted side chain C3-C 20 Alkyl group. In some embodiments, T 1 C3-C as an optional substitute 20 Alkenyl. In some embodiments, T 1 C is an optional replacement 10 -C 20 Alkenyl. In some embodiments, T 2 For C3-C 30 Aliphatic. In some implementations, T 2 C3-C as an optional substitute 30 Alkyl group. In some embodiments, T 2 C3-C as an optional substitute 20 Alkyl group. In some embodiments, T 2 C5-C as an optional replacement 20 Alkyl group. In some embodiments, T 2 For optional substitution of linear C3-C 20 Alkyl group. In some embodiments, T 2 optional substituted side chain C3-C 20 Alkyl group. In some embodiments, T 2 C3-C as an optional substitute 20 Alkenyl. In some embodiments, T 1 C is an optional replacement 10 -C 20 Alkenyl. In some embodiments, T 1 and T 2 For the same reason. In some implementations, T 1 and T 2 For different reasons.

[0161] In some implementations, T 1 and T 2 Each is selected independently from: , , , , , , , , , ; , , , ; , , and .

[0162] In some implementations, part-L 1 -X 1 -T 1 Choose from the following groups: , , , , , , , and .

[0163] In some implementations, part-L 2 -X 2 -T 2 Choose from the following groups: , , , , , , , and .

[0164] In some implementations, part-L 1 -X 1 -T 1 and part-L 2 -X 2 -T 2 Each is selected independently from: .

[0165] As described in this article, L 3 C1-C, which are the bonds and can be arbitrarily substituted 10 An aliphatic group or optionally substituted 2- to 10-membered heteroaliphatic group comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, L 3 As a key. In some implementations, L 3 C1-C is an optional substitute 10 An aliphatic group, or optionally substituted 2- to 10-membered heteroaliphatic group comprising 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, L 3 C1-C is an optional substitute10 Aliphatic. In some implementations, L 3 C1-C is an optional substitute 10 Alkylene. In some embodiments, L 3 C1-C is an optional substitute 10 Alkenyl group. In some embodiments, L 3 The substituted ynylene group is optional. In some embodiments, L 3 It is a C1-C6 alkylene group. In some embodiments, L 3 -(CH2) 1-6 - In some implementations, L 3 -(CH2) 2-4 - In some implementations, L 3 It is -(CH2)2-. In some implementations, L 3 It is -(CH2)3-. In some implementations, L 3 It is -(CH2)4-. In some implementations, L 3 It is -(CH2)5-. In some implementations, L 3 for , or .

[0166] In some implementations, L 3 It is a 2- to 10-membered heteroaliphatic compound containing 1 to 4 heteroatoms selected from N, O, and S, with optional substitution. In some embodiments, L 3 It is a 2- to 10-membered heteroaliphatic compound containing 1 to 4 heteroatoms selected from N, O, and S, with optional substitution. In some embodiments, L 3 It is a 2- to 8-membered heteroaliphatic compound containing 1 to 4 heteroatoms selected from N, O, and S, with optional substitution. In some embodiments, L 3 It is a 2- to 6-membered heteroaliphatic compound containing 1 to 2 heteroatoms selected from N, O, and S, with optional substitution. In some embodiments, L 3 It is a 5-membered heteroaliphatic compound containing one or two heteroatoms selected from N, O, and S, with optional substitution. In some embodiments, L 3 for: , , or As described in this article, G is -N(R) 2 )C(S)N(R 2 )2、-OH、-N(R 2 )2、-N + (R 3 )3、-N(R5 )C(O)R 3 -N(R) 5 )S(O)2R 3 -N(R) 5 )C(O)N(R 3 )2、-CH(NR 2 -R 4 or -S(O)2R 3 In some implementations, G is -N(R) 2 )C(S)N(R 2 )2、-OH、-N(R 2 )2、-N(R 5 )C(O)R 3 -N(R) 5 )S(O)2R 3 -N(R) 5 )C(O)N(R 3 )2、-CH(NR 2 -R 4 or -S(O)2R 3 .

[0167] In some implementations, G is -N(R) 2 )C(S)N(R 2 2. In some implementations, G is -N(H)C(S)N(R) 2 2. In some implementations, G is -N(H)C(S)N(H)(R 2 ) 。 In some implementations, G is -N(CH3)C(S)N(R) 2 2. In some implementations, G is -N(OH)C(S)N(R) 2 )2.

[0168] In some implementations, G is -N(H)C(S)N(R) 2 )2, where each R 2 Selected from optionally substituted C1-C6 aliphatic and OH groups. In some embodiments, G is -N(H)C(S)N(OH)(R 2 ), where R 2 It is an optionally substituted C1-C6 aliphatic compound. In some embodiments, G is -N(H)C(S)N(R) 2 )2, where each R 2 Selected from C1-C6 aliphatic molecules with optional substitutions. In some embodiments, G is -N(H)C(S)N(R) 2 )2, where each R 2It is methyl, ethyl, propyl, or butyl. In some embodiments, G is -N(H)C(S)N(CH3)2. In some embodiments, G is -N(H)C(S)N(CH3)(OCH3). In some embodiments, G is -N(H)C(S)N(CH3)(OH). In some embodiments, G is -N(OH)C(S)N(CH3)2.

[0169] In some implementations, G is -N(H)C(S)N(R) 2 )2, where R 2 Two instances, together with the atoms they are attached to, form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is -N(H)C(S)N(R) 2 )2, where R 2 Two instances, together with the atoms they are attached to, form optionally substituted 4- to 12-membered heterocycles containing 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is -N(H)C(S)N(R) 2 )2, where R 2 Two instances, together with the atoms they are attached to, form optionally substituted azaheptanine, pyrrolidine, piperidine, piperazine, or azaheptanine rings. In some embodiments, G is -N(R 2 )C(S)N(H)(R 2 ) ， Where R 2 Two instances of G, together with the atoms they are attached to, form optionally substituted 4- to 12-membered heterocycles containing 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is: or .

[0170] In some implementations, G is -OH.

[0171] In some implementations, G is -N(R) 2 2. In some implementations, G is -N(R) 2 )2, where each R 2 Independently H or optionally substituted C1-C6 aliphatic. In some embodiments, G is -N(R 2 )2, where each R 2 The C1-C6 aliphatic group is optionally substituted. In some embodiments, G is -N(R 2 )2, where each R 2 It can be methyl, ethyl, propyl, butyl, pentyl, or hexyl independently. In some embodiments, G is -N(CH3)2.

[0172] In some implementations, G is -N + (R 3 2. It should be understood that when G is -N + (R 3 At 2, G reacts with suitable equilibrium ions, such as halogens (e.g., Cl). - F - ,Br - Or I - Pairing is used to provide compounds that are chemically stable at neutral or physiological pH. In some embodiments, G is -N + (CH3)3.

[0173] In some implementations, G is -N(R) 5 )C(O)R 3 In some implementations, G is -N(H)C(O)R 3 In some embodiments, G is -N(H)C(O)-C1-C6 aliphatic. In some embodiments, G is -N(H)C(O)-CH3.

[0174] In some implementations, G is -N(R) 5 )S(O)2R 3 In some implementations, G is -N(H)S(O)2R 3 In some embodiments, G is -N(H)S(O)2-C1-C6 aliphatic. In some embodiments, G is -N(H)S(O)2-CH3.

[0175] In some implementations, G is -N(R) 5 )C(O)N(R 3 2. In some implementations, G is -N(H)C(O)N(R) 3 2. In some implementations, G is -N(H)C(O)N(H)(R 3 G is -N(H)C(O)N(H)(CH3). G is -N(H)C(O)N(CH3)2.

[0176] In some implementations, G is -CH(NR) 2 In some embodiments, G is -CH(N-C1-C6 aliphatic). In some embodiments, G is -CH(N-CH3). In some embodiments, G is... .

[0177] In some implementations, G is R 4 And R 4G is an optionally substituted 4- to 12-membered heterocycle. In some embodiments, G is an optionally substituted 4- to 6-membered monocyclic heterocycle. In some embodiments, G is an optionally substituted nitrogen-containing heterocyclic butane, pyrrolidine, piperidine, piperazine, or nitrogen-containing heptane. In some embodiments, G is a 6- to 12-membered bicyclic heterocycle containing 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, G is a 4- to 12-membered heterocycle containing 1 to 4 heteroatoms selected from N, O, and S, and is optionally -(CH2). 0-4 N(R°)2 or -(CH2) 0-4 OR° substitution, wherein R° is hydrogen or C1-C6 aliphatic.

[0178] In some implementations, G is R 4 And R 4 G is a 5- to 12-membered heteroaryl group containing 1 to 4 heteroatoms selected from N, O, and S, with optional substitution. In some embodiments, G is a 5- to 6-membered monocyclic heteroaryl group containing 1 to 3 heteroatoms selected from N, O, and S, with optional substitution. In some embodiments, G is a 6- to 12-membered bicyclic heteroaryl group containing 1 to 4 heteroatoms selected from N, O, and S, with optional substitution by -(CH2). 0-4 SR° substitution, wherein R° is hydrogen or C1-C6 aliphatic. In some embodiments, G is... .

[0179] In some implementations, G is R 4 , where R 4 For -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C6-C in )2 are replaced 12 Aryl 。 In some implementations, G is -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 )2 is a phenyl group that has one or more of its components substituted.

[0180] In some implementations, G is R 4 , where R 4 For the oxygenated group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C3-C in )2 are substituted 12Cyclic aliphatic. In some embodiments, G is an oxy-substituted group, -(CH2). 0-6 -OH or -(CH2) 0-6 -N(R 5 C3-C6 cyclic aliphatic compounds substituted with one or more of the radicals in 2. In some embodiments, G is optionally replaced by an oxo group, -OH, or -N(R). 5 One or more of the cyclobutyl, cyclopentyl or cyclohexyl groups in )2 are substituted.

[0181] In some implementations, G is -S(O)2R 3 In some embodiments, G is -S(O)2-C1-C6 aliphatic. In some embodiments, G is -S(O)2-CH3.

[0182] In some implementations, G is selected from: , , , , , , , , , , , , and .

[0183] It should be understood that some -L 3 -G can be a cation or an ionizable group, making some -L 3 The specific atom in -G (e.g., a nitrogen atom) may have a positive charge at neutral pH or physiological pH. In some embodiments, such a head group also contains a suitable balancing ion (e.g., a halogen atom, such as Cl). - ,Br - I - F - wait).

[0184] In some implementations, part-L 3 -G is selected from: , , , , , , , , , , , , , , , , , , , , , , and .

[0185] In some implementations, -L 3 -G is selected from: , , , , , , , , , , , , , , , , , , , and .

[0186] As described in this article, each R 2 In each case, H, arbitrarily substituted C1-C6 aliphatic and OR are independently selected. 3 The group formed; or R 2 Two instances, together with the atoms they are attached to, form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, each R 2 In each case, H, arbitrarily substituted C1-C6 aliphatic and OR are independently selected. 3 A group consisting of [groups]. In some implementations, R [is used]. 2 Two instances, together with the atoms to which they are attached, form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O, and S.

[0187] As described in this article, each R 3 In each case, H and the arbitrarily substituted C1-C are chosen independently. 10 A group composed of aliphatic groups.

[0188] As described in this article, R 4It is a 4- to 12-membered heterocycle with 1 to 4 heteroatoms selected from N, O, and S, optionally substituted, and a 4- to 12-membered heteroaryl group with 1 to 4 heteroatoms selected from N, O, and S, substituted with -(CH2). 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C6-C in )2 are replaced 12 Aryl group, or with an oxy group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C3-C in )2 are substituted 12 Cyclic aliphatic.

[0189] As described in this article, each R 5 Independently selected from H and optionally substituted C1-C6 aliphatic compounds.

[0190] In some embodiments, the compounds described herein are compounds represented by formula IIa: IIa Or its pharmaceutically acceptable salts, wherein G, L 1 L 2 and L 3 As described in the categories and subclasses of this article.

[0191] In some embodiments, the compounds described herein are compounds represented by formula IIb: IIb Or its pharmaceutically acceptable salts, wherein G, L 1 L 2 and L 3 As described in the categories and subclasses of this article.

[0192] In some embodiments, the compounds described herein are compounds represented by formula IIc: IIc Or its pharmaceutically acceptable salts, wherein G, L 1 L 2 and L 3 As described in the categories and subclasses of this article.

[0193] In some embodiments, the compounds described herein are represented by formula IIIa: IIIa Or its pharmaceutically acceptable salt, wherein L1 L 2 X 1 X 2 T 1 and T 2 As described in the categories and subclasses of this article.

[0194] In some embodiments, the compounds described herein are represented by formula IIIb: IIIb Or its pharmaceutically acceptable salt, wherein L 1 L 2 X 1 X 2 R 1 T 1 and T 2 As described in the categories and subclasses of this article.

[0195] In some embodiments, the compounds described herein are represented by formula IIIc: IIIc Or its pharmaceutically acceptable salt, wherein L 1 L 2 X 1 X 2 T 1 and T 2 As described in the categories and subclasses of this article.

[0196] In some embodiments, the compounds described herein are represented by formula IIId: IIId Or its pharmaceutically acceptable salt, wherein L 1 L 2 X 1 X 2 T 1 and T 2 As described in the categories and subclasses of this article.

[0197] In some embodiments, the compounds described herein are represented by formula IIId-1: IIId-1 Or its pharmaceutically acceptable salt, wherein L 1 L 2 X 1 X 2 T 1 and T 2As described in the categories and subclasses of this article.

[0198] In some embodiments, the compounds described herein are represented by formula IIId-2: IIId-2 Or its pharmaceutically acceptable salt, wherein L 1 L 2 X 1 X 2 T 1 and T 2 As described in the categories and subclasses of this article.

[0199] In some embodiments, the compounds described herein are represented by formula IIIe: IIIe Or its pharmaceutically acceptable salt, wherein L 1 L 2 R 1 T 1 and T 2 As described in the categories and subclasses of this article.

[0200] In some embodiments, the compounds described herein are represented by formula IIIf: IIIf Or its pharmaceutically acceptable salt, wherein L 1 L 2 T 1 and T 2 As described in the categories and subclasses of this article.

[0201] In some embodiments, the compounds described herein are represented by formula IIIg: IIIg Or its pharmaceutically acceptable salt, wherein L 1 L 2 X 1 X 2 T 1 and T 2 As described in the categories and subclasses of this article.

[0202] In some embodiments, the compounds described herein are represented by formula IIIg-1: IIIg-1 Or its pharmaceutically acceptable salt, wherein L 1L 2 R 1 T 1 and T 2 As described in the categories and subclasses of this article.

[0203] In some embodiments, the compounds described herein are represented by formula IIIh-1: IIIh-1 Or a pharmaceutically acceptable salt thereof, wherein X 50 For -S-, -S(O)-, or -S(O)2-, n' is 1-6, and R 100 C1-C is an optional substitute 16 Lipids.

[0204] In some embodiments, the compounds described herein are represented by formula IIIh-2: IIIh-2 Or a pharmaceutically acceptable salt thereof, wherein X 50 It is -S-, -S(O)- or -S(O)2-, and n' is 1-6.

[0205] In some embodiments, the compounds described herein are represented by formula IIIh-3: IIIh-3 Or a pharmaceutically acceptable salt thereof, wherein X 50 It is -S-, -S(O)- or -S(O)2-, and n' is 1-6.

[0206] In some embodiments, the compounds described herein are represented by formula IIIh-4: IIIh-4 Or a pharmaceutically acceptable salt thereof, wherein n' is 1-6 and R 100 C1-C is an optional substitute 16 Lipids.

[0207] In some embodiments, the compounds described herein are represented by formula IIIh-5: IIIh-5 Or a pharmaceutically acceptable salt thereof, wherein n' is 1-6.

[0208] In some embodiments, the compounds described herein are selected from Table 1: Table 1

[0209] In some embodiments, the provided compounds are provided and / or used in salt form (e.g., pharmaceutically acceptable salt form). Unless otherwise indicated, references to compounds provided herein should be understood to include references to their salts.

[0210] Methods for preparing thiolipin The compounds described herein are particularly useful compared to previous lipid compounds, at least in part, because of their ease of preparation. Specifically, different fragments can be coupled via sulfur bonds to provide a variety of sulfur-lipid compounds that can be used to prepare nucleic acid particles. For example, the sulfur-lipid compounds disclosed herein can be prepared according to the following conditions: Figure 79 , 80 Preparations were carried out using general schemes 1, 2, and 3 as described in 81.

[0211] The tail connector is any divalent connector, such as an aliphatic or heteroaliphatic group; the tail end is a hydrophobic group, such as an aliphatic group; the head group is a polar or cationic or ionizable head group; the tail junction is a biodegradable group, such as an ester or a sulfur-containing moiety (e.g., thioether, sulfonyl, or sulfonamide); and the head-tail junction is a central atom or functional group that connects one or more tails to a head group (e.g., a tertiary amine group).

[0212] As described herein, in some embodiments, this disclosure provides for the preparation of compounds represented by formula IV: IV A method using a pharmaceutically acceptable salt thereof, said method comprising: Compounds represented by formula V V Compounds represented by one of formulas VIa-VIc and compounds represented by one of formulas VIIa-VIIc Contact in the presence of a reducing agent in: L 4 and L 5 Each of them is independently an optional substitution of C1-C. 30 Aliphatic groups; L 6 C1-C, which are the bonds and can be arbitrarily substituted 10 Aliphatic groups or optionally substituted 2- to 10-membered heteroaliphatic groups comprising 1 to 4 heteroatoms selected from N, O, and S; X 3 and X 4 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 40 )-、-N(R 40 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 40 )2-、-OC(S)C(R 40 )2-、-C(R 40 )2C(S)O- or -S-, where X 3 or X 4 One or both of them are selected from -S(O)2N(R) 40 )-、-N(R 40 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 40 )2-、-OC(S)C(R 40 )2-、-C(R 40 )2C(S)O- or -S-; Each R 40 Independently for each case the optionally substituted C1-C 20 Aliphatic or H; T 3 and T 4 Each of the C3-Cs can be substituted independently. 20 aliphatic; G 1 -N(R)6 )C(S)N(R 6 )2、-OH、-N(R 6 )2、-N(R 9 )C(O)R 7 -N(R) 9 )S(O)2R 7 -N(R) 9 )C(O)N(R 7 )2、-CH(NR 7 ) or -R 8 ; Each G 2 Independently, it can be either O or N2; Each G 3 Independently halogens (e.g., Cl, Br, or I), -OTs, or OTf; Each R 6 In each case, H, arbitrarily substituted C1-C6 aliphatic or OR are independently selected. 7 The group formed; or R 6 Two instances together with the atoms to which they are attached form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O and S; Each R 7 In each case, the group consisting of free H and arbitrarily substituted C1-C6 aliphatic molecules is selected independently; R 8 It is a 4- to 12-membered heterocycle with 1 to 4 heteroatoms selected from N, O, and S, optionally substituted, and a 4- to 12-membered heteroaryl group with 1 to 4 heteroatoms selected from N, O, and S, substituted with -(CH2). 0-6 -OH or -(CH2) 0-6 -N(R 9 One or more of the C6-C in )2 are replaced 12 Aryl group, or with an oxy group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 9 One or more of the C3-C in )2 are substituted 12 Cycloaliphatic; and Each R 9 Independently selected from H and optionally substituted C1-C6 aliphatic compounds.

[0213] In some embodiments, the reducing agent is NaBH3CN or NaBH(OCOCH3)3. In some embodiments, the reducing agent is NaBH3CN. In some embodiments, the reducing agent is NaBH(OCOCH3)3.

[0214] In some embodiments, the method for preparing the compounds described herein further includes preparing a compound represented by formula VIa or a compound represented by formula VIIa, wherein G 1 For O, It is achieved by using compounds represented by formula VIII or formula IX. It is carried out by contact with an oxidizing agent.

[0215] In some embodiments, the oxidant is DMSO, PCC, or DMP. In some embodiments, the oxidant is DMSO, and the method further includes contacting the compound represented by Formula VIII or Formula IX and DMSO with a sulfur trioxide pyridine complex (SO3•pyridine).

[0216] In some embodiments, the method for preparing the compounds described herein further includes preparing compounds represented by formula VIII or formula IX: It works by making a compound represented by formula X or a compound represented by formula XI. With compounds represented by formula XII or compounds represented by formula XIII The process is carried out by contact in the presence of H2O2 and SOCl2; and each Y... 1 It is a halogen.

[0217] Particles for nucleic acid delivery In some embodiments, the particles of this disclosure comprise one or more of the compounds described herein (e.g., compounds of formulas I-IIIh-5), nucleic acids (such as RNA (e.g., mRNA), DNA, or mixtures thereof), and one or more of polymer-conjugated lipids, auxiliary lipids, and steroids. In some embodiments, the particles of this disclosure comprise one or more of the compounds described herein (e.g., compounds of formulas I-IIIh-5), nucleic acids (such as RNA (e.g., mRNA), DNA, or mixtures thereof), auxiliary lipids, and steroids. In some embodiments, the particles of this disclosure comprise one or more of the compounds described herein (e.g., compounds of formulas I-IIIh-5), nucleic acids (such as RNA (e.g., mRNA), DNA, or mixtures thereof), polymer-conjugated lipids, auxiliary lipids, and steroids.

[0218] The thiolipin compounds described herein contain cationic or cationically ionizable moieties, which are capable of forming electrostatic interactions between positively charged moieties and negatively charged nucleic acids, thereby forming particles.

[0219] In some implementations, the particles described herein (e.g., nucleic acid particles, such as ribonucleic acid particles or deoxyribonucleic acid particles) comprise more than one type of nucleic acid molecule, wherein the molecular parameters of the nucleic acid molecules may be similar or different from each other with respect to molar mass or basic structural elements (such as molecular architecture, capping, coding regions or other features).

[0220] In some embodiments, the nucleic acid particles described herein are nanoparticles. As used in this disclosure, "nanoparticle" means a particle with an average diameter suitable for parenteral administration and a diameter less than 1000 nm. In some embodiments, the composition comprising nanoparticles may have an average nanoparticle size (e.g., average diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the composition comprising nanoparticles may have an average nanoparticle size (e.g., average diameter) of about 50 nm to about 100 nm. In some embodiments, the composition comprising nanoparticles may have an average nanoparticle size (e.g., average diameter) of about 50 nm to about 150 nm. In some embodiments, the composition comprising nanoparticles may have an average nanoparticle size (e.g., average diameter) of about 60 nm to about 120 nm. In some embodiments, the composition comprising nanoparticles may have an average nanoparticle size (e.g., average diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0221] Compositions comprising the nucleic acid particles described herein (e.g., ribonucleic acid particles or deoxyribonucleic acid particles) may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or smaller than the nanoparticles. As an example, compositions comprising the nucleic acid particles described herein (e.g., ribonucleic acid particles or deoxyribonucleic acid particles) may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[0222] The nucleic acid particles (e.g., ribonucleic acid particles or deoxyribonucleic acid particles) described herein are characterized by an "N / P ratio," which is the molar ratio of cationic (nitrogen) groups ("N" in N / P) in the cationic polymer to anionic (phosphate) groups ("P" in N / P) in the RNA. It should be understood that the cationic groups are in cationic form (e.g., N...). + The group is a cation or a group that can be ionized to become a cation. The use of a single value in the N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to the value relative to 1; for example, an N / P ratio of about 4 is intended to refer to about 4:1. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N / P ratio greater than or equal to 4. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N / P ratio of about 4 to about 16. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N / P ratio of about 6 to about 12. In some embodiments, the nucleic acid particles described herein have an N / P ratio of about 4 to about 12. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N / P ratio of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N / P ratio of about 6. In some implementations, the N / P ratio of the nucleic acid particles (e.g., ribonucleic acid particles) described herein is about 12.

[0223] The compounds described herein are also referred to as "ionizable" or "cationic" lipids. In some embodiments, such lipids are intended to refer to compounds that can become cations (i.e., become positively charged) at physiological pH.

[0224] The term "average diameter" or "mean diameter" refers to the average hydrodynamic diameter of a particle, as measured by dynamic laser light scattering (DLS), using a so-called cumulant algorithm for data analysis, which provides a so-called Z-mean with a length dimension and a dimensionless polydispersity index (PDI) as the result (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the terms "average diameter," "mean diameter," "diameter," or "size" of a particle are used synonymously with this value of the Z-mean.

[0225] The "polydispersity index" is preferably calculated based on dynamic light scattering measurements through so-called cumulative analysis, as mentioned in the definition of "mean diameter." Under certain prerequisites, it can serve as a measure of the size distribution of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles) as a whole.

[0226] Different types of nucleic acid particles have previously been described as suitable for delivering nucleic acids in particulate form (e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery mediators, the encapsulation of nucleic acids in nanoparticles physically protects the nucleic acids from degradation and, depending on specific chemistry, can facilitate cellular uptake and endosome escape.

[0227] Some embodiments described herein relate to compositions, methods, and uses involving more than one, such as two, three, four, five, six, or even more types of nucleic acids. The nucleic acid types may be RNA and / or DNA. For example, the particles described herein may contain one type of RNA (e.g., one type of mRNA) and one type of DNA.

[0228] In nucleic acid particle compositions, it is possible to formulate each nucleic acid species individually as a separate nucleic acid particle formulation. In this case, each individual nucleic acid particle formulation will contain one nucleic acid species. Individual nucleic acid particle formulations can exist as separate entities, for example, in separate containers. Such formulations can be obtained by separately providing each nucleic acid species (usually in the form of a solution containing nucleic acid) and a particle-forming agent, thereby allowing particle formation. The corresponding particles will contain only the specific nucleic acid species provided during particle formation (individual microparticle formulations).

[0229] In some embodiments, the composition (e.g., a pharmaceutical composition) comprises more than one individual nucleic acid particle formulation. The corresponding pharmaceutical composition is referred to as a "mixed microparticle formulation." A mixed microparticle formulation according to this disclosure can be obtained by separately forming individual nucleic acid particle formulations as described above, and then mixing the individual nucleic acid particle formulations. Through the mixing step, a formulation comprising a mixed population of nucleic acid particles is obtained. The individual nucleic acid particle populations may be together in a container containing the mixed population of individual nucleic acid particle formulations.

[0230] Alternatively, it is possible to formulate different nucleic acid species together as a "combined microparticle formulation." Such formulations are obtained by providing a combination of different nucleic acid species and a particle-forming agent (typically a combined solution), thereby allowing particle formation. Unlike "mixed microparticle formulations," "combined microparticle formulations" typically contain particles containing more than one nucleic acid species. In combined microparticle compositions, different nucleic acid species are usually present together within a single particle.

[0231] In some implementations, when present in the provided nucleic acid particles, the nucleic acid is resistant to degradation by nucleases in aqueous solution.

[0232] Lipid nanoparticles In some embodiments, the nucleic acid particles are lipid nanoparticles. In some embodiments, the lipid nanoparticles are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., the cationic lipids described herein), nucleic acids (e.g., RNA and / or DNA), polymer-conjugated lipids, accessory lipids, and sterols. Lipid nanoparticles (LNPs) have been proven useful for delivering nucleic acid cargoes to tissues of interest. LNPs are used, for example, in certain commercial vaccines for treating COVID-19.

[0233] In some embodiments, the LNP of this disclosure comprises i) cationic lipids (e.g., thiolipin compounds described herein, such as compounds of any one of formulas I-IIIh-5); ii) accessory lipids; iii) polymer-conjugated lipids (e.g., polyethylene glycol-bound lipids, “PEG lipids”); and iv) steroids. In some embodiments, the LNP of this disclosure may comprise i) cationic lipids (e.g., thiolipin compounds described herein, such as compounds of any one of formulas I-IIIh-5); ii) accessory lipids; and iii) steroids. In some embodiments, the LNP described herein may also comprise additional additives as described herein. The LNP of this disclosure can be used in a variety of environments. For example, the nucleic acid-containing LNP described herein can be used to deliver said nucleic acid to the cells of a subject. In some embodiments, the nucleic acid-containing LNP described herein can be used to increase the expression of a protein in a subject. In some embodiments, the nucleic acid-containing LNP described herein can be used to induce a pharmacological effect induced by the expression of a protein in a subject. The lipid nanoparticles described herein are characterized by the molar percentage (mol%) of the components in the lipid nanoparticles. The mol% used for lipid components in lipid nanoparticles is relative to the total other lipid components in the lipid nanoparticles.

[0234] (i) Auxiliary lipids As described herein, the LNP of this disclosure comprises an accessory lipid. In some embodiments, the accessory lipid is a phospholipid. In some embodiments, the accessory lipid is or contains 1,2-distearate- sn1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamine (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)), sphingomyelin (SM), 1,2-diacylglycerol-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS), ceramides, cholesterol, steroids (such as sterols and their derivatives).

[0235] In some embodiments, the cofactor lipid is or comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. In some embodiments, the cofactor lipid is or comprises diacylphosphatidylcholine, such as distearatel phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bispentadecanylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibenzylphosphatidylcholine (DBPC), and di-di-... Tridecanoylphosphatidylcholine (DTPC), bis(tetracosanoylphosphatidylcholine) (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16) LysoPC) and phosphatidylethanolamines, including, for example, diacylphosphatidylethanolamines such as dioleoylphosphatidylethanolamine (DOPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphyranoylphosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate choline (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1′-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), and N-palmitoyl-D-erythrosphoylphosphatidylcholine (SM). In some embodiments, the auxiliary lipids are selected from the group consisting of: DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipids are selected from the group consisting of: DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC.

[0236] The assisting lipids may be synthetic or naturally derived. Other assisting lipids suitable for use in lipid nanoparticles are described in WO2021 / 026358, WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference in their entirety.

[0237] In some embodiments, the lipid nanoparticles contain about 5 mol% to about 15 mol% of phospholipids. In some embodiments, the lipid nanoparticles contain about 8 mol% to about 12 mol% of phospholipids. In some embodiments, the lipid nanoparticles contain about 10 mol% of phospholipids. In some embodiments, the lipid nanoparticles contain about 5 mol% to about 15 mol% of DSPC. In some embodiments, the lipid nanoparticles contain about 8 mol% to about 12 mol% of DSPC. In some embodiments, the lipid nanoparticles contain about 10 mol% of DSPC.

[0238] (ii) Polymer-conjugated lipids As described herein, the LNP of this disclosure comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a lipid conjugated to polyethylene glycol (PEG-lipid). In some embodiments, the PEG lipid is selected from polyethylene glycol-modified diacylglycerols (PEG-DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), e.g., 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (PEG2000-DMG)), polyethylene glycol-modified phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinates (PEG-S-DAG) (such as 4-O-(2',3'-bis(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)-2000]), and PEG-S-DAG (PEG-S-DAG). (DSPE-PEG2000 amine), polyethylene glycol-modified ceramide (PEG-cer) or PEG diexoxypropyl carbamate (such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecoxy)propyl)carbamate, 2,3-di(tetradecoxy)propyl-1-N-(ω-methoxy(polyethoxy)ethyl)carbamate) and N-palmitoyl-sphingosine-1-{succinoyl[methoxy(polyethylene glycol)2000]} (C16-PEG2000 ceramide or C16Cer-PEG2K).

[0239] In some implementations, the PEG-lipid is PEG2000-DMG: .

[0240] In some implementations, the PEG-lipid is DMG-PEG.

[0241] In some embodiments, PEG-lipids are provided in WO2021 / 026358, WO 2017 / 075531 or WO 2018 / 081480, each of which is incorporated herein by reference in its entirety.

[0242] In some embodiments, the polymer-conjugated lipid is 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159). In some embodiments, the polymer-conjugated lipid is: Or a pharmaceutically acceptable salt thereof, wherein n' is an integer from about 45 to about 50.

[0243] In some embodiments, the polymer-conjugated lipid is C16 PEG2000: .

[0244] In some implementations, the PEG-lipid has the following structure: In the above formula, n is 30 to 60, such as about 50. In one embodiment, the PEG-conjugated lipid (polyethylene glycol-modified lipid) is PEG. 2000 -C-DMA, which preferably refers to 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoxypropylamine (MPEG-(2 kDa)-C-DMA) or methoxypolyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).

[0245] In some embodiments, the PEG-lipid is selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, PEG-S-DMG, PEG-cer, PEG dialkoxypropyl carbamate (e.g., w-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecoxy)propyl)carbamate or 2,3-di(tetradecoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate), ALC-0159, and combinations thereof. In some embodiments, the PEG-lipid is ALC-0159 or PEG2000-DMG. In some embodiments, the PEG-lipid is ALC-0159. In some embodiments, the PEG-lipid is PEG2000-DMG. In some embodiments, the PEG-lipid is PEG-DAG. In some embodiments, the PEG-lipid is PEG-PE. In some embodiments, the PEG-lipid is PEG-S-DAG. In some embodiments, the PEG-lipid is PEG-cer. In some implementations, the PEG-lipid is PEG dialkoxypropyl carbamate.

[0246] In some embodiments, the PEG group, which is part of the PEG-lipid, has an average weight-average molecular weight (M) of about 2000 g / mol in a composition comprising one or more PEG-lipid molecules. w ).

[0247] In some embodiments, the polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosine-modified lipid or pSar-lipid. The term "sarcosine-modified lipid" refers to a molecule comprising both a lipid moiety and a polysarcosine (poly(N-methylglycine)) moiety.

[0248] In some embodiments, the polymer-conjugated lipid is a polyoxazoline (POX)-conjugated lipid and / or a polyoxazide (POZ)-conjugated lipid, also referred herein as a conjugate of POX and / or POZ polymers and one or more hydrophobic chains, or as oxazoline- and / or oxazin-conjugated lipids or POX- and / or POZ-lipids. The terms "oxazoline-conjugated lipid" or "POX-lipid" refer to a molecule comprising both a lipid moiety and a polyoxazoline moiety. The terms "oxazin-conjugated lipid" or "POZ-lipid" refer to a molecule comprising both a lipid moiety and a polyoxazide moiety. The terms "oxazoline- / oxazin-conjugated lipid," "POX / POZ-lipid," or "POXZ-lipid" refer to a molecule comprising both a lipid moiety and a portion of a copolymer of polyoxazoline and polyoxazide.

[0249] In some embodiments, the LNPs described herein may comprise sarcosinated lipids. In some embodiments, the nucleic acid compositions described herein (such as DNA or RNA compositions, especially mRNA compositions) comprise sarcosinated lipids and are substantially free of PEGylated lipids (or contain no PEGylated lipids).

[0250] In some embodiments, the nucleic acid compositions described herein (such as DNA or RNA compositions) comprise cationic / cationically ionizable lipids and sarcosinate lipids (pSAR-conjugated lipids) as described herein. In some embodiments, the nucleic acid compositions described herein (such as DNA or RNA compositions, especially mRNA compositions) may also comprise neutral lipids (e.g., phospholipids, cholesterol, or derivatives thereof) or combinations of neutral lipids (e.g., phospholipids and cholesterol, or derivatives thereof). In some embodiments, the nucleic acid compositions described herein (such as DNA or RNA compositions, especially mRNA compositions) comprise cationic / cationically ionizable lipids, sarcosinate lipids, neutral lipids (e.g., phospholipids), and cholesterol, or derivatives thereof, as described herein. In some embodiments, the phospholipid is DSPC.

[0251] In some embodiments of the nucleic acid compositions (such as DNA or RNA compositions, especially mRNA compositions) described herein that contain sarcosinated lipids, the compositions are substantially free of PEGylated lipids (or contain no PEGylated lipids).

[0252] In some embodiments, the sarcosinated lipid comprises 2 to 200 sarcosine units, such as 5 to 100 sarcosine units, 10 to 50 sarcosine units, 15 to 40 sarcosine units, for example about 23 sarcosine units.

[0253] In some embodiments, the sarcosinated lipids comprise the structure of the following general formula (XVII): XVII Where s represents the number of sarcosine units.

[0254] In some embodiments, the sarcosinated lipid comprises the structure of the following general formula (XVIII): XVIII Where R 21 and R 22 One of them contains a hydrophobic group and the other is an H, a hydrophilic group, or optionally a functional group containing a targeting portion; and x is the number of sarcosine units.

[0255] In some embodiments, the LNPs described herein may comprise oxazolined and / or oxazinized lipids. In some embodiments, the nucleic acid compositions described herein (such as DNA or RNA compositions, especially mRNA compositions) comprise oxazolined and / or oxazinized lipids and are substantially free of polyethylene glycol-modified lipids (or free of polyethylene glycol-modified lipids).

[0256] In some embodiments, the polymer-conjugated lipid comprises a monomer of 2-(2-(2-aminoethoxy)ethoxy)acetic acid. In some embodiments, the polymer of the polymer-conjugated lipid is or comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA) or derivatives thereof, as defined herein.

[0257] In some embodiments, the polymer comprises the following general formula: Where X 11 and X 12 Together they are optionally substituted amides, optionally substituted thioamides or esters; Y is -CH2-, -(CH2)2- or -(CH2)3-; z is 2 to 24; and n is 1 to 100.

[0258] In some implementations, (i) when X 11 When it is -C(O)-, then X 12 For -NR 10 -;(ii) when X 11 For -NR 10 - when, then X 12 (iii) When X is -C(O)-; 11 When X is -C(S)-, then 12 For -NR 1 -;(iv) When X 11 For -NR 1 - when, then X 12 For -C(S)-; (v) when X 11 When it is -C(O)-, then X 12 For -O-; or (vi) when X 11 When it is -O-, then X 12 It is -C(O)-; where R 10 It is hydrogen or C 1-8 Alkyl group. In some embodiments, X 11 For -C(O)- and X 12 For -NR 10 -, where R 10It is hydrogen or C 1-8 Alkyl group. In some embodiments, X 11 For -C(O)- and X 12 For -NR 10 -, where R 10 It is hydrogen or methyl. In some embodiments, X 11 For -C(O)- and X 12 For -NR 10 -, where R 10 It is hydrogen. In some embodiments, Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0259] In some embodiments, the polymer comprises the following general formula: Where R 10 It is hydrogen or a C1-8 alkyl group; z is 2 to 24; and n is 1 to 100. In some embodiments of the above formula, z is 2 to 10. In some embodiments, z is 2 to 5. In some embodiments, z is 2. In some embodiments of the above formula, R 10 It is hydrogen or methyl. In some embodiments, R 10 It is hydrogen.

[0260] In some embodiments, the polymer-conjugated lipid comprises "n" monomers with the following structure: In some embodiments of the above formula, n is 5 to 50. In some embodiments, n is 5 to 25. In some embodiments, n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.

[0261] In some embodiments, the polymer-conjugated lipids comprise monomers with the following structures: .

[0262] In some implementations, the polymer-conjugated lipids are selected from the following table:

[0263] In some embodiments, the polymer-conjugated lipids comprise about 0.5 mol% to about 5 mol% of the total lipids in the LNP. In some embodiments, the LNP comprises about 1.0 mol% to about 2.5 mol% of polymer-conjugated lipids. In some embodiments, the LNP comprises about 1.5 mol% to about 2.0 mol% of polymer-conjugated lipids. In some embodiments, the LNP comprises about 1.5 mol% to about 1.8 mol% of polymer-conjugated lipids.

[0264] In some embodiments, the molar ratio of total cationic lipids to total polymer-conjugated lipids is from about 100:1 to about 20:1. In some embodiments, the molar ratio of total cationic lipids to total polymer-conjugated lipids is from about 50:1 to about 20:1. In some embodiments, the molar ratio of total cationic lipids to total polymer-conjugated lipids is from about 40:1 to about 20:1. In some embodiments, the molar ratio of total cationic lipids to total polymer-conjugated lipids (e.g., PEG-conjugated lipids) is from about 35:1 to about 25:1.

[0265] In some embodiments, the LNP of this disclosure may also comprise a compound of formula (A1): Wherein P comprises a polymer; L comprises a hydrophobic portion (e.g., lipid) connected to a first end of the polymer; B comprises a binding portion connected to a second end of the polymer; X10 is absent or is a first binding portion; and X20 is absent or is a second binding portion.

[0266] In some embodiments of formula (A1), X10 comprises a carbonyl group. In some embodiments of formula (A1), X20 comprises a reaction product of a thiol or cysteine ​​reactive group (e.g., a maleimide group) with a thiol or cysteine ​​group of a compound comprising the binding moiety.

[0267] In some embodiments of formula (A1), L comprises lipids as described above. In some embodiments of formula (A1), L comprises DSPE (distearylphosphatidylethanolamine), DPPE (dispalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleoylphosphatidylethanolamine) which can be linked to P via an amide group.

[0268] In some embodiments of formula (A1), P comprises the polymer as described above. In some embodiments of formula (A1), P comprises a polymer selected from the group consisting of: poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), poly-2-(2-(2-aminoethoxy)-ethoxy)acetic acid (pAEEA), and poly-2-(2-(2-methylaminoethoxy)-ethoxy)acetic acid (pMAEEA) (including derivatives thereof). In some embodiments of formula (A1), P comprises poly(ethylene glycol) (PEG); for example, PEG as described above.

[0269] In some embodiments of Formula (A1), the binding portion B comprises a portion (e.g., an antibody or an antigen-binding fragment thereof) that binds to a cell surface antigen (e.g., a receptor, antigen, or marker displayed on the surface of a target cell, such as a T cell, B cell, or cancer cell). In some embodiments of Formula (A1), the binding portion B comprises or is a polypeptide (e.g., an epitope tag), such as an ALFA-tag as described herein. In some embodiments of Formula (A1), the binding portion B comprises or is an ALFA-tag that comprises or consists of a sequence selected from or composed of the group consisting of SEQ.ID.NO. 1, SEQ.ID.NO. 2, SEQ.ID.NO. 3, and SEQ.ID.NO. 4.

[0270] In some embodiments, the compound of formula (A1) may be a peptide-conjugated lipid. In some embodiments, the compound of formula (A1) may be DSPE-PEG2K-α. Therefore, in some embodiments, the LNP of this disclosure may also comprise a peptide-conjugated lipid, optionally DSPE-PEG2K-α. In some embodiments, the lipid nanoparticles comprise about 0.05 mol% to about 5 mol%, optionally about 0.1 mol% to about 2 mol% of the compound of formula (A1) (wherein the mol% can be provided by alternative steroids).

[0271] As described in the examples, the LNP containing the formula (A1) compound (wherein the binding portion B comprises or is a peptide) can be further functionalized by binding to a peptide (a “docking compound”), which is typically a bispecific polypeptide (such as a bispecific antibody or its antigen-binding fragment), wherein the docking compound comprises (i) a portion of the peptide that binds to the binding portion B, and (ii) a portion that binds to a cell surface antigen on a target cell. In some embodiments, the LNP of the comprising (A1) compound (wherein binding portion B comprises or is an ALFA-tag (as described herein)) can be further functionalized by binding to an ALFA-tag (a “docking compound”) comprising (i) an NbAlfa VHH domain comprising the CDR1 sequence SEQ.ID.NO. 5, the CDR2 sequence SEQ.ID.NO. 6, and the CDR3 sequence SEQ.ID.NO. 7; and (ii) an anti-CD3 binding VHH comprising the CDR1 sequence SEQ.ID.NO. 8, the CDR2 sequence SEQ.ID.NO. 9, and the CDR3 sequence SEQ.ID.NO. 10.

[0272] (iii) Steroids As described herein, in some embodiments, the nucleic acid particles further comprise steroids. In some embodiments, the steroid is a sterol. In some embodiments, the sterol is β-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulinum toxin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or α-tocopherol. In some embodiments, the sterol is β-sitosterol. In some embodiments, the sterol is stigmasterol. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is cholecalciferol. In some embodiments, the sterol is ergocalciferol. In some embodiments, the sterol is calcipotriol. In some embodiments, the sterol is botulinum toxin. In some embodiments, the sterol is lupeol. In some embodiments, the sterol is ursolic acid. In some embodiments, the sterol is oleanolic acid. In some embodiments, the sterol is cycloartenol. In some embodiments, the sterol is lanosterol. In some implementations, the sterol is α-tocopherol.

[0273] In some embodiments, the lipid nanoparticles contain about 30 mol% to about 50 mol% of steroids. In some embodiments, the lipid nanoparticles contain about 35 mol% to about 45 mol% of steroids. In some embodiments, the lipid nanoparticles contain about 38 mol% to about 40 mol% of steroids. In some embodiments, the lipid nanoparticles contain about 38.5 mol% of steroids. In some embodiments, the lipid nanoparticles contain about 40 mol% of steroids.

[0274] In some embodiments, the lipid nanoparticles contain about 30 mol% to about 50 mol% cholesterol. In some embodiments, the lipid nanoparticles contain about 35 mol% to about 45 mol% cholesterol. In some embodiments, the lipid nanoparticles contain about 38 mol% to about 41 mol% cholesterol. In some embodiments, the lipid nanoparticles contain about 38.5 mol% cholesterol. In some embodiments, the lipid nanoparticles contain about 40.7 mol% cholesterol.

[0275] In some implementations, the lipid nanoparticles comprise: Approximately 40 mol% to approximately 50 mol% of sulfur lipid compounds; Steroids ranging from approximately 30 mol% to approximately 50 mol%; Approximately 5 mol% to approximately 15 mol% of auxiliary lipids; and Polymer-conjugated lipids of about 1 mol% to about 5 mol%.

[0276] In some implementations, the lipid nanoparticles comprise: Approximately 45 mol% to approximately 50 mol% of thiolipin compounds; Steroids, approximately 35 mol% to approximately 45 mol%. Approximately 5 mol% to approximately 15 mol% of auxiliary lipids; and Polymer-conjugated lipids of about 1 mol% to about 5 mol%.

[0277] In some implementations, the lipid nanoparticles comprise: Approximately 47.5 mol% of sulfur lipid compounds; Approximately 40.5 mol% steroids; Approximately 10 mol% of auxiliary lipids; and Approximately 2 mol% of polymer-conjugated lipids.

[0278] (iv) Methods for manufacturing lipid nanoparticles Lipids and nucleic acid-containing lipid nanoparticles, as well as methods for their preparation, are known in the art, including, for example, U.S. Patent Nos. 8,569,256 and 5,965,542, and U.S. Patent Publications Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, and 2014 / 0308304. No., No. 2014 / 0200257, No. 2013 / 086373, No. 2013 / 0338210, No. 2013 / 0323269, No. 2013 / 0245107, No. 2013 / 0195920, No. 2013 / 0123338, No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 0295832, No. 2012 / 0183581, No. 2012 / No. 0172411, No. 2012 / 0027803, No. 2012 / 0058188, No. 2011 / 0311583, No. 2011 / 0311582, No. 2011 / 0262527, No. 2011 / 0216622, No. 2011 / 0117125, No. 2011 / 0091525, No. 2011 / 0076335, No. 2011 / 0060032, No. 2010 / 0130588 No. 2007 / 0042031, No. 2006 / 0240093, No. 2006 / 0083780, No. 2006 / 0008910, No. 2005 / 0175682, No. 2005 / 017054, No. 2005 / 0118253, No. 2005 / 0064595, No. 2004 / 0142025, No. 2007 / 0042031, No. 1999 / 009076 and PCT Publication No. WO The entire disclosure of the patents described in No. 99 / 39741, No. 2018 / 081480, No. 2017 / 004143, No. 2017 / 075531, No. 2015 / 199952, No. 2014 / 008334, No. 2013 / 086373, No. 2013 / 086322, No. 2013 / 016058, No. 2013 / 086373, No. WO2011 / 141705 and No. 2001 / 07548 is incorporated herein by reference for the purposes described herein.

[0279] For example, in some embodiments, cationic lipids, auxiliary lipids, and steroids are dissolved in ethanol at a predetermined weight or molar ratio / percentage (e.g., the weight or molar ratio / percentage described herein). In some embodiments, lipid nanoparticles (LNPs) are prepared at a total lipid to RNA or DNA molar ratio of approximately 10:1 to 30:1. In some embodiments, this RNA or DNA may be diluted to 0.2 mg / mL in acetate buffer.

[0280] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion containing nucleic acids (e.g., RNA or DNA) can be formed as follows: an ethanol solution containing lipids (such as thiolipins, accessory lipids, steroids and polymer-conjugated lipids as described herein) is injected into an aqueous solution containing nucleic acids (e.g., RNA or DNA).

[0281] In some embodiments, the lipid and nucleic acid solutions can be mixed at room temperature by, for example, using a piston pump to pump each solution (e.g., a lipid solution containing the sulfur lipid compound, auxiliary lipid, steroid, and any other additives described herein) at a controlled flow rate into a mixing unit. In some embodiments, the flow rates of the lipid and nucleic acid solutions entering the mixing unit are maintained at a 1:3 ratio. After mixing, when the ethanol lipid solution is diluted with aqueous RNA, nucleic acid-lipid particles are formed. Lipid solubility decreases, while the positively charged cationic lipids interact with the negatively charged nucleic acids.

[0282] In some implementations, solutions containing lipid nanoparticles encapsulating nucleic acids (e.g., RNA) can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0283] In some embodiments, the compositions or complexes described herein further comprise a pharmaceutically acceptable surfactant. In some embodiments, the pharmaceutically acceptable surfactant is selected from polysorbates (e.g., polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80)), poloxamer, and amphiphilic groups comprising a portion selected from polyalkylene glycols (e.g., polyethylene glycol), poly(2-oxazoline), poly(2-methyl-2-oxazoline), polysarcosine, polyvinylpyrrolidone, and poly[N-(2-hydroxypropyl)methacrylamide], wherein said portion is bound to one or more C 12 -C 20 Aliphatic groups.

[0284] RNA In some embodiments, the particles described herein comprise one or more oligosaccharide compositions and nucleic acids. In some embodiments, the nucleic acid is RNA.

[0285] In some embodiments, the RNA suitable for the techniques described herein is single-stranded RNA. In some embodiments, the RNA disclosed herein is linear RNA. In some embodiments, the single-stranded RNA is non-coding RNA, wherein its nucleotide sequence does not include an open reading frame (or its complementary sequence). In some embodiments, the single-stranded RNA has a nucleotide sequence encoding one or more polypeptides (e.g., epitopes) of the disclosed polypeptide or multiple polypeptides (or a complementary sequence to the sequence encoding said polypeptide or multiple polypeptides).

[0286] In some implementations, the RNA is or contains siRNA, miRNA, or other non-coding RNA.

[0287] In many embodiments, the associated RNA contains at least one open reading frame (ORF) (e.g., mRNA); in some embodiments, the associated RNA contains a single ORF; in some embodiments, the associated RNA contains more than one ORF.

[0288] In some embodiments, the RNA comprises, for example, an ORF encoding a polypeptide of interest or multiple polypeptides of interest. In some embodiments, the RNA generated according to the techniques provided herein comprises multiple ORFs (e.g., encoding multiple polypeptides). In some embodiments, the RNA generated according to the techniques provided herein comprises a single ORF encoding multiple polypeptides. In some such embodiments, the polypeptide is or contains an antigen or an epitope thereof (e.g., an associated antigen).

[0289] In some embodiments, the ORF used according to this disclosure encodes a polypeptide comprising, for example, a signal sequence (such as an intrinsic or heterologous signal sequence) that functions in mammalian cells. In some embodiments, the signal sequence directs the secretion of the encoded polypeptide, and in some embodiments, the signal sequence directs the delivery of the encoded polypeptide to a defined cell compartment, preferably the cell surface, the endoplasmic reticulum (ER), or an endosome-lysosome compartment.

[0290] In some embodiments, the ORF encodes a polypeptide containing multiple elements (e.g., intrinsic or heterologous multiple elements). In some embodiments, the ORF encoding a surface polypeptide (e.g., a polypeptide containing a signal sequence that guides surface localization) contains multiple elements.

[0291] In some implementations, the ORF encodes a polypeptide containing transmembrane elements or domains.

[0292] In some implementations, the ORF is codon-optimized for expression in cells of a specific host (e.g., a mammalian host, such as a human).

[0293] In some embodiments, the RNA contains unmodified uridine residues; RNA containing only unmodified uridine residues may be referred to as "uRNA". In some embodiments, the RNA contains one or more modified uridine residues; in some embodiments, such RNA (e.g., RNA containing fully modified uridine residues) is referred to as "modRNA". In some embodiments, the RNA may be self-amplifying RNA (saRNA). In some embodiments, the RNA may be trans-amplifying RNA (taRNA) (see, for example, WO2017 / 162461).

[0294] In some embodiments, the associated RNA comprises one or more polypeptide-coding moieties. In some specific embodiments, one or more such moieties may encode one or more polypeptides that are or comprise a bioactive polypeptide or a portion thereof (e.g., an enzyme or cytokine or a therapeutic protein, such as a substitute protein or an antibody or portion thereof). In some specific embodiments, one or more such moieties may encode one or more polypeptides that are or comprise an antigen (or an epitope thereof), cytokine, enzyme, etc. In some embodiments, one or more encoded polypeptides may be or comprise one or more neoantigens or neoepitaxes associated with tumors. In some embodiments, one or more encoded polypeptides may be or comprise one or more antigens (or epitopes thereof) of an infectious agent (e.g., bacteria, fungi, viruses, etc.). In some embodiments, the encoded polypeptide may be a variant of a wild-type polypeptide.

[0295] In some embodiments, the single-stranded RNA (e.g., mRNA) may include a secretion signal coding region (e.g., a secretion signal coding region that allows one or more encoded target entities to be secreted post-translationally by the cell). In some embodiments, such a secretion signal coding region may be or contain a non-human secretion signal. In some embodiments, such a secretion signal coding region may be or contain a human secretion signal.

[0296] In some embodiments, the single-stranded RNA (e.g., mRNA) may contain at least one non-coding element (e.g., to enhance RNA stability and / or translation efficiency). Examples of non-coding elements include, but are not limited to, a 3' untranslated region (UTR), a 5' UTR, a cap structure (e.g., an enzyme-added cap in some embodiments; a co-transcriptional cap in some embodiments), a polyadenine (polyA) tail (e.g., in some embodiments, it may be or contain 100 or more A residues, and / or in some embodiments, it may include one or more "break" [i.e., non-A] sequence elements) and any combination thereof. Exemplary embodiments of such non-coded elements can be found in, for example, WO2011015347, WO2017053297, US 10519189, US10494399, WO2007024708, WO2007036366, WO2017060314, WO2016005324, WO2005038030, WO2017036889, WO2017162266 and WO2017162461, each of which is incorporated herein by reference in its entirety.

[0297] Format At least four formats have been developed for use in RNA pharmaceutical compositions (e.g., immunogenic compositions or vaccines): mRNA containing unmodified uridine (uRNA), nucleoside-modified mRNA (modRNA), self-amplified mRNA (saRNA), and trans-amplified RNA.

[0298] Unmodified uridine platforms may be characterized by, for example, inherent adjuvant effects, good tolerability and safety, and one or more of strong antibody and T-cell responses.

[0299] The characteristics of a modified uridine (e.g., pseudouridine) platform may include a reduced adjuvant effect, desensitized innate immune sensing activation and thus enhanced antigen expression, good tolerability and safety, and strong antibody and CD4-T cell responses. As described herein, this disclosure provides insights into the potential use of such strong antibody and CD4-T cell responses, particularly for vaccination.

[0300] Features of self-amplification platforms may include, for example, long duration of peptide (e.g., protein) expression, good tolerability and safety, and a high probability of producing efficacy using extremely low vaccine doses.

[0301] In some implementations, self-amplification platforms (e.g., saRNA comprises a nucleic acid molecule encoding both a replicase (e.g., a viral replicase) and the gene of interest, wherein the nucleic acid molecule is capable of cis-replication by the replicase (cis-replication system). In some embodiments, a trans-amplification platform (e.g., ta RNA comprises two nucleic acid molecules, one of which encodes a replicase (e.g., a viral replicase) and the other nucleic acid molecule is capable of being trans-replicated by said replicase (e.g., a replicon) (trans-replication system). In some embodiments, the self-amplification / trans-amplification platform (e.g., RNA) comprises multiple nucleic acid molecules, wherein said nucleic acids encode multiple replicases and / or replicons.

[0302] In some implementations, the trans-replication system includes the presence of two nucleic acid molecules in a single host cell.

[0303] In some such embodiments, the nucleic acid encoding a replicase (e.g., a viral replicase) cannot self-replicate in target cells and / or target organisms. In some such embodiments, the nucleic acid encoding a replicase (e.g., a viral replicase) lacks at least one conserved sequence element essential for the synthesis of the (-) strand based on the (+) strand template and / or the synthesis of the (+) strand based on the (-) strand template.

[0304] In some implementations, the self-amplifying RNA contains a 5' cap; in some trans-replication systems, at least the RNA encoding the replicase is capped. Not wishing to be limited to either theory, it has been found that the 5' cap may be crucial for high trans-level expression of the gene of interest.

[0305] In some implementations, the self-amplification / trans-amplification platform does not require the reproduction of viral particles (e.g., is unrelated to the undesirable formation of viral particles). In some implementations, the self-amplification / trans-amplification platform cannot form viral particles.

[0306] In some embodiments, the RNA may contain an internal ribosome entry site (IRES) element. In some embodiments, the RNA does not contain an IRES site; specifically, in some embodiments, saRNA does not contain an IRES site. In some such embodiments, the translation of the gene of interest and / or replicase is not driven by an IRES element. In some embodiments, the IRES element is replaced with a 5' cap. In some such embodiments, 5' cap substitution does not affect the sequence of the polypeptide encoded by the RNA.

[0307] In some embodiments, the complex described herein comprises modRNA, saRNA, taRNA, or uRNA. In some embodiments, the complex comprises modRNA. In some embodiments, the complex comprises saRNA. In some embodiments, the complex comprises taRNA. In some embodiments, the complex comprises uRNA.

[0308] How to use The particles described herein can be used to treat and prevent the diseases, conditions, and disorders described herein in a subject. In some embodiments, this disclosure provides a method of treating a disease, condition, or disorder, the method comprising administering to a patient a composition comprising the particles described herein. In some embodiments, this disclosure provides the use of a composition comprising the particles described herein for treating a disease, condition, or disorder. In some embodiments, the disease, condition, or disorder is an infectious disease, cancer, an autoimmune disease, or a rare disease.

[0309] In some implementations, the infectious disease is caused by or associated with a viral pathogen. In some implementations, the viral pathogen belongs to a family selected from the following: poxviridae, rhabdoviridae, filoviridae, paramyxoviridae, hepadnaviridae, coronaviridae, caliciviridae, picornaviridae, reoviridae, retroviridae, and orthomyxoviridae. In some implementations, the infectious disease is caused by or associated with a virus selected from the following: SARS-CoV-2, influenza virus, Crimean-Congo Hemorrhagic Fever (CCHF), Ebola virus, Lassa virus, Marburg virus, HIV, Nipah virus, and MERS-CoV.

[0310] In some implementations, infectious diseases are defined as those caused by or related to bacterial pathogens. In some implementations, the bacterial pathogen belongs to a genera selected from the following: *Actinomyces israelii*, *Bacillus anthracis*, *Bacteroides fragilis*, *Borrelia pertussis*, *Borrelia burgdorferi*, *Borrelia garinii*, *Borrelia afzelii*, *Borrelia recurrentis*, *Brucella abortus*, *Brucella canis*, *Brucella melitensis*, *Brucella suis*, *Campolobacter jejuni*, *Chlamydia pneumoniae*, *Chlamydia trachomatis*, and *Chlamydophila psittaci*. Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila *Pneumophila*, *Leptospira*, *Listeria monocytogenes*, *Mycobacterium leprae*Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroids, Rickettsia ricektssii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0311] In some implementations, the infectious disease is caused by or related to a parasite. In some implementations, the parasite belongs to a family selected from the following genera: Plasmodium, Leishmania, Cryptosporidium, Entamoeba, Trypanosomas, Schistosomas, Ascaris, Echinococcus, and Taeniidae.

[0312] In some implementations, the disease, symptom, or ailment is cancer. In some implementations, cancer is selected from bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral / oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

[0313] In some implementations, the disease, symptom, or disorder is a genetic condition. In some implementations, the genetic condition is associated with a gain-of-function mutation or a loss-of-function mutation.

[0314] In some implementations, the disease, symptom, or ailment is an autoimmune disease. In some implementations, the autoimmune disease is selected from Addison disease, celiac disease, rheumatoid arthritis, lupus, inflammatory bowel disease, dermatomyositis, multiple sclerosis, diabetes, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, pernicious anemia, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and Sjörgen syndrome.

[0315] In some implementations, the disease, symptom, or ailment is defined as a rare disease. As described herein, a rare disease is a life-threatening or chronically debilitating condition with such a low prevalence (e.g., less than 1 in 2000 people) that it requires a special, collaborative effort to address.

[0316] In some embodiments, this disclosure provides a complex that can selectively target a specific system in the body. As used herein, reference to “targeting” a specific system means causing an increase in the expression of RNA derived from the cargo in the complex in the desired system. For example, in some embodiments, the complex described herein can selectively target the lung, liver, spleen, heart, brain, lymph nodes, bladder, kidney, and pancreas. As described herein, the complex “selectively targets” an organ (e.g., 65% or more of the mRNA expressed by a single target is expressed in 65% or more of the mRNA expressed in other organs after administration) (i.e., 65% or more of the mRNA expressed throughout the body is expressed from a single organ, with the remaining 35% distributed among one or more different organs). In some embodiments, the complex described herein selectively targets the lung. In some embodiments, the complex described herein selectively targets the liver. In some embodiments, the complex described herein selectively targets the spleen. In some embodiments, the complex described herein selectively targets the heart.

[0317] delivery method This disclosure particularly provides particles incorporated into compositions (e.g., pharmaceutical compositions or formulations as mentioned herein) for administration to a subject. For example, in some embodiments, the composition comprising the particles described herein is administered as a single therapy. In some embodiments, the composition comprising the particles described herein is administered as part of a combination therapy. In some embodiments, the concentration of total RNA in the composition described herein (e.g., the total concentration of all one or more RNA molecules) is from about 0.01 mg / mL to about 0.5 mg / mL, or from about 0.05 mg / mL to about 0.1 mg / mL.

[0318] The composition (also known as a pharmaceutical composition) may additionally contain pharmaceutically acceptable excipients, including, as used herein, any and all solvents, dispersion media, diluents or other liquid media, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for the desired specific dosage form. (Remington's The Science and Practice of Pharm) ac y, 21st edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; which is incorporated herein by reference in its entirety) discloses various excipients for formulating pharmaceutical compositions and known techniques for preparing them. Unless any conventional excipient medium is incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a harmful manner with any one or more other components of the pharmaceutical composition, its use is contemplated within the scope of this disclosure.

[0319] In some implementations, the excipient is approved for human and veterinary use. In some implementations, the excipient is approved by the United States Food and Drug Administration (FDA). In some implementations, the excipient is pharmaceutical grade. In some implementations, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0320] 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 the pharmaceutical formulation. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents and / or aromatizers may be present in the composition at the formulator's discretion.

[0321] For general considerations in the formulation and / or manufacture of pharmaceutical preparations, see, for example, Remington: The Science and Practice of Pharmaceuticals. ma cy, 21st edition, Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety).

[0322] In some embodiments, the pharmaceutical compositions provided herein can be developed using conventional techniques (such as Remington: The Science and Practice of Pharmaceutical Sciences). ma The techniques disclosed in Lippincott Williams & Wilkins, 2005 (which are incorporated herein by reference in their entirety) are formulated with one or more pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients.

[0323] The pharmaceutical complexes and compositions described herein may be administered by appropriate methods known in the art. As those skilled in the art will understand, the route and / or mode of administration may depend on a variety of factors, including, but not limited to, the stability and / or pharmacokinetics and / or pharmacodynamics of the pharmaceutical compositions described herein.

[0324] In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration, including administration modalities other than enteric and local administration, typically by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0325] In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers suitable for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions.

[0326] In some specific embodiments, the pharmaceutical compositions described herein are formulated for subcutaneous (sc) administration. In some specific embodiments, the pharmaceutical compositions described herein are formulated for intramuscular (im) administration.

[0327] Therapeutic compositions must generally be sterile and stable under manufacturing and storage conditions. Compositions may be formulated as solutions, dispersions, powders (e.g., lyophilized powders), microemulsions, lipid nanoparticles, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by using coatings (such as lecithin), in the case of dispersions by maintaining the desired particle size, and by using surfactants. In many cases, the composition should preferably include isotonic agents such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride. In some embodiments, prolonged absorption of the injectable composition may be achieved by incorporating an agent that delays absorption (e.g., monostearate and gelatin) into the composition.

[0328] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound with one or a combination of the ingredients listed above (as needed) into a suitable solvent, followed by sterilization and microfiltration.

[0329] In some embodiments, the dispersion is prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and any other desired components from the components listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which produce a powder containing the active ingredient plus any other desired components from its previously sterile filtered solution.

[0330] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Appropriate flowability can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0331] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of microorganisms can be prevented by sterilization procedures and by incorporating various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.). Incorporation of isotonic agents (such as sugars, sodium chloride, etc.) into the pharmaceutical compositions described herein may also be desirable. Furthermore, prolonged absorption of injectable drug forms can be achieved by incorporating agents that delay absorption (such as aluminum monostearate and gelatin).

[0332] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in or subsequently developed in pharmacological techniques. Generally, such preparation methods include the following steps: associating one or more active ingredients with a diluent or another excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or required, shaping and / or packaging the product into desired single-dose or multi-dose units.

[0333] The pharmaceutical compositions disclosed herein can be prepared, packaged, and / or sold as a whole, in a single unit dose, and / or in multiple single unit doses. As used herein, "unit dose" means a discrete amount of a pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using the systems and / or methods described herein.

[0334] In some embodiments, the active agent that may be included in the pharmaceutical composition described herein is or is included in the combination therapy described herein. The pharmaceutical composition described herein may be administered in combination therapy, i.e., in combination with other agents. In some embodiments, such therapeutic agents may include agents that cause regulatory T cell clearance or functional inactivation. For example, in some embodiments, the combination therapy may include the provided pharmaceutical composition with at least one immune checkpoint inhibitor.

[0335] In some implementations, the pharmaceutical compositions described herein may be administered in combination with radiotherapy and / or autologous peripheral stem cell or bone marrow transplantation.

[0336] In some embodiments, the pharmaceutical compositions described herein can be frozen to allow for long-term storage.

[0337] Although the description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all species of animals. Modifications to the compositions to make them suitable for administration to humans suitable for administration to a variety of animals are well known in the art, and ordinary skilled veterinary pharmacologists can design or implement such modifications using only ordinary (if any) experimental design.

[0338] Exemplary Implementation Although not restrictive, the following numbered embodiments are illustrative of certain aspects of this disclosure: Implementation Scheme 1. A compound represented by Formula I: I Or its pharmaceutically acceptable salt, wherein: L 1 and L 2 Each of the C1-Cs can be substituted independently. 30Aliphatic groups; L 3 C1-C, which are the bonds and can be arbitrarily substituted 10 Aliphatic groups or optionally substituted 2- to 10-membered heteroaliphatic groups comprising 1 to 4 heteroatoms selected from N, O, and S; X 1 and X 2 Each is independently selected from the bond, -OC(O)-, -C(O)O-, -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-, where X 1 or X 2 One or both of them are selected from -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-; Each R 1 Independently for each case the optionally substituted C1-C 20 Aliphatic or H; T 1 and T 2 Each of the C3-Cs can be substituted independently. 30 aliphatic; G is -N(R) 2 )C(S)N(R 2 )2、-OH、-N(R 2 )2、-N + (R 3 )3、-N(R 5 )C(O)R 3 -N(R) 5 )S(O)2R 3 -N(R) 5 )C(O)N(R 3 )2、-CH(NR 2 -R 4 and -S(O)2R 3 ; Each R 2 In each case, H, arbitrarily substituted C1-C6 aliphatic and OR are independently selected. 3 The group formed; or R2 Two instances together with the atoms to which they are attached form optional substituted 4- to 12-membered heterocycles or optional substituted 4- to 12-membered heteroaryl rings containing 1 to 4 heteroatoms selected from N, O and S; Each R 3 In each case, H and the arbitrarily substituted C1-C are chosen independently. 10 A group composed of aliphatic groups; and R 4 It is a 4- to 12-membered heterocycle with 1 to 4 heteroatoms selected from N, O, and S, optionally substituted, and a 4- to 12-membered heteroaryl group with 1 to 4 heteroatoms selected from N, O, and S, substituted with -(CH2). 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C6-C in )2 are replaced 12 Aryl group, or with an oxy group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C3-C in )2 are substituted 12 Cycloaliphatic; Each R 5 Independently selected from H and optionally substituted C1-C6 aliphatic compounds.

[0339] Implementation Scheme 2. The compound of Implementation Scheme 1, wherein L 1 and L 2 Each is C1-C 30 Alkylene.

[0340] Implementation Scheme 3. The compound as described in Implementation Scheme 1 or 2, wherein L 1 and L 2 Each is independently -(CH2) 6-12 -

[0341] Implementation Scheme 4. The compound of any one of Implementation Schemes 1-3, wherein L 1 and L 2 They are the same.

[0342] Implementation Scheme 5. The compound of any one of Implementation Schemes 1-3, wherein L 1 and L 2 For different reasons.

[0343] Implementation Scheme 6. A compound as described in any one of Implementation Schemes 1-5, wherein X 1 and X 2 Each is independently selected from -S(O)2N(R) 1 )-、-N(R 1)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- and -S-.

[0344] Implementation Scheme 7. A compound as described in any one of Implementation Schemes 1-5, wherein X 1 and X 2 One of them is a bond, -OC(O)- or -C(O)O-, and X 1 and X 2 The other one is -S(O)2N(R) 1 )-、-N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-、-OC(S)C(R 1 )2-、-C(R 1 )2C(S)O- or -S-.

[0345] Implementation Scheme 8. A compound as described in any one of Implementation Schemes 1-5, wherein X 1 -S(O)2N(R) 1 )-, where R 1 For C1-C 10 Aliphatic, and X 2 It is -C(O)O-.

[0346] Implementation Scheme 9. A compound as described in any one of Implementation Schemes 1-5, wherein X 1 and X 2 Each is -S(O)2N(R) 1 )-, where each R 1 Independently for R 1 For C1-C 10 Lipids.

[0347] Implementation Scheme 10. A compound as described in any one of Implementation Schemes 1-5, wherein X 1 and X 2 They are the same.

[0348] Implementation Scheme 11. A compound as described in any one of Implementation Schemes 1-5, wherein X 1 and X 2 For different reasons.

[0349] Implementation Scheme 12. The compound of any one of Implementation Schemes 1-11, wherein T 1 and T 2 Each is independently selected from the arbitrarily substituted C3-C 20 alkyl.

[0350] Implementation Scheme 13. The compound of any one of Implementation Schemes 1-11, wherein T 1 and T 2 Each is selected independently from: , , , , , , , , , ; , , , ; , , and .

[0351] Implementation Scheme 14. The compound of any of the foregoing implementation schemes, wherein a portion of -L 1 -X 1 -T 1 Choose from the following groups: , , , , , , , and .

[0352] Implementation Scheme 15. The compound of any of the foregoing implementation schemes, wherein a portion of -L 2 -X 2 -T 2 Choose from the following groups: , , , , , , , and .

[0353] Implementation Scheme 16. The compound of any of the foregoing implementation schemes, wherein a portion of -L 1 -X 1 -T 1 and part-L 2 -X2 -T 2 Each is selected independently from: .

[0354] Implementation Scheme 17. A compound as described in any of Implementation Schemes 1-16, wherein L 3 C1-C is an optional substitute 10 Lipids.

[0355] Implementation Scheme 18. A compound as described in any of Implementation Schemes 1-17, wherein G is -N(R 2 )C(S)N(R 2 )2 or -N(R 5 )S(O)2R 3 .

[0356] Implementation Scheme 19. The compound of any of the foregoing implementation schemes, wherein G is -N(R 2 )C(S)N(R 2 )2.

[0357] Implementation Scheme 20. A compound as described in any of the foregoing implementation schemes, wherein G is -N(H)C(S)N(R) 2 )2, where each R 2 Selected from optional substituted C1-C6 aliphatic and OH groups.

[0358] Implementation Scheme 21. The compound of any of the preceding implementation schemes, wherein G is -OH.

[0359] Implementation Scheme 22. The compound of any of the preceding implementation schemes, wherein G is an optionally substituted 4- to 12-membered heterocycle.

[0360] Implementation Scheme 23. The compound of any of the preceding implementation schemes, wherein G is selected from: , , , , , , , , , , and .

[0361] Implementation Scheme 24. The compound of any of the foregoing implementation schemes, wherein a portion of -L 3 -G is selected from: , , , , , , , , , , , , , , , , , , , , , , and .

[0362] Implementation Scheme 25. The compound of any of the foregoing implementation schemes, wherein -L 3 -G is selected from: , , , , , , , , , and .

[0363] Implementation Scheme 26. The compound of any of the foregoing implementation schemes, wherein the compound is represented by formula IIa: IIa Or its pharmaceutically acceptable salt.

[0364] Implementation Scheme 27. A compound as described in any of the preceding implementation schemes, wherein the compound is represented by formula IIb: IIb Or its pharmaceutically acceptable salt.

[0365] Implementation Scheme 28. The compound of any of the foregoing implementation schemes, wherein the compound is represented by formula IIc: IIc Or its pharmaceutically acceptable salt.

[0366] Implementation Scheme 29. A compound as described in any of the foregoing implementation schemes, wherein the compound is represented by formula IIIa: IIIa Or its pharmaceutically acceptable salt.

[0367] Implementation Scheme 30. A compound as described in any of the foregoing implementation schemes, wherein the compound is represented by formula IIIb: IIIb Or its pharmaceutically acceptable salt.

[0368] Implementation Scheme 31. The compound of any of the foregoing implementation schemes, wherein the compound is represented by formula IIIc: IIIc Or its pharmaceutically acceptable salt.

[0369] Implementation Scheme 32. The compound of any of the foregoing implementation schemes, wherein the compound is represented by formula IIId: IIId Or its pharmaceutically acceptable salt.

[0370] Implementation Scheme 33. The compound of any of the foregoing implementation schemes, wherein the compound is represented by formula IIIe: IIIe Or its pharmaceutically acceptable salt.

[0371] Implementation Scheme 34. The compound of any of the foregoing implementation schemes, wherein the compound is represented by formula IIIf: IIIf Or its pharmaceutically acceptable salt.

[0372] Implementation Scheme 35. The compound of any of the foregoing implementation schemes, wherein the compound is represented by formula IIIg: IIIg Or its pharmaceutically acceptable salt.

[0373] Implementation Scheme 36. The compound of any of the preceding implementation schemes, wherein the compound is selected from Table 1.

[0374] Implementation Scheme 37. A particle comprising a compound and a nucleic acid from any one of Implementation Schemes 1-36.

[0375] Implementation scheme 38. The particles of implementation scheme 37, wherein the nucleic acid is RNA, DNA or a mixture thereof.

[0376] Implementation Scheme 39. As in Implementation Scheme 38, the RNA is mRNA.

[0377] Implementation scheme 40. The particles of implementation scheme 39, wherein the RNA is modRNA, circRNA, saRNA, taRNA or uRNA.

[0378] Implementation Scheme 41. The particles of Implementation Scheme 37, wherein the DNA is linear DNA, plasmid DNA, microcircular DNA, nanoplasmid DNA, dog bone DNA, or a transposon.

[0379] Implementation Scheme 42. Particles of any of Implementation Schemes 37-41, wherein the particles further comprise one or more of an accessory lipid, a polymer-conjugated lipid, or a sterol.

[0380] Implementation scheme 43. The particles of implementation scheme 42, wherein the auxiliary lipid is phospholipid.

[0381] Implementation Scheme 44. The particles of Implementation Scheme 43, wherein the phospholipids are selected from: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, more preferably from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), bispentadecanylphosphatidylcholine, dilaurylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), and diarachidonicylphosphatidylcholine (DAPC). Dithaneylphosphatidylcholine (DBPC), bis(tridecyl)phosphatidylcholine (DTPC), bis(tetradecyl)phosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphyranoylphosphatidylethanolamine (DPyPE), and combinations thereof.

[0382] Implementation Scheme 45. Particles of any of Implementation Schemes 42-44, wherein the polymer-conjugated lipids are selected from the group consisting of: poly(ethylene glycol) (PEG) conjugated lipids, poly(sarcosine) (pSar) conjugated lipids, poly(aminoethoxyethoxyacetic acid) (pAEEA) conjugated lipids; and poly(2-methylaminoethoxyethoxyacetic acid) (pMAEEA) conjugated lipids.

[0383] Implementation Scheme 46. Particles as in any of Implementation Schemes 42-45, wherein the polymer-conjugated lipid is selected from the following PEG-lipids: PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, PEG-cer, PEG dialkoxypropyl carbamate, and combinations thereof.

[0384] Implementation Scheme 47. Particles of any of Implementation Schemes 42-46, wherein the sterol is selected from β-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulinum toxin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or α-tocopherol.

[0385] Implementation Scheme 48. Particles as described in any of Implementation Schemes 42-47, wherein the particles are characterized by an N / P ratio of about 4 to about 16.

[0386] Implementation Scheme 49. A method for increasing or causing an increase in RNA expression in a target of a subject, the method comprising administering to the subject a composition comprising particles of any one of Implementation Schemes 42-48.

[0387] Implementation scheme 50. The method of implementation scheme 49, wherein the target is selected from the lung, liver, spleen, heart, brain, lymph nodes, bladder, kidney and pancreas.

[0388] Implementation Scheme 51. A method for treating a disease, symptom, or ailment of a subject, the method comprising administering to the subject a composition comprising particles of any one of Implementation Schemes 42-48.

[0389] Implementation scheme 52. The method of implementation scheme 51, wherein the disease, symptom or ailment is an infectious disease, cancer, genetic disease, autoimmune disease or rare disease.

[0390] Implementation Scheme 53. The method of any of Implementation Schemes 48-52, wherein the particles are administered parenterally or intranasally.

[0391] Implementation Scheme 54. The method of Implementation Scheme 53, wherein the particles are administered intramuscularly, subcutaneously, intradermally, or intravenously.

[0392] Implementation scheme 55. Particles of any of implementation schemes 42-48, which are used as pharmaceuticals.

[0393] Implementation Scheme 56. Particles of any of Implementation Schemes 42-48, used for the treatment and / or prevention of diseases or conditions, wherein the diseases or conditions are infectious diseases, cancer, genetic conditions, autoimmune diseases, or rare diseases.

[0394] Example The embodiments provided herein record and support certain aspects of this disclosure, but are not intended to limit the scope of any claim. The following non-limiting embodiments are provided to further illustrate certain teachings provided in this disclosure. Those skilled in the art will recognize from this application that various changes can be made to the specific embodiments described in the present invention without departing from the spirit and scope of these teachings.

[0395] The following abbreviations may be used in the examples below: aq. (aqueous solution); ACN (acetonitrile); BLI (bioluminescence imaging); BM (standard); CHOL (cholesterol); d (day); Da / kDa (Dalton / kilodalton); DCM (dichloromethane); DLS (dynamic light scattering); DMF (N,N-dimethylformamide); DIPEA (N,N-diisopropylethylamine); DMAP (4-dimethylaminopyridine); DODMA (1,2-dioleoyloxy-3-dimethylaminopropane); DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine); DMSO (dimethyl sulfoxide); DSPC (distearate phosphatidylcholine); EA (ethyl acetate); ee (enantiomer excess); equiv. (equivalent); ELSD (evaporative light scattering detector); EtOH (ethanol); h or hr (hour); Hex (hexane); HPLC (High-performance liquid chromatography); intramuscular; intravenous; KHMDS (Kapok bis(trimethylsilyl)amino)amino; LAH (lithium aluminum hydride); LCMS (Liquid chromatography-mass spectrometry); LDA (lithium diisopropylamino); LiHMDS (lithium bis(trimethylsilyl)amino); LNP (lipid nanoparticles); MeOH (methanol); min; NMR (nuclear magnetic resonance); PBS (phosphate-buffered saline); Pd / C (carbohydrate palladium); PEG-DMG (1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000); PEI (polyethyleneimine); PPh3O (triphenylphosphine oxide); Pt / C (carbohydrate platinum); rb (round bottom); Rf (retention factor); rt or RT (room temperature); sc (subcutaneous); SM (starting material); TEA (triethylamine); THF (tetrahydrofuran); THP (Tetrahydropiperan); TLC (thin-layer chromatography); TsOH (p-toluenesulfonic acid or tosylic acid); and UV (ultraviolet light).

[0396] The α-tag or α-peptide refers to the peptide with sequence SEQ.ID.NO.1.

[0397] The reference to NbAlfa refers to the VHH domain containing the CDR1 sequence SEQ.ID.NO.5, the CDR2 sequence SEQ.ID.NO.6, and the CDR3 sequence SEQ.ID.NO.7. The reference to aCD3-VHH refers to the anti-CD3 binding VHH, which contains the CDR1 sequence SEQ.ID.NO.8, the CDR2 sequence SEQ.ID.NO.9, and the CDR3 sequence SEQ.ID.NO.10.

[0398] Example 1: Preparation and characterization of sulfonamide lipids Fragment synthesis: This embodiment provides a method for preparing a tail-joint fragment via an unoxidized binding residue "-S-" having a -2 oxidation state. Figure 79 ).

[0399] Compounds BNT-21, BNT-46, and BNT-20 of the examples were prepared according to the methods provided above.

[0400] Thiolipids with linker residue -S- General procedure for synthesizing thioethers from thiols and α-o-bromools 1.1 equivalents of thiol (33 mmol) and 30 mmol of appropriate α-o-bromool were sequentially added to a solution of 1M NaOEt (40 mL, 40 mmol, 1.33 equivalents) in anhydrous ethanol, prepared by adding NaH (60% suspension in mineral oil) and EtOH. The reaction mixture was stirred at room temperature under dry nitrogen for 48 hours. The reaction mixture was evaporated to an oil / solid mixture and then absorbed, depending on the solubility of the product, into an emulsion of n-hexane or diethyl ether (150 mL) and 10% NaH₂PO₄ aqueous solution (150 mL). The organic phase was washed with brine (150 mL) and dried over anhydrous Na₂SO₄. After filtration of the salt, the crude product was purified by rapid chromatography on silica gel using ethyl acetate / n-hexane. The product fraction was identified using ELSD and UV detectors of the eluent. Evaporation of the product fraction yielded a colorless oily thiolipin alcohol representing the precursor of the tail aldehyde.

[0401] 9-(Octylthio)non-1-ol 1 H-NMR (CDCl3) δ [ppm]: 3.64 t, 2H; 2.48 m, 4H; 1.58 m, 4H; 1.2-1.4 m 22H; 0.96 t, 3H; 13 C-NMR (CDCl3) δ [ppm]: 63.01;32.74;32.14;32.12;31.79;29.69;29.66;29.44;29.32;29.20;29.17;29.15;28.93;28.89;25.68;22.62;14.08 (Me).

[0402] 11-((2-ethylhexyl)thio)undecyl-1-ol: 1 H-NMR (CDCl3) δ [ppm]: 3.63 t, J (6.7 Hz), 2H; 2.47 m, 4H); 1.6 m, 1.2-1.5 m, 28H; 0.88, 2t, 6H; 13 C-NMR (CDCl3) δ [ppm]: 63.08; 39.34; 36.75; 32.87; 32.75; 29.76; 29.56; 29.49; 29.40; 29.24; 28.93; 28.88; 25.71; 25.54; 22.99; 14.11 (Me); 10.80 (Me); Rf: 0.15 (n-hexane / EtOAc: 9 / 1, v / v).

[0403] General procedure for synthesizing thioethers from thioacetates and α-o-bromools At 0 °C, a solution of 1 M NaOMe (26 mL, 6.1 mmol, 1.2 equivalents) prepared by adding NaH (60% suspension in mineral oil) and MeOH in anhydrous methanol was continuously supplemented with 1 equivalent of thioacetate (5.1 mmol) and 1.2 equivalents (6.1 mmol) of appropriate α-o-bromool. The reaction mixture was heated to room temperature and then refluxed under dry nitrogen for 20 h. The reaction mixture was evaporated into an oil / solid mixture and then absorbed into an emulsion of CHCl3 and 10% citric acid (100 mL). The organic phase was washed with brine (100 mL) and then dried over anhydrous Na2SO4. After filtration of the salt, the crude product was purified by rapid chromatography on silica gel using ethyl acetate / n-hexane. The product fraction was identified using ELSD and UV detectors of the eluent. The evaporation product fraction yielded a colorless oily thiolipin alcohol representing the precursor of the tail aldehyde.

[0404] 9-((3,7-dimethyloctyl)thio)non-1-ol This embodiment also provides a one-pot reaction sequence for the synthesis of thiolipin alcohols from two alcohols: Methanesulfonation of 2-butyl-octanol A racemic mixture of 2-butyl-octanol (5.05 mmol, 941 mg, 10 mL), DMAP (10 mg), and trimethylamine (15 mmol, 2.09 mL) in anhydrous THF (10 mL) was added to methanesulfonic anhydride (7.74 mmol, 1.348 g). The reaction mixture was stirred at room temperature and gradually turned pale yellow. After maintaining at room temperature for 2 hours, TLC (n-hexane / ethyl acetate 4 / 1 v / v) revealed conversion to a product that could be stained with molybdate. Conversely, in an R-phase almost identical to the product... f 2-Butyl-octanol inhibited staining at (0.54) and therefore remained as white spots on TLC.

[0405] The methanesulfonate product can then be further converted into thioacetate without further purification.

[0406] Potassium thioacetate (11.99 mmol, 1.37 g) and 10 mg tetraethylammonium tetrafluoroborate were added to the methanesulfonation reaction mixture. After the addition of the thioacetate, the reaction mixture was stirred for 3 hours. Then, TEA (15 mmol) and EtOH (15 mL) were added to the reaction mixture to improve the solubility of the thioacetate. The reaction mixture was stirred at room temperature for 16 hours, then maintained at 70 °C for 3 hours. TLC revealed that the original methanesulfonate band was transformed into a high Rf spot (Rf = 0.86, n-hexane / ethyl acetate 4 / 1 (v / v)), which was readily stained by molybdate and absorbed UV at 254 nm.

[0407] The product was evaporated into a solid under vacuum at a maximum temperature of 40°C. It was then absorbed into an emulsion of n-hexane (50 mL) and 1M NaOH aqueous solution (50 mL). The n-hexane was concentrated into a pale yellow oil, which was subsequently converted primarily into branched-chain thiolipin alcohols according to a standard procedure.

[0408] 9-((2-Butyloctyl)thio)non-1-ol 1 H-NMR (CDCl3) δ [ppm]: 3.62 t, 2H; 2.46 m, 4H; 1.44-1.52 m, 4H; 1-1.43m, 28H; 1.5 m, 6H; 13C-NMR (CDCl3) δ [ppm]: 63.03;37.93;37.23;33.20;32.89;32.77;31.86;29.75;29.62;29.46;2 9.34;29.17;28.89;28.85;26.59;26.69;22.99;22.66;14.10(Me);14.09 (Me); IR (Pure): [cm -1 ] 3300 (s); 2922 (l); 2853 (l); 1458 (s); 1055 (s); 723 (s); MS (APCI) m / z 345.3291 (100%) (C 21 H 44 OS + H) + .

[0409] General procedure for forming tetramethylthiourea onion salt 1.1 equivalents of tetramethylurea (48.7 mmol) and 0.015 equivalents of LiI (0.7 mmol) were added to 1 equivalent of a primary aliphatic halide (44.3 mmol) and heated to 60 °C. The tetramethylurea dissolved within 1 hour, forming a yellow liquid. Prolonged reaction resulted in the formation of a white precipitate, which accumulated throughout subsequent heating periods. After 23 hours, the entire reaction mixture became a solid. The precipitate was suspended in n-hexane and passed through a Schlenk glass frit. The tetramethylurea was washed away with hexane, indicated by the absence of UV absorption (l: 256 nm) in the filtrate. The product was obtained as a white bromide.

[0410] 2-(3,7-Dimethyloctyl)-1,1,3,3-Tetramethylthiourea bromide 1 H-NMR (CDCl3) δ [ppm]: 5.6 s, 12H; 3.12 m, 2H; 1.69 m, 2H; 1.53 m, 2H; 1.1-1.35 m, 6H; 0.93 d, J(6.3 Hz), 3H; 0.87 d, J(6.6 Hz), 6H.

[0411] General methods for the formation of thiols from tetramethylthiourea salts Thiourea onium salt (30 mmol) was added to an aqueous solution of 5N NaOH (80 mL, 400 mmol). The suspension formed an emulsion during the exothermic reaction. After the exothermic reaction was stopped, the reaction mixture was stirred at room temperature for 1 hour. The organic phase was extracted with diethyl ether (150 mL). The aqueous phase was neutralized with 10% NaH₂PO₄ and extracted with diethyl ether (100 mL). The combined organic phases were washed with 10% NaH₂PO₄ and dried over anhydrous Na₂SO₄. After removing the salt by filtration, a colorless, oily thiol with an odor was obtained.

[0412] General procedure for synthesizing tail-based aldehydes Add 15 mL of DCM or an amount sufficient to homogenize the emulsion as possible to a stirred emulsion / solution of thiolipin tail alcohol (1 equivalent; 25 mmol), 10 equivalents of diisopropylethylamine (250 mmol), and 10 equivalents of anhydrous DMSO (250 mmol). Add 7 equivalents of a SO3 / pyridine complex (176 mmol) fractionally to this solution under dry nitrogen. Stir the mixture magnetically under dry nitrogen. The addition of the SO3 complex produces a slightly exothermic reaction, so the reaction mixture can be temporarily cooled by an ice bath. After continuous stirring at room temperature for more than 2 hours, the reaction was found to be complete by TLC (hexane, ethyl acetate).

[0413] The reaction mixture was transferred to a 10% NaH₂PO₄ aqueous solution (200 mL) and diethyl ether (200 mL). After phase separation, the turbid organic phase was removed. The turbid aqueous phase was washed twice consecutively with 100 mL of diethyl ether each time. The organic phases were combined and passed through a silica 60-cell plug (height: 30 mm, diameter: 55 mm) to remove turbidity caused by residual SO₃ complexes. The filtrate was concentrated into a clear oil, which can be chromatographically obtained on silica or used directly for the formation of thiolipins using the following multicomponent reaction.

[0414] 11-((2-ethylhexyl)thio)undecaldehyde 1 H-NMR (CDCl3) δ [ppm]: 9.78 s, 1H; 2.46 m, 4H; 2.43 t 2H; 1.6 m, 4H; 1.2-1.45 m; 0.9 m, 6H; 13C-NMR (CDCl3) δ [ppm]: 202.79 (CHO); 43.86; 39.32; 36.72; 32.83; 32.38; 29.71; 29.30; 29.28; 29.17; 29.10; 28.86; 25.53; 22.95; 22.07; 14.07 (Me); 10.78 (Me);); MS (APCI) m / z 315.2718 (100%) (M+H) + .

[0415] 9-((2-Butyloctyl)thio)nonanal 1 H-NMR (CDCl3) δ [ppm]: 9.78s, 1H; 2.48 m, 4H; 2.42 t, 2H; 1.43-1.66 m4H; 1.2-1.4 m 25H; 0.92 m, 6H; 13 C-NMR (CDCl3) δ [ppm]: 202.82 (CHO);43.87;37.94;37.24;33.20;32.89;32.87;31.86;29.70;29.62;29.22;29.08;29.02;28.86;28.81;26.59;22.99;22.66;22.03;14.10 (Me); 14.09 (Me); IR (Pure): [cm -1 ] 2923 (l); 2853 (l); 1726 (l) 1456 (s); 756 (s); MS (APCI) m / z 343.3035 (100%) (M+H) + .

[0416] Thiolipids are generally synthesized from caustic amines and two tail aldehydes. This procedure can also provide pure sulfur lipids from impure aldehydes prepared in previous procedures.

[0417] Add 1 equivalent (1 mmol) of cephaloamine (as a cephaloamine in 2 mL of 0.5 M DCM) to a magnetically stirred solution of 2–2.8 equivalents (2–2.8 mmol) of tailed aldehyde and anhydrous sodium sulfate (500 mg, 3.51 mmol) in anhydrous dichloromethane (2 mL). The initially clear solution became turbid. One hour later, dichloromethane (10 mL) was first added under dry nitrogen at room temperature, followed by a small amount of solid N[H(B(OAc)3] (2 g, 9.44 mmol). The turbid solution was stirred at room temperature for 16 hours. The reaction mixture was evaporated to a white paste. The paste was resuspended in an emulsion of diethyl ether / saturated sodium bicarbonate solution. The organic phase was removed; the aqueous phase was extracted twice with 20 mL of diethyl ether each time. The ether layers were combined and washed with brine (100 mL), then dried over anhydrous sodium sulfate. Salts were removed by filtration, and the filtrate was evaporated to a pale yellow or white oil, which could be absorbed in hexane before undergoing chromatography on silica. Chromatography on silica was performed using a gradient of 1% triethylamine in hexane / chloroform: 100 / 0-0 / 100 (v / v); and 1% triethylamine in chloroform / methanol: 100 / 0-93 / 7 (v / v). The products were detected in the effluent by ELSD.

[0418] The collected products fractionated and evaporated into a colorless oily substance.

[0419] 2-(bis(9-(octylthio)nonyl)amino)ethane-1-ol 1 H-NMR (CDCl3) δ [ppm]: 3.55m, 2H; 2.61 m 2H; 2.49 m 12H; 1.56n, 8H; 1.2-1.5 m; 0.87 m; 6H; 13 C-NMR (CDCl3) δ [ppm]: 53.82; 53.82; 32.18; 32.16; 31.81; 29.72; 29.70; 29.21, 29.19; 28.96; 28.92; 26.89; 21.04; 14.09.

[0420] 3-(bis(9-(octylthio)nonyl)amino)prop-1-ol 1H-NMR (CDCl3) δ [ppm]: 3.79 m, 2H; 2.64 m 2H; 2.49 m 8H; 2.40 m, 4H; 1.67m, 2H; 1.56 m, 8H; 6H 1.56 2H; 1.45m; 2H; 1.2-1.41 m; 0.86 m, 6H; 13 C-NMR (CDCl3) δ [ppm]: 54.11; 32.17; 31.81; 29.72; 29.52; 29.48; 29.22; 29.19; 28.96; 28.92; 27.47; 26.68; 22.64; 14.09.

[0421] 2-(bis(11-((2-ethylhexyl)thio)undecyl)amino)ethanol-1-ol 1 H-NMR (CDCl3) δ [ppm]: 3.57 m, 2H; 2.64 m, 2H; 2.47 m 12H; 1.58 m 4H; 1.2-1.5 m, 51H; 0.88 m, 12H; MS (APCI) m / z 658.5992 (100%) (M+H) + .

[0422] 3-(bis(11-((2-ethylhexyl)thio)undecyl)amino)-1,1,1-trifluoroprop-2-ol: 1 ¹H-NMR (CDCl₃) δ [ppm]: 4.2 broad singlet, 1H; 2.9 broad singlet, 2H; 2.76 broad singlet, 4H; 2.49 m 8H; 1.59 m, 4H; 1.2–1.5 m, 51H; 0.85 m, 12H; 13 C-NMR (CDCl3) δ [ppm]: 65.7q, J CF (31 Hz); 54.70; 39.30; 36.70; 32.82; 32.37; 29.71; 29.40; 29.23; 28.88; 28.85; 26.92: 25.50; 22.96; 14.08 (Me); 10.77 (Me); 19 F-NMR (CDCl3) δ [ppm]: -79; MS(APCI) m / z 726.5855 (100%) (M+H) + .

[0423] (2R)-3-(bis(11-((2-ethylhexyl)thio)undecyl)amino)-2-fluoroprop-1-ol 1 ¹H-NMR (CDCl₃) δ [ppm]: 4.65 doublet triplet, 1H; 3.82 doublet multiplet, 2H; 2.79 doublet multiplet, 2H; 2.5 m, 8H; 1.58 m, 4H; 1.2–1.5 m; 0.88 m, 12H; 13 C-NMR (CDCl3) δ [ppm]:55.28;39.32;36.73;32.85;32.38;29.75;29.55;29.51;29.47;29.23;28.92;28.87;94.8;45.7;25.52;22.97;14.10;10.79; 19 F-NMR (CDCl3) δ [ppm]: -92; LC / MS: MeCN-10-55 min. C8-RP column Rf: 34.66 min; MS (APCI) m / z (100%) 690.6062 (M+H) + .

[0424] (2S)-3-(bis(11-((2-ethylhexyl)thio)undecyl)amino)-2-fluoroprop-1-ol 1 H-NMR (CDCl3) δ [ppm]: 19 F-NMR (CDCl3) δ [ppm] MS (APCI), see (2R)-3-(bis(11-((2-ethylhexyl)thio)undecyl)amino)-2-fluoroprop-1-ol LC / MS: MeCN-10-55 min. Column: Rf: 34.65 min. 13 C-NMR (CDCl3) δ [ppm]: MS (ESI) m / z (100%) 690.6063 (M+H) + .

[0425] 3-(bis(11-((2-ethylhexyl)thio)undecyl)amino)-2,2-difluoroprop-1-ol: 1 H-NMR (CDCl3) δ [ppm]: 3.88 t, J HF (17 Hz), 2H; 2.96 t, J HF (17 Hz), 2H; 2.52 m, 4H; 2.5 m, 8H; 1.56 m, 4H; 1.2-1.5 m, 50 H; 0.88 m, 12H; 13 C-NMR (CDCl3) δ[ppm]: 120.29 t, J CF (246 Hz);55.08;39.33;36.72;32.85;32.39;29.74;29.52;29.49;29.47;29.22;28.91;28.87;27.20;26.51;25.53;22.97;14.09;10.79 19 F-NMR (CDCl3) δ[ppm] -107; MS (ESI) m / z (100%) (M+H) + .

[0426] 3-(bis(11-((2-ethylhexyl)thio)undecyl)amino)prop-1-ol: (BNT-74) 1 H-NMR (CDCl3) δ [ppm]: 3.79 m, 2H; 2.72 m, 2H; 2.47 m 8H; 2.41m, 4H; 1.68 m, 2H; 1.56 m, 4H; 1.2-1.5 m, 51H; 0.9 m, 12H; 13 C-NMR (CDCl3) δ [ppm]:]39.39;39.37;36.76;32.90;32.43;29.80;29.61;29.58;29.53;29.28;28.97;28.91;27.74;27.54;26.82;25.57;23.01;14.15 (Me);10.84 (Me);MS (APCI) m / z (100%):672.6125 (M+ H) + .

[0427] 4-(bis(11-((2-ethylhexyl)thio)undecyl)amino)but-1-ol (BNT-75) 1 H-NMR (CDCl3) δ [ppm]: 3.56 m, 2H; 2.47 m, 14H; 1.67 m, 4H; 1.57m, 6H; 1.2-1.5 m, 56H; 0.88 m, 12H; MS (APCI) m / z: 686.6267 (M+H) + .

[0428] 2-(bis(9-((2-butyloctyl)thio)nonyl)amino)ethane-1-ol) (BNT-24) 1 H-NMR (CDCl3) δ [ppm] 3.68 m, 2H; 2.61 m, 2H; 2.48 m, 8H; 1.48-1.6 m, 6H; 1.2-1.47 m, 60H; 0.89 m, 12 H; 13 C-NMR (CDCl3) δ [ppm] 58.18;55.55;53.82;37.93;37.23;33.20;32.89;31.86;29.76;29.62 ;29.50;29.22;28.91;28.85;27.38;26.94;26.58;22.99;22.66;14.11 (Me);14.10 (Me) MS (APCI) m / z: 714.6787(M) + .

[0429] 3-(bis(9-((2-butyloctyl)thio)nonyl)amino)prop-1-ol (BNT-71) 1 H-NMR (CDCl3) δ [ppm] 3.82 m, 2H; 2.63 m, 2H; 2.48 m, 8H; 2.4 m, 4H; 1.67 m, 2H; 1.4-1.6 m, 10H; 1.2-1.4 m, 52H; 0.88 m, 12 H; 13C-NMR (CDCl3) δ[ppm] 54.10;37.93;37.23;33.19;32.88;31.85;29.76;29.60;29.50;29.47;2 9.21;28.90;28.84;27.69;27.46;26.57;22.98;22.65;14.09;14.08;MS (APCI) m / z (100%):728.6767 (M) + .

[0430] General procedure for synthesizing thiolipin from thioether alcohols and amines Synthesis of 11-((2-ethylhexyl)thio)undecyl ester of propen-2-ene-1-sulfonic acid Allyl sulfonyl chloride (1.2 equivalents, MW: 139.97; d: 1.31 mg / mL; 650 µL, 6 mmol) was added to 11-((2-ethylhexyl)thio)undecyl-1-ol (1 equivalent, 5 mmol, 1582 mg), dichloromethane (4 mL), and pyridine (1 mL). The reaction mixture was magnetically stirred at room temperature and turned dark during this period. The reaction mixture was poured into ice-water (50 mL). The product was extracted from the resulting emulsion with chloroform (30 mL). The dark extract was treated with activated charcoal and then dried with sodium sulfate. After filtration through diatomaceous earth 545, the filtrate was evaporated to a black solid and a yellow oil. Only the latter was soluble in n-hexane / EtOAc: 9 / 1, v / v, which could be purified using the same solvent mixture.

[0431] 1 H-NMR (CDCl3) δ [ppm] 5.90 m, J (17.4 Hz, 10.3 Hz, 7.2H z) 1H; 5.45 m, 2H); 4.22 t, J (6.6 Hz Hz); 3.82 d, J (6.6 Hz); 2.47 m, 4H; 1.71 m, 2H; 1.56 m, 2H; 1.2-1.5 m, 23H; 0.88 m, 6H; 13C-NMR (CDCl3) δ [ppm] 142.60, 142.38, 70.93,54.83, 39.37, 36.77, 32.89, 32.43, 29.77, 29.45, 29.25, 29.02, 28.94, 28.91,25.57, 25.40, 23.01, 14.14, 10.83; TLC: Rf: 3.13 (Hexane / EtOAc: 9 / 1, v / v); MS(APCI) m / z (100%): 421.2805 (M+H) + .

[0432] 11-((2-ethylhexyl)thio)undecyl methanesulfonate At 0 °C, dichloromethane (5 mL), triethylamine (3.04 equivalents, 5.3 mL, 38.0 mmol), and methanesulfonic anhydride (1.53 equivalents, 3.2 g, 18.7 mmol) were added to 11-((2-ethylhexyl)thio)undecyl-1-ol (1 equivalent, 12.5 mmol, 4 g). The reaction mixture was magnetically stirred while simultaneously heating to room temperature for 2 hours. The reaction mixture was quenched with saturated aqueous NaHCO3 solution (50 mL), extracted with chloroform (3 × 30 mL), and dried over sodium sulfate. After filtration, the sulfonate ester could be used without further purification.

[0433] MS (APCI) m / z (100%): 395.2650 (M+H) + .

[0434] Replacement of sulfonate groups with amines: 1-(11-((2-ethylhexyl)thio)undecyl)azacycloheptane One equivalent of 1-propen-1-sulfonic acid 11-((2-ethylhexyl)thio)undecyl ester (MW: 420.71, 102 mg, 0.24 mmol) was heated with hexamethyleneimine (14.8 equivalents, 400 µL, 3.55 mmol) at 80 °C for 24 hours. The resulting biphasic reaction mixture was absorbed into chloroform and washed twice with saturated sodium bicarbonate solution. The latter was dried over sodium sulfate. After salt removal, the residual hexamethyleneimine was co-evaporated four times with toluene. The resulting oil was subjected to chromatography with n-hexane / ethyl acetate / TEA to provide the oil.

[0435] 1H-NMR (CDCl3) δ [ppm]: 2.77 m, 4H; 2.56 m, 2H; 2.47 m, 4H; 1.97 m, 4H; 1.72 m 4 H; 1.59 m, 3H; 1.2-1.5 m, 24 H; 0.87 2 t J (11 Hz), 6H; 13 C-NMR (CDCl3) δ[ppm]: 58.17; 55.20; 39.34; 36.74; 32.86; 32.40; 29.76; 29.52; 29.49; 29.44; 28.93; 28.88; 27.44; 26.99; 26.65: 26.55;25.54;22.98;14.11;10.80;MS (ESI) m / z (100%):398.3809 (M + H) + .

[0436] N-(cyclopentylmethyl)-11-((2-ethylhexyl)thio)undecyl-1-amine One equivalent of 1-((2-ethylhexyl)thio)undecyl methanesulfonate (MW: 394.67, 788 mg, 2 mmol) was heated to 60 °C for 24 hours with DIPEA (1.5 equivalent, 538 mg, 3 mmol) and cyclopentylmethylamine (1.05 equivalent, 208 mg, 2.1 mmol). The resulting reaction mixture was purified on a silica 60 column using a hexane / EtOAc 100-80 / 0-20 (v / v) gradient, followed by a hexane / EtOAc / TEA 80-76 / 20-19 / 0-5 (v / v / v) gradient. The product fractions were combined.

[0437] 1 H-NMR (CDCl3) δ [ppm]: 2.61 t, J(7 Hz), 2H; 2.55 d, J(7 Hz), 2H; 2.48m, 4H; 2.03 p, J(7 Hz), 1H; 1.78 m, 2H; 1.60 m, 5H; 1.5 m, 4H; 1.21-1.42 m 24H; 1.17 m, 2H; 0.82 2 t; 6H; 13C-NMR (CDCl3) δ [ppm]: 55.93, 50.35;40.028.91;27.42;25.57;28.91;7;39.37;36.77;32.90;32.44;30.59;30.11;2 9.80; 29.60; 29.56; 29.53; 29.28; 28.97; 217.42; 25.57; 25.32; 23.02; 14.14; 10.84; MS (ESI) m / z (100%):398.3809 (M + H) + .

[0438] The following compounds were prepared using methods similar to those described above.

[0439] MS (APCI) m / z (100%): 461.3921 (M + H) + Chemical formula: C29H52N2S Precision mass: 460.3851 Molecular weight: 460.8090 Preparation of bis(11-((2-ethylhexyl)thio)undecyl)amine via ammonium chloride and Na2SO4 A suspension of 11-((2-ethylhexyl)thio)undecaldehyde (2 equivalents; 618 mg; 2 mmol), ammonium chloride (1 equivalent, 1 mmol, 53.5 mg), anhydrous sodium sulfate (50 mg), and diisopropylethylamine (1 equivalent, 1 mmol, 170 µL) in dichloromethane (5 mL) was magnetically stirred at room temperature for 30 min. Triacetoxyborohydride (4 equivalents, 847 mg, 4 mmol) was added fractionally over 30 min under nitrogen cover. The reaction mixture was stirred at room temperature for 12 hours and then quenched by adding saturated sodium bicarbonate aqueous solution (20 mL). The product was extracted with chloroform (2 × 20 mL). The organic phase was washed with brine and then dried over anhydrous sodium sulfate. After salt removal, the crude product containing primary, secondary, and tertiary amines was purified by chromatography.

[0440] Preparation of bis(11-((2-ethylhexyl)thio)undecyl)amine via ammonium chloride and tetraisopropoxytitanium A suspension of 11-((2-ethylhexyl)thio)undecaldehyde (2 equivalents; 3 g; 9.7 mmol), ammonium chloride (1 equivalent, 19.4 mmol, 1039 mg), tetraisopropoxytitanium (4 equivalents, 19.4 mmol, 5.51 g, 5.74 mL), and triethylamine (2 equivalents, 19.4 mmol, 1.96 g, 2.7 mL) in anhydrous ethanol (40 mL) was magnetically stirred under nitrogen at room temperature for 30 min. Sodium borohydride (2.25 equivalents, 21.82 mmol, 0.825 g) was added under nitrogen cover. After stirring the reaction mixture at room temperature for 12 hours, it was quenched by adding an aqueous ammonia solution (35%, 50 mL). The reaction mixture was extracted with diethyl ether (2 × 100 mL).

[0441] The combined organic phases were washed with brine (2 × 100 mL) and Na₂SO₄. After salt removal, the crude product containing primary, secondary, and tertiary amines was purified by chromatography.

[0442] Major product: MS (ESI) m / z (100%): 630.5654 (M + O + H) + Retention time: 35.02 min HPLC Synthesis and oxidation of various sulfur-containing lipid fragments 2-Butyloctyl mesylate A solution of 2-butyloctyl-1-ol (10.0 g, 53.6 mmol, 1.00 equivalent) and triethylamine (16.2 g, 160 mmol, 3.00 equivalent) in 100 mL DCM was stirred under argon at 0 °C. Methanesulfonic anhydride (14.0 g, 80.4 mmol, 1.5 equivalent) was added fractionally, and the reaction mixture was heated and stirred at room temperature for 2 h. The reaction progress was monitored by TLC using a solvent mixture (n-hexane / EtOAc; 4:1). The reaction mixture was neutralized with (200 mL) NaHCO3, the organic layer was separated, and the aqueous layer was further extracted with (2 × 100 mL) DCM. The combined organic phases were washed with (2 × 100 mL) brine, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was used in the next step without any further purification.

[0443] 2-Butyloctyl ethanethioacetic acid A solution of 2-butyloctyl methanesulfonate (5.00 g, 20.5 mmol, 1.00 equivalent) in 10 mL of DMF was stirred under argon at room temperature. Potassium thioacetate (7.00 g, 61.5 mmol, 3.00 equivalent) was added fractionally, and the reaction mixture was stirred under argon at 85 °C for 12 h. The resulting reaction mixture was then diluted with (200 mL) EtOAc and washed with (100 mL) 1 MHCL, (100 mL) NaHCO3, (100 mL) H2O, and (2 × 100 mL) brine. The organic phase was collected, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (SiO2; n-hexane / EtOAc = 95 / 5).

[0444] 2-Butyloctane-1-thiol A solution of NaOH (2.00 g, 50.0 mmol, 2.00 equivalent) in 2.5 mL of H₂O was added dropwise to a solution of 2-butyloctyl ethanethioacetate (5.00 g, 24.7 mmol, 1.00 equivalent) in 20 mL of EtOH. The reaction mixture was stirred at 40 °C for 2 hours. The reaction progress was monitored by TLC using a solvent mixture (n-hexane). The reaction mixture was neutralized with 2 M HCl at 0 °C, diluted with (100 mL) diethyl ether, and washed with (2 × 100 mL) H₂O and (2 × 100 mL) brine. The organic layer was collected, dried over Na₂SO₄, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (SiO₂; n-hexane).

[0445] 9-((2-Butyloctyl)thio)non-1-ol In a three-necked round-bottom flask, 60 mL of MeOH was added under argon. Then, 60% NaH (780 mg, 19.7 mmol, 1.00 equivalent) was added fractionally over 10 minutes at 0 °C under argon. Next, 2-Butyloctane-1-thiol (4.00 g, 19.7 mmol, 1.00 equivalent) was added under argon at room temperature, and the reaction mixture was stirred for 10 minutes. Subsequently, 9-bromonon-1-ol (4.40 g, 19.7 mmol, 1.00 equivalent) was added under argon, and the reaction mixture was stirred at 65 °C for 3 hours. The solvent was evaporated under reduced pressure, the mixture was diluted with (200 mL) n-hexane, the NaH was filtered off, and the filtrate was collected and washed with (2 × 100 mL) brine. The organic layer was collected, dried over Na₂SO₄, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (SiO₂; n-hexane / EtOAc; 4 / 1).

[0446] 9-((2-Butyloctyl)sulfonyl)non-1-ol A solution of 9-((2-butyloctyl)thio)non-1-ol (4.00 g, 11.5 mmol, 1.00 equivalent) in 10 mL of ice-cold AcOH was stirred at room temperature. 30% H₂O₂ (2.00 mL, 92.0 mmol, 8.00 equivalent) was added dropwise to the reaction mixture, followed by the addition of Na₂WO₄·2H₂O (5%, as a catalyst), and the reaction mixture was stirred at 50 °C for 2 hours. The reaction mixture was neutralized at 0 °C with (10 mL) NaHCO₃, diluted with (100 mL) EtOAc, washed with (2 × 50 mL) H₂O, and then washed with (2 × 50 mL) brine. The organic layer was collected, dried over Na₂SO₄, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (SiO₂; n-hexane / EtOAc; 1 / 1).

[0447] 9-((2-Butyloctyl)sulfonyl)nonanal A solution of 9-((2-butyloctyl)sulfonyl)non-1-ol (3.00 g, 8.00 mmol, 1.00 equivalent) in 10 mL of DCM was stirred at room temperature. DMP (5.00 g, 12.0 mmol, 1.50 equivalent) was added to the reaction mixture, followed by dropwise addition of H2O (159 µl, 8.80 mmol, 1.10 equivalent) over 30 minutes. The reaction was quenched with 10 mL of H2O, diluted with (100 mL) DCM, and washed with (2 × 100 mL) NaHCO3 and (2 × 100 mL) brine. The organic layer was collected, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (SiO2; n-hexane / EtOAc; 4 / 1).

[0448] BNT-72, BNT-73 and BNT-76 A solution of 9-((2-butyloctyl)sulfonyl)nonanal (2.00 equivalents) and NH2-R (1.00 equivalents) in 5 ml DCM was stirred under argon at room temperature for 30 min. Then, NaBH(OAc)3 (4.00 equivalents) was added to the reaction mixture under argon, and the reaction mixture was stirred at room temperature for another 12 h. The reaction mixture was diluted with (50 ml) DCM and washed with (3 × 50 ml) NaHCO3 and (2 × 50 ml) brine. The organic layer was collected, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (SiO2; n-hexane / EtOAc / TEA; 4% / 1% / 1% to n-hexane / EtOAc / TEA; 1% / 1% / 1%, for BNT-72, BNT-73, and BNT-76).

[0449] Synthesis of certain sulfonamide lipids Octylhexylamine (CAS: 82223-69-6; CAS: 86933-36-0 (HCl salt)) is available from Enamine US Inc., Aldlab Chemicals, LLC and Bridgewater Pharmtech, Inc. 7-Octen-1-sulfonyl chloride (CAS: 923279-52-1) is available from Hong Kong Chemhere Co., Ltd. or otherwise prepared according to Kirschberg, Thorsten A. et al., Bioorganic & Medicinal Chemistry Letters (2014), 24(3), 969-972. Specifically, 7-octen-1-sulfonyl chloride can be prepared in high yield from the acid obtained by treatment of 7-bromo-1-octene with phosphoryl chloride using sodium sulfite.

[0450] 7-Octen-1-sulfonyl chloride (CAS: 923279-52-1) was added to a solution of octylhexylamine (CAS: 82223-69-6) in THF and DEA.

[0451] The terminal olefin (compound 3) is converted into aldehydes via ozone decomposition to provide compound 4.

[0452] The general procedure for synthesizing sulfonamides from terminal aldehydes is carried out in a manner similar to that reported in WO2021250102.

[0453] Ozone was added to a solution of 7-octenesulfonamide dissolved in a 1 / 1 mixture of DCM / MeOH at -78°C. It was allowed to bubble through the reaction mixture until the blue color persisted. Then, N2 was bubbled through the solution to remove excess ozone (removal of the blue color), followed by the addition of triphenylphosphine (Ph3P, 5 equivalents). The reaction mixture was then slowly heated to room temperature over a 1-hour period. The resulting heterogeneous mixture was filtered through a diatomaceous earth pad. The pad was washed with DCM. The filtrate was concentrated under reduced pressure and purified by rapid column chromatography.

[0454] An aldehyde compound (e.g., compound 4 in the above scheme) is reacted with a primary amine with a head group in a DCM at room temperature in the presence of Na[HB(OAc)3] and Na2SO4 to obtain a protected precursor of a lipid or final lipid with a functional head group.

[0455] The compounds described herein can be prepared according to the following scheme: BNT51 reacts 4-amino-butanol (CAS: 13325-10-5) with the aldehyde of compound 4. The remaining head group in the above scheme can be prepared using the following commercially available reagents: CAS: 127346-48-9: N-tert-butoxycarbonyl-1,3-diaminopropane hydrochloride CAS: 75178-96-0: tert-butyl 3-aminopropylcarbamate CAS: 68076-36-8: tert-butyl 4-aminobutylcarbamate CAS: 33545-98-1: N-tert-butoxycarbonyl-1,4-diaminobutane hydrochloride BOC-derived lipids can be deprotected using TFA. After removing the protecting group, the terminal amino group is deprotonated to provide thiourea. The amino group is then acylated with a thioacyl halide in the presence of DMAP and TEA.

[0456] Use the following reagents: CAS: 16420-13-6: Dimethylthiocarbamoyl chloride, commercially available.

[0457] CAS: 2241238-65-1: 1-Acetidine thiocyanate chloride, prepared according to the method described in WO2018140730 CAS: 19009-42-8: 1-pyrrolidine thiocyanate chloride, commercially available.

[0458] CAS: 16420-13-6: Dimethylthiocarbamoyl chloride, commercially available.

[0459] CAS: 2241238-65-1: 1-Acetidine thioyl chloride, prepared according to the method described in WO2018140730.

[0460] CAS: 19009-42-8: 1-pyrrolidine thiocyanate chloride, commercially available.

[0461] The sulfur lipid compounds disclosed herein can be prepared according to methods similar to those known to those skilled in the art.

[0462] Example 2: Manufacturability and in vitro studies of LNP formulations composed of novel ionizable lipids BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59 This embodiment describes a method for preparing lipid nanoparticles (LNPs) using specific sulfur lipid compounds described herein (e.g., BNT-51, BNT-52, BNT-54, BNT-56, BNT-57, or BNT-59), and their in vitro biological effects. The LNPs of this embodiment were prepared by microfluidic mixing of a lipid mixture in ethanol and an RNA solution in an acidic aqueous buffer. Details of the formulation are given in the table below.

[0463] LNP size was analyzed using DLS. Figure 1 It was found that all formulations had hydrodynamic diameters less than 111 nm, except for BNT-56. The formulations exhibited narrow size distributions (PDI ≤ 0.2). Lipids with uniform sulfonamide tails (tail 1: BNT-51, BNT-52, and BNT-54; tail 2: BNT-56, BNT-57, and BNT-59) showed slightly different sizes due to different head groups. Lipids with thiourea head groups (BNT-52 and BNT-54 from tail 1; BNT-57 and BNT-59 from tail 2) produced smaller formulations compared to lipids with hydroxyl head groups (BNT-51 from tail 1, and BNT-56 from tail 2). For both tail groups: the hydroxyl-containing thiourea head (BNT-54 from tail 1; BNT-59 from tail 2) showed the lowest size in their respective groups. Tail base 1 shows a smaller size compared to its counterpart tail base 2, indicating that the branching location and branching length affect the hydrodynamic diameter.

[0464] The zeta potential of the formulation was studied by electrophoretic light scattering. Figure 2 The formulation exhibits neutral to negative zeta potential values.

[0465] The mRNA encapsulation efficiency of the study formulation was determined using Ribogreen assays. Figure 3AAll formulations except BNT-56 showed extremely high encapsulation efficiency (≥87%). No free mRNA was observed, as indicated by AGE. Figure 3B ).

[0466] The integrity of the formulation's mRNA was determined by capillary electrophoresis and fragment analysis. Figure 4 ).like Figure 4 As can be seen, negligible amounts of mRNA fragmentation were observed for all formulations.

[0467] The cytotoxicity and transfection efficiency of the formulation were tested on three different cell lines (C2C12, HepG2, and RAW 264.7) at three different mRNA concentrations (12.5 ng, 25 ng, and 50 ng). Figures 5A-5C and Figures 6A-6C The formulations did not show significant cytotoxicity under the test conditions. All formulations were able to effectively transfect the tested cell lines. Figures 6A-6C BNT-51 showed the highest transfection efficiency at all concentrations on all tested cell lines.

[0468] Example 3: Manufacturability and in vitro studies of LNP formulations composed of ionizable lipids BNT-sulfon-01, BNT-sulfon-02, BNT-sulfon-03 or BNT-sulfon-04.

[0469] This example illustrates the manufacturability and in vitro biological effects of LNPs composed of the following lipids: BNT-sulfon-O1, BNT-sulfon-O2, BNT-sulfon-O3, or BNT-sulfon-O4. BNT-51 and BNT-52 have also been formulated for use in this example. The LNPs of this example were prepared by microfluidic mixing of the lipid mixture in ethanol and an RNA solution in an acidic aqueous buffer. Details of the formulation are given in the table below.

[0470] LNP size was analyzed using DLS. Figure 7 It was found that the hydrodynamic diameter of all formulations was less than 100 nm, except for BNT-sulfonyl-04. PDI values ​​≤ 0.2 were found, indicating a narrow size distribution. The cyclic amine head groups (BNT-sulfonyl-02 and BNT-sulfonyl-03) were adequately adapted and did not significantly alter the size and size distribution compared to the parent lipid BNT-52. BNT-sulfonyl-01 produced smaller sizes (55 nm and 79 nm, respectively) compared to BNT-52.

[0471] The zeta potential of the formulation was studied by electrophoretic light scattering. Figure 8 All formulations showed very similar (neutral to negative) zeta potential values.

[0472] The mRNA encapsulation efficiency of the study formulation was determined using Ribogreen assays. Figure 9A The formulation effectively encapsulates mRNA (encapsulation efficiency ≥ 84%). No free mRNA was observed, as shown by AGE assay. Figure 9B ).

[0473] The integrity of the formulation's mRNA was determined by capillary electrophoresis and fragment analysis. Figure 10 ).like Figure 10 As can be seen, negligible amounts of mRNA fragmentation were observed for all formulations.

[0474] The cytotoxicity and transfection efficiency of the formulation were tested on four different cell lines (C2C12, HepG2, RAW 264.7, and Hek 293) at three different mRNA concentrations (12.5 ng, 25 ng, and 50 ng). Figures 11A-11D and Figures 12A-12D Generally, the formulation did not show significant cytotoxicity under the test conditions. The formulation was able to effectively transfect the tested cell lines. Figures 12A-12D BNT-51 showed the highest in vitro transfection efficiency at all concentrations on all tested cell lines. BNT-sulfon-04 showed the lowest transfection efficiency, most likely attributable to reduced efficacy due to the short tail. BNT-52 showed slightly better expression than BNT-sulfon-01. Formulations with cyclic amine head groups (BNT-sulfon-02 and BNT-sulfon-03) showed similar in vitro performance to the parent lipid BNT-52.

[0475] The in vitro hemolytic effects of BNT-sulfonyl-O2 and BNT-sulfonyl-O3 were investigated at an mRNA dose of 16 mg / kg (in vivo matrix, the mRNA dose was equal to 16 mg / kg). Figure 13A Even at extremely high mRNA doses, the formulation showed negligible hemolysis, indicating that it does not affect the integrity of red blood cell membranes (PBS as a negative control, Triton (0%–2%) as a positive control). Additionally, the effect of the formulation on complement activation has been determined. Figure 13B ).

[0476] Example 4: In vitro study of the effects of auxiliary lipids on LNP formulations composed of novel ionizable lipid BNT-51 This embodiment investigates the manufacturability and in vitro biological effects of LNPs composed of BNT-51 and two different accessory lipids (DSPC or DOPE). Specifically, in this embodiment, LNPs with the same composition but different accessory lipids were prepared, characterized, and tested in vitro. The LNPs were prepared by microfluidically mixing a lipid mixture in ethanol with an RNA solution in an acidic aqueous buffer. Details of the formulation are given in the table below.

[0477] LNP size was analyzed using DLS. Figure 14 The hydrodynamic diameter of the formulation with DSPC was found to be higher than that of the BNT-51 formulation with DOPE (106 nm and 72 nm, respectively). However, BNT-51_DOPE has shown a higher PDI (0.28) than BNT-51_DSPC (0.13).

[0478] The zeta potential of the formulation was studied by electrophoretic light scattering. Figure 15 The auxiliary lipids do not significantly affect the zeta potential value. The formulation exhibits a neutral zeta potential.

[0479] The mRNA encapsulation efficiency of the study formulation was determined using Ribogreen assays. Figure 16A Both formulations exhibited extremely high encapsulation efficiency (≥90%) regardless of the type of adjuvant lipids. High encapsulation efficiency has also been confirmed by AGEs. Figure 16B ).

[0480] The integrity of the formulation's mRNA was determined by capillary electrophoresis and fragment analysis. Figure 17 ).like Figure 17 As can be seen, no mRNA fragmentation was observed in the formulation.

[0481] The cytotoxicity and transfection efficiency of the formulation were tested on four different cell lines (C2C12, HepG2, RAW 264.7, and HEK-293) at three different mRNA concentrations (12.5 ng, 25 ng, and 50 ng). Figures 18A-18D and Figures 19A-19D The formulations did not significantly alter cell viability under the test conditions (no cytotoxicity). Luciferase expression levels showed that all formulations were able to effectively transfect the test cell lines (RLU approximately 10). 6 - 10 8 ).

[0482] Example 5: In vitro study of the effects of stealth lipids and stealth lipid anchors on LNP formulations composed of novel ionizable lipid BNT-51. This report describes the manufacturability and in vitro biological effects of LNPs composed of the same novel ionizable lipid (BNT-51) but different stealth lipids (PEG-DMG, VE-(AEEA)14_AC_2, or DMG-(AEEA)14_AC_2). For this purpose, LNPs with the same composition but different stealth lipids were prepared, characterized, and tested in vitro. The LNPs were prepared by microfluidic mixing of the lipid mixture in ethanol and an RNA solution in an acidic aqueous buffer. Details of the formulation are given in the table below.

[0483] The size of LNP was studied using DLS. Figure 20 It is possible to obtain well-defined formulations with a diameter ≤ 100 nm and a PDI ≤ 0.2 (narrow polydispersity). The hydrodynamic diameter and PDI of the formulation are substantially similar.

[0484] The zeta potential of the formulation was studied by electrophoretic light scattering. Figure 21 All formulations showed very similar neutral to slightly negative zeta potential values.

[0485] The mRNA encapsulation efficiency of the formulation was assessed using the Ribogreen assay. Figure 22A Formulations with the same hidden lipid anchor (BNT-51_PEG-DMG and BNT_51_DMG-AEEA) showed similar levels of encapsulation efficiency (82% and 88%, respectively). However, the formulation with a vitamin E lipid anchor (BNT-51_VitE-AEEA) showed lower encapsulation efficiency, indicating that the hidden lipid anchor, rather than the hidden lipid itself, has an effect on mRNA encapsulation efficiency. To determine the amount of free mRNA, agarose gel electrophoresis (AGE) was performed. As can be seen from the electrophoresis results, no free mRNA was observed in either the BNT-51_PEG-DMG or BNT_51_DMG-AEEA formulations. Figure 22B BNT-51_VitE-AEEA showed negligible amounts of free mRNA (8.5%, semi-quantitative by ImageJ analysis).

[0486] The integrity of the formulation's mRNA was determined by capillary electrophoresis and fragment analysis. Figure 23 As shown in the figure, no mRNA fragmentation was observed in the formulation.

[0487] The cytotoxicity and transfection efficiency of the formulation were tested on four different cell lines (C2C12, HepG2, RAW 264.7, and HEK-293) at three different mRNA concentrations (12.5 ng, 25 ng, and 50 ng). Figures 24A-24D and Figures 25A-25DThe formulations did not show any cytotoxicity under the test conditions. Luciferase expression levels showed that all formulations were able to effectively transfect the test cell lines (RLU approximately 10). 6 - 10 8 ) ( Figures 25A-25D Dose- and cell line-dependent differences in transfection efficiency were observed. Generally, BNT-51 formulations with a DMG hidden lipid anchor (BNT-51_PEG-DMG and BNT_51_DMG-AEEA formulations) showed slightly higher transfection efficiency than BNT-51 formulations with a vitamin E lipid anchor (BNT-51_VitE-AEEA).

[0488] Example 6: Preparation and characterization of RNA / DNA-lipid nanoparticles (BNT51 / Ac-AEEA14-DMA / DSPE-PEG2k-α-tagged lipids) Preformed lipid nanoparticles (BNT51 / Ac-AEEA14-DMA / DSPE-PEG2k-α) were fabricated via ethanol injection. (Lipids) Pre-formed α-tagged lipid nanoparticles were prepared using a lipid mixture consisting of BNT51, DSPC, cholesterol, Ac-AEEA14-DMA, and DSPE-PEG2k-α peptide (a DSPE-PEG2K polymer functionalized with an α-peptide having the sequence of SEQ.ID.NO. 1) in a molar ratio of 47.5:10:40.5:1.8:0.2. The lipid mixture was dissolved in an organic solvent (ethanol) at a total lipid concentration of 20 mM and mixed with an aqueous phase (5 mM AcOH) at a total flow rate of 90 mL / min (45 mL / min for the nucleic acid phase, 15 mL / min for the lipid phase, and 30 mL / min for online dilution) at a volume ratio of 3:1 (aqueous phase:organic phase). Organic solvents in the obtained pristine colloidal nanoparticles were removed by dialysis against 5 mM AcOH using a Slide-A-Lyzer dialysis kit (Thermo Fisher Scientific, Waltham, MA, USA) with a molecular weight cutoff value (MCWO) of 10K. The nanoparticles were then concentrated to a 2-fold concentration using an Amico Ultra-15 dialysis unit (Merck, Darmstadt, Germany) with a molecular weight cutoff value (MCWO) of 30K, by centrifugation at 3000 rpm × 10 min at 4 °C. After concentration, the nanoparticles were diluted to a 10% sucrose concentration.

[0489] Freeze-drying of pre-formed lipid (BNT51 / Ac-AEEA14-DMA / DSPE-PEG2k-α-tagged lipid) nanoparticles Qualitative The freeze stability of preformed lipid nanoparticles was evaluated by cycling the formulation from -20°C and -80°C (overnight) to +25°C (2 hours) at least three times. Particle size and polydispersity index of the formulation were measured on the freeze-thawed samples. The formulation was gently inverted and mixed between the freeze and thaw cycles before the next freezing cycle. Colloidal stability of the nanoparticles was observed to be maintained at at least three freeze-thaw cycles at -20°C and -80°C. Figure 26 As depicted in the text.

[0490] Manufacturing functionalized RNA / DNA using the aqueous-aqueous protocol (LNP2) lipid particles RNA / DNA-lipid particles were prepared using an aqueous-aqueous protocol (LNP2) as described herein. Reporter gene Thy1.1 RNA and reporter gene Venus DNA were used for characterization experiments. Briefly, RNA (Thy1.1) in aqueous buffer (HEPES 10 mM, EDTA 0.1 mM, pH 7.0) and DNA (Venus) in water were mixed with preformed lipid nanoparticles at a 1:1 volume ratio in an aqueous solution (5 mM acetic acid, 10% sucrose). The preformed lipid nanoparticles consisted of BNT51, DSPC, cholesterol, Ac-AEEA14-DMA, and DSPE-PEG2k-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2. Mixing was achieved using a T-mixing element (2.4 mm T-mixer, 1.6 mm tube) at a total flow rate of 360 ml / min (180 ml / min per phase) based on a standard syringe pump. The resulting RNA / DNA-lipid particles were then functionalized with aCD3-VHH ligand at a ligand / cargo ratio of 0.48 w / w and further diluted to a final nucleic acid concentration of 0.1 mg / mL with 60 mM HEPES (pH 6.0) and 20% (w / v) sucrose (final buffer composition: 22 mM HEPES, 9% sucrose, pH approximately 5). RNA / DNA-lipid particles were prepared at an N:P ratio of 6:1 and filtered through a 0.22 µm polyethersulfone (PES) filter.

[0491] Manufacturing functionalized RNA / DNA-lipid particles using an aqueous-organic approach (LNP1) Alternatively, RNA / DNA-lipid particles were prepared using an aqueous-ethanol mixing protocol (LNP1), as described herein. Reporter gene Thy1.1 RNA and reporter gene Venus DNA were also used for characterization experiments. Briefly, RNA (Thy1.1) in an aqueous buffer (HEPES 10 mM, EDTA 0.1 mM, pH 7.0) and DNA (Venus) in water were mixed with an ethanol-lipid mixture at a total lipid concentration of 14.4 mM, at a volume ratio of 3 parts nucleic acid to 1 part lipid mixture, wherein the ethanol-lipid mixture contained BNT51, DSPC, cholesterol, Ac-AEEA14-DMA, and DSPE-PEG2k-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2. Mixing was achieved using a T-mixing element (0.5 mm T-mixer) with a standard syringe pump-based device equipped with online dilution and a total flow rate of 90 ml / min (45 ml / min for nucleic acid phase, 15 ml / min for lipid phase, and 30 ml / min for online dilution). Organic solvents in the obtained pristine colloidal nanoparticles were removed by dialysis against 20 mM HEPES (pH 5.5) using a Slide-A-Lyzer dialysis cartridge G2 (ThermoFisher Scientific, Waltham, MA, USA) with a molecular weight cutoff (MCWO) of 10K. The resulting RNA / DNA-lipid particles were then functionalized with aCD3-VHH ligand at a ligand / cargo ratio of 0.48 w / w and further diluted to a final nucleic acid concentration of 0.1 mg / mL and a storage matrix of 20 mM HEPES (pH 5.5) and 10% (w / v) sucrose. Typically, RNA / DNA-lipid particles are prepared at an N:P ratio of 6:1 and filtered through a 0.22µm polyethersulfone (PES) filter.

[0492] Characterization of formulation Particle size was determined by dynamic light scattering using a DynaPro plate reader II (Wyatt, Dernbach, Germany). Based on the measurements, size (Z-mean) and polydispersity index (PDI) were calculated using Dynamics 7.8.1.3 software based on cumulative analysis. For the measurements, samples were diluted 1:10 in water and analyzed in triplicate. The pH was measured on 150 μL samples after calibrating the apparatus with standards at pH 4, 7, and 10. Successful inclusions were verified by agarose gel electrophoresis.

[0493] The size and PDI of functionalized, sterile-filtered RNA / DNA-LNP1 and RNA / DNA-LNP2 (prepared as described above) stored at a final nucleic acid concentration of 0.1 mg / mL were determined by DLS measurements. It was observed that aCD3-VHH-functionalized RNA / DNA-LNP1 and RNA / DNA-LNP2 could be successfully fabricated using the Ac-AEEA14-DMA recessive moiety, resulting in particles <100 nm in size and with a PDI ≤ 0.3. Figure 27A The inclusion of the cargo was successfully verified by agarose gel electrophoresis: no RNA or DNA bands were present in the sample, indicating that the formulation contained no free cargo. Additionally, when the sample was treated with the release solution (control), all corresponding RNA and DNA bands were clearly visible, confirming successful encapsulation of all cargo. Figure 27B ).

[0494] Stability and cold-chain properties of functionalized RNA / DNA-lipid nanoparticles (BNT51 / Ac-AEEA14-DMA / α-tagged lipids) Freeze-thaw study The stability of RNA / DNA functionalized nanoparticles was evaluated at 2°C, -8°C, and 25°C. Freeze-thaw studies were performed by cycling the formulation from -20°C and -80°C (overnight) to +25°C (2 hours) at least three times. Particle size and polydispersity index of the formulation were measured on the frozen-thawed samples. The formulation was thawed and mixed by gentle inversion between freezing and thawing cycles before the next freezing cycle.

[0495] Both LNP1 and LNP2 formulations showed freezing stability for up to three freeze-thaw cycles, such as Figure 27C As described in the study, for the functionalized RNA / DNA-LNP2, the colloidal stability of the nanoparticles was maintained at 2℃–8℃ and 25℃ (currently evaluated for 2 weeks), while for the functionalized RNA / DNA-LNP1, an increase in size was observed. Figure 27C ).

[0496] In vitro transfection studies For transfection studies, 1 µl or 10 μl (100 ng or 1000 ng dose) of the appropriate formulation was pre-diluted in 50 μL of X-Vivo 15 in an ultra-low adhesion 96-well plate. 0.3e6 thawed human T cells were diluted in 100% PHS and added to the nanoparticle dilution. After incubation for 30 min (37°C, 5% CO2), 100 µl of X-Vivo 15 containing 200 IU / ml IL-2 was added to each well, and the cells were cultured for another 96 h (37°C, 5% CO2). Thy1.1 RNA was analyzed by flow cytometry after the samples were labeled with cell type-specific antibodies. Figure 29 A) and Venus DNA ( Figure 29B) Cell type-specific transfection.

[0497] Figures 28A-28B This displays the percentage of transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) in all transfected PBMCs (transfected, y-axis) for RNA (Thy1.1) and DNA (Venus) under each test formulation condition (as indicated). The LNP2 sample showed higher RNA and DNA transfection rates compared to the LNP1 sample; expected variations depend on the donor and dosage used.

[0498] Example 7: Fabrication of RNA / DNA lipid nanoparticles (BNT51 / Ac-AEEA14-VitE / DSPE-PEG2k-α-tagged lipids) Preformed lipid nanoparticles (BNT51 / Ac-AEEA14-VitE / DSPE-PEG2k-α) were fabricated via ethanol injection. (Label lipids) To prepare α-tagged preformed lipid nanoparticles, a lipid mixture consisting of BNT51, DSPC, cholesterol, Ac-AEEA14-VitE, and DSPE-PEG2k-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2 was dissolved in an organic solvent (ethanol) at a total lipid concentration of 20 mM. The mixture was then mixed with an aqueous phase (5 mM AcOH) at a total flow rate of 90 mL / min (45 mL / min for the nucleic acid phase, 15 mL / min for the lipid phase, and 30 mL / min for online dilution) using a standard syringe pump-based device equipped with an online dilution and T-mixing element (0.5 mm T-mixer). The organic solvent in the obtained pristine colloidal nanoparticles was removed by dialysis against 5 mM AcOH using a Slide-A-Lyzer dialysis kit (Thermo Fisher Scientific, Waltham, MA, USA) with a molecular weight cutoff of 10K (MCWO). Using an Amico Ultra-15 (Merck, Darmstadt, Germany) with a molecular weight cutoff (MCWO) of 30K, the nanoparticles were concentrated to twice their original size by centrifugation at 3000 rpm for 10 minutes at 4°C. After up-concentration, the nanoparticles were diluted to a 10% sucrose concentration.

[0499] Freeze-drying of pre-formed lipid (BNT51 / Ac-AEEA14-VitE / DSPE-PEG2k-α-tagged lipid) nanoparticles Qualitative The freeze-thaw stability of preformed lipid nanoparticles was investigated by cycling the formulation from -20°C and -80°C (overnight) to +25°C (2 hours) at least three times. Particle size and polydispersity index of the formulation were measured on the freeze-thawed samples. The formulation was gently inverted and mixed between the freeze-thaw and freeze-thaw cycles before the next freezing cycle. Colloidal stability of the nanoparticles was observed to be maintained at at least three freeze-thaw cycles at -20°C and -80°C. Figure 29 As depicted in the text.

[0500] Manufacturing functionalized RNA / DNA-lipid particles using an aqueous-to-aqueous process RNA / DNA-lipid particles were prepared using an aqueous-aqueous protocol (LNP2) as described herein. Briefly, RNA (Thy1.1) in an aqueous buffer (HEPES 10 mM, EDTA 0.1 mM, pH 7.0) and DNA (Venus) in water were mixed with preformed lipid nanoparticles in an aqueous solution (5 mM acetic acid, 10% sucrose) at a 1:1 volume ratio. The preformed lipid nanoparticles consisted of BNT51, DSPC, cholesterol, Ac-AEEA14-VitE, and DSPE-PEG2k-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2. Mixing was achieved using a T-mixing element (2.4 mm T-mixer, 1.6 mm tube) at a total flow rate of 360 ml / min (180 ml / min per phase) based on a standard syringe pump. The resulting RNA / DNA-lipid particles were then functionalized with aCD3-VHH ligand at a ligand / cargo ratio of 0.48 w / w and further diluted to a final nucleic acid concentration of 0.1 mg / mL with 60 mM HEPES (pH 6.0) and 20% (w / v) sucrose (final buffer composition: 22 mM HEPES, 9% sucrose, pH approximately 5). RNA / DNA-lipid particles were prepared at an N:P ratio of 6:1 and filtered through a 0.22 µm polyethersulfone (PES) filter.

[0501] Manufacturing functionalized RNA / DNA-lipid particles using an aqueous-organic approach RNA / DNA-lipid particles were prepared using an aqueous-ethanol mixing protocol (LNP1), as described herein. Briefly, RNA (Thy1.1) in an aqueous buffer (HEPES 10 mM, EDTA 0.1 mM, pH 7.0) and DNA (Venus) in water were mixed with an ethanol-lipid mixture at a total lipid concentration of 14.4 mM, at a volume ratio of 3 parts nucleic acid to 1 part lipid mixture. The ethanol-lipid mixture contained BNT51, DSPC, cholesterol, Ac-AEEA14-VitE, and DSPE-PEG2k-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2. Mixing was achieved using a T-mixing element (0.5 mm T-mixer) with a standard syringe pump-based device equipped with online dilution and a total flow rate of 90 ml / min (45 ml / min for the nucleic acid phase, 15 ml / min for the lipid phase, and 30 ml / min for online dilution). Organic solvents in the obtained pristine colloidal nanoparticles were removed by dialysis with 20 mM HEPES (pH 5.5) using a Slide-A-Lyzer dialysis kit G2 (Thermo Fisher Scientific, Waltham, MA, USA) with a molecular weight cutoff value (MCWO) of 10K. The resulting RNA / DNA-lipid particles were then functionalized with aCD3-VHH ligand at a ligand / cargo ratio of 0.48 w / w and further diluted to a final nucleic acid concentration of 0.1 mg / mL and a storage matrix of 20 mM HEPES (pH 5.5) and 10% (w / v) sucrose. RNA / DNA-lipid particles were prepared at a 6:1 N / P ratio and filtered through a 0.22 µm polyethersulfone (PES) filter.

[0502] Characterization of formulation Particle size was determined by dynamic light scattering using a DynaPro plate reader II (Wyatt, Dernbach, Germany). Based on the measurements, size (Z-mean) and polydispersity index (PDI) were calculated using Dynamics 7.8.1.3 software based on cumulative analysis. For the measurements, samples were diluted 1:10 in water and analyzed in triplicate. The pH was measured on 150 μL samples after calibrating the apparatus with standards at pH 4, 7, and 10. Successful inclusions were verified by agarose gel electrophoresis.

[0503] The size and PDI of functionalized sterile filtered RNA / DNA-LNP1 and RNA / DNA-LNP2 stored at a final nucleic acid concentration of 0.1 mg / mL were determined by DLS measurements. It was observed that aCD3-VHH functionalized RNA / DNA-LNP1 and RNA / DNA-LNP2 could be successfully fabricated using the Ac-AEEA14-VitE recessive moiety, resulting in particles <100 nm in size and with a PDI ≤ 0.3. Figure 30A The inclusion of the cargo was successfully verified by agarose gel electrophoresis: no RNA or DNA bands were present in the sample, indicating that the formulation contained no free cargo. Additionally, when the sample was treated with the release solution (control), all corresponding RNA and DNA bands were clearly visible, confirming successful encapsulation of all cargo. Figure 30B ).

[0504] Functionalized RNA / DNA-lipid nanoparticles (BNT51 / Ac-AEEA14-VitE / DSPE-PEG2k-α-tagged lipids) Stability and freeze-thaw studies The stability of RNA / DNA functionalized nanoparticles was investigated at 2℃–8℃ and 25℃. Freeze-thaw studies were performed by cycling the formulation from -20℃ and -80℃ (overnight) to +25℃ (2 hours) at least three times. Particle size and polydispersity index of the formulations were measured on the frozen-thawed samples. The formulations were gently inverted and mixed between the thaw and freezing cycles before the next freezing cycle. Both LNP1 and LNP2 formulations showed freezing stability for up to three freeze-thaw cycles, as shown in the figure. Figure 7 As described in C. It was observed that for functionalized LNP2, the colloidal stability of the nanoparticles was maintained at 2℃–8℃ and 25℃ (currently evaluated over 2 weeks), while for functionalized LNP1, an increase in size was observed (…). Figure 30C ).

[0505] In vitro transfection studies For transfection studies, 1 µl or 10 µl (100 ng or 1000 ng dose) of the appropriate formulation was pre-diluted in 50 µl of X-Vivo 15 in an ultra-low adhesion 96-well plate. 0.3e6 thawed human T cells were diluted in 100% PHS and added to the nanoparticle dilution. After incubation for 30 min (37°C, 5% CO2), 100 µl of X-Vivo 15 containing 200 IU / ml IL-2 was added to each well, and the cells were cultured for another 96 h (37°C, 5% CO2). Cell type-specific transfections (Thy1.1 RNA and Venus DNA) were analyzed by flow cytometry after labeling the samples with cell type-specific antibodies.

[0506] Figures 31A-31BThis displays the percentage of transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) in all transfected PBMCs (transfected, y-axis) for RNA (Thy1.1) and DNA (Venus) under each test formulation condition (as indicated). The LNP2 sample showed higher RNA and DNA transfection rates compared to those observed in the LNP1 sample; the expected changes are related to the donor and dosage used.

[0507] Example 8: Fabrication of RNA / DNA lipid nanoparticles (BNT51 / Ac-AEEA14-DMA / DSPE-pAEEA14-α-tagged lipids) Preformed lipid nanoparticles (BNT51 / Ac-AEEA14-DMA / DSPE-pAEEA14-α) were fabricated via ethanol injection. (Label lipids) To prepare α-tagged preformed lipid nanoparticles, a lipid mixture consisting of BNT51, DSPC, cholesterol, Ac-AEEA14-DMA, and DSPE-pAEEA14-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2 was dissolved in an organic solvent (ethanol) at a total lipid concentration of 20 mM. The mixture was then mixed with an aqueous phase (5 mM AcOH) at a total flow rate of 90 mL / min (45 mL / min for the nucleic acid phase, 15 mL / min for the lipid phase, and 30 mL / min for online dilution) using a standard syringe pump-based device equipped with an online dilution and T-mixing element (0.5 mm T-mixer). The organic solvent in the obtained pristine colloidal nanoparticles was removed by dialysis against 5 mM AcOH using a Slide-A-Lyzer dialysis kit (Thermo Fisher Scientific, Waltham, MA, USA) with a molecular weight cutoff (MCWO) of 10K. Using Amico Ultra-15 (Merck, Darmstadt, Germany) with a molecular weight cutoff (MCWO) of 30K, the nanoparticles were concentrated to twice their original size by centrifugation at 3000 rpm for 10 minutes at 4°C. After concentration, the nanoparticles were diluted to a sucrose concentration of 10%.

[0508] Freezing of pre-formed lipid (BNT51 / Ac-AEEA14-DMA / DSPE-pAEEA14-α-tagged lipid) nanoparticles stability The freeze stability of preformed lipid nanoparticles was investigated by cycling the formulation from -20°C and -80°C (overnight) to +25°C (2 hours) at least twice. Particle size and polydispersity index of the formulation were measured on the freeze-thawed samples. The formulation was gently inverted and mixed between the freeze and thaw cycles before the next freezing cycle. Colloidal stability of the nanoparticles was observed to be maintained at at least two freeze-thaw cycles at -20°C and -80°C. Figure 32A As depicted in the text.

[0509] Manufacturing functionalized RNA / DNA-lipid particles using an aqueous-to-aqueous process RNA / DNA-lipid particles were prepared using an aqueous-aqueous protocol (LNP2) as described herein. Briefly, RNA in an aqueous buffer of 10 mM HEPES and 0.1 mM EDTA (pH 7.0) and DNA in water were mixed with preformed lipid nanoparticles at a 1:1 volume ratio in a 5 mM acetic acid, 10% sucrose aqueous solution. The preformed lipid nanoparticles consisted of BNT51, DSPC, cholesterol, Ac-AEEA14-DMA, and DSPE-pAEEA14-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2. Mixing was achieved using a T-mixing element (2.4 mm T-mixer, 1.6 mm tube) at a total flow rate of 360 ml / min (180 ml / min per phase) based on a standard syringe pump. The resulting RNA / DNA-lipid particles were then functionalized with aCD3-VHH ligand at a ligand / cargo ratio of 0.48 w / w and further diluted to a final nucleic acid concentration of 0.1 mg / mL and a storage matrix of 22 mM HEPES (pH 5.5) and 10% (w / v) sucrose. RNA / DNA-lipid particles were prepared at an N:P ratio of 12:1 and filtered through a 0.22 µm polyethersulfone (PES) filter.

[0510] Characterization and freeze-thaw studies of the formulation Particle size was determined by dynamic light scattering using a DynaPro plate reader II (Wyatt, Dernbach, Germany). Based on the measurements, size (Z-mean) and polydispersity index (PDI) were calculated using Dynamics 7.8.1.3 software based on cumulative analysis. For the measurements, samples were diluted 1:10 in water and analyzed in triplicate. The pH was measured on 150 μL samples after calibrating the apparatus with standards at pH 4, 7, and 10. Successful inclusions were verified by agarose gel electrophoresis.

[0511] Freeze-thaw studies were conducted by cycling the formulation from -20°C and -80°C (overnight) to +25°C (2 hours) at least twice. Particle size and polydispersity index of the formulation were measured on the frozen-thawed samples. The formulation was thawed between freezing cycles by gentle inversion mixing before the next freezing cycle.

[0512] The size and PDI of functionalized sterile filtered RNA / DNA-LNP2 stored at a final nucleic acid concentration of 0.1 mg / mL were determined by DLS measurements. It was observed that aCD3-VHH-functionalized LNP2 could be successfully fabricated using the Ac-AEEA14-DMA recessive moiety and DSPE-pAEEA14-α lipids, resulting in particles <100 nm in size and with a PDI ≤ 0.3. Figure 32B Goods successfully included, as confirmed by agarose gel electrophoresis. Freezing stability was observed over two freeze-thaw cycles, such as… Figure 32B As depicted in the text.

[0513] Example 9: RNA / DNA lipid nanoparticles (BN) T Manufacturing of 52 / Ac-AEEA14-DMA / DSPE-pAEEA14-α-tagged lipids Preformed lipid nanoparticles (BNT52 / Ac-AEEA14-DMA / DSPE-pAEEA14-α) were fabricated via ethanol injection. (Label lipids) To prepare α-tagged preformed lipid nanoparticles, a lipid mixture consisting of BNT52, DSPC, cholesterol, Ac-AEEA14-DMA, and DSPE-pAEEA14-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2 was dissolved in an organic solvent (ethanol) at a total lipid concentration of 20 mM. The mixture was then mixed with an aqueous phase (5 mM AcOH) at a total flow rate of 90 mL / min (45 mL / min for the nucleic acid phase, 15 mL / min for the lipid phase, and 30 mL / min for online dilution) using a standard syringe pump-based device equipped with an online dilution and T-mixing element (0.5 mm T-mixer). The organic solvent in the obtained pristine colloidal nanoparticles was removed by dialysis against 5 mM AcOH using a Slide-A-Lyzer dialysis kit (Thermo Fisher Scientific, Waltham, MA, USA) with a molecular weight cutoff of 10K (MCWO). Using Amico Ultra-15 (Merck, Darmstadt, Germany) with a molecular weight cutoff (MCWO) of 30K, the nanoparticles were concentrated to twice their original size by centrifugation at 3000 rpm for 10 minutes at 4°C. After concentration, the nanoparticles were diluted to a sucrose concentration of 10%.

[0514] Freezing of pre-formed lipid nanoparticles (BNT52 / Ac-AEEA14-DMA / DSPE-pAEEA14-α-tagged lipids) stability The freeze stability of preformed lipid nanoparticles was investigated by cycling the formulation from -20°C and -80°C (overnight) to +25°C (2 hours) at least twice. Particle size and polydispersity index of the formulation were measured on the freeze-thawed samples. The formulation was gently inverted and mixed between the freeze and thaw cycles before the next freezing cycle. Colloidal stability of the nanoparticles was observed to be maintained at at least two freeze-thaw cycles at -20°C and -80°C. Figure 33A As depicted in the text.

[0515] Manufacturing functionalized RNA / DNA-lipid particles using an aqueous-to-aqueous process RNA / DNA-lipid particles were prepared using an aqueous-aqueous protocol (LNP2) as described herein. Briefly, RNA in an aqueous buffer (HEPES 10 mM, EDTA 0.1 mM, pH 7.0) and DNA in water were mixed with preformed lipid nanoparticles at a 1:1 volume ratio in a 5 mM acetic acid, 10% sucrose aqueous solution. The preformed lipid nanoparticles consisted of BNT52, DSPC, cholesterol, Ac-AEEA14-DMA, and DSPE-pAEEA14-α peptide in a molar ratio of 47.5:10:40.5:1.8:0.2. Mixing was achieved using a T-mixing element (2.4 mm T-mixer, 1.6 mm tube) at a total flow rate of 360 ml / min (180 ml / min per phase) based on a standard syringe pump. The resulting RNA / DNA-lipid particles were then functionalized with aCD3-VHH ligand at a ligand / cargo ratio of 0.48 w / w and further diluted to a final nucleic acid concentration of 0.1 mg / mL and a storage matrix of 22 mM HEPES (pH 5.5) and 10% (w / v) sucrose. RNA / DNA-lipid particles were prepared at an N:P ratio of 12:1 and filtered through a 0.22 µm polyethersulfone (PES) filter.

[0516] Characterization and freeze-thaw studies of the formulation Particle size was determined by dynamic light scattering using a DynaPro plate reader II (Wyatt, Dernbach, Germany). Based on the measurements, size (Z-mean) and polydispersity index (PdI) were calculated using Dynamics 7.8.1.3 software based on cumulative analysis. For the measurements, samples were diluted 1:10 in water and analyzed in triplicate. pH was measured on 150 μL samples after calibrating the apparatus with standards at pH 4, 7, and 10. Successfully included goods were validated by agarose gel electrophoresis. Freeze-thaw studies were performed by cycling the formulation from -20°C and -80°C (overnight) to +25°C (2 hours) at least twice. Particle size and polydispersity index of the formulation were measured on the frozen-thawed samples. The formulation was gently inverted and mixed between the thawed and frozen cycles before the next freezing cycle.

[0517] The size and PDI of functionalized sterile filtered RNA / DNA-LNP2 stored at a final nucleic acid concentration of 0.1 mg / mL were determined by DLS measurements. It was observed that aCD3-VHH-functionalized LNP2 could be successfully fabricated using the Ac-AEEA14-DMA recessive moiety and DSPE-pAEEA14-α lipids, resulting in particles <100 nm in size and with a PDI ≤ 0.3. Figure 33B Goods successfully included, as confirmed by agarose gel electrophoresis. Freezing stability was observed over two freeze-thaw cycles, such as… Figure 33B As depicted in the text.

[0518] Example 10: Target transfection of T cells using thiolipin This embodiment evaluates the applicability of LNPs containing the sulfur lipids described herein for cell-specific targeting by introducing a targeting system specifically targeting primary T cells.

[0519] This embodiment compares the sulfur lipids BNT51 and BNT52 formulated in functionalized LNPs with the known ionizable lipid DODMA. The composition of the LNPs produced at an N / P ratio of 6 is shown in the table below.

[0520] To test RNA and DNA delivery, a cargo mixture containing Thy1.1 RNA and Venus nanoparticle DNA was formulated into LNPs using methods known to those skilled in the art, together with the aforementioned lipid mixture. The LNPs containing the tagged lipid DSPE-PEG2k-ALFA were incubated with an anti-CD3 specific VHH_NbAlfa construct, which binds to an α-tag (with sequence SEQ.ID.NO. 1) present on the surface of the LNP. The resulting particles exhibited acceptable particle properties (by DLS: diameter <200 nm, PDI <0.5) and negligible amounts of free RNA / DNA, as assessed by agarose gel electrophoresis. The particles thus obtained were tested in a PBMC assay.

[0521] For transfection studies, 10 μl of the appropriate formulation was pre-diluted in 50 μl of X-Vivo15 in an ultra-low adhesion 96-well plate. 1e 6 One thawed human PBMC was diluted in 50 μl of condensed PHS and added to the nanoparticle dilution (1000 ng of prepared nucleic acid cargo / 1e6 PBMCs). After incubation for 30 minutes (37°C, 5% CO2), 3e 5 Transfected PBMCs were transferred to new plates and cultured for 4 days in 200 µl X-Vivo15 + 5% PHS + 100 U / ml IL-2 (37℃, 5% CO2). Cell type-specific Thy1.1-RNA and Venus nanoparticle DNA transfection of PBMCs were analyzed by flow cytometry.

[0522] Compared to LNPs containing DODMA, LNPs containing the sulfur lipids described herein showed greater RNA and DNA transfection in T cells.

[0523] Figures 33A-33B Bar graph illustrating the targeted transfection of T cells using the thiolipin described herein. In vitro evaluation in hPBMCs. Comparison of three different LNPs containing three different ionizable lipids shows the superior RNA and DNA transfection efficiency of the thiolipin described herein formulated in LNP2 and LNP3. The top graph depicts the percentage (y-axis) of Thy1.1-expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD19+ B cells) in all single cells and live cells. The bottom graph depicts the percentage of CD4+ T cells and CD8+ T cells expressing the Venus-nanoplasmid.

[0524] Example 11: Targeted transfection of T cells using additional sulfur lipid compounds This embodiment evaluates lipids BNT52 and BNT76 formulated in functionalized LNPs. The composition of LNPs produced at an N / P ratio of 12 is shown in the table below.

[0525] To test RNA and DNA delivery, a cargo mixture containing Thy1.1 RNA and Venus nanoparticle DNA was formulated into LNPs using methods in the art, together with the aforementioned lipid mixture. The LNPs containing the tagged lipid DSPE-PEG2k-ALFA were incubated with an anti-CD3 specific VHH_NbAlfa construct that binds to an α-tag present on the surface of the LNP. The resulting particles exhibited acceptable particle properties (by DLS: diameter <200 nm, PDI <0.5) and negligible amounts of free RNA / DNA, as assessed by agarose gel electrophoresis. The particles thus obtained were tested in a PBMC assay.

[0526] For PBMC transfection studies, 1 µl of the corresponding nanoparticle formulation (total cargo concentration = 0.1 µg / µL) was pre-diluted in 50 µl of X-Vivo15 in an ultra-low adhesion 96-well plate. 1e 6 Thawed human PBMCs were diluted in 50 µl of human serum from male AB coagulated whole blood and added to the nanoparticle diluent. After incubation for 30 min (37°C, 5% CO2), 100 µl of X-Vivo15 containing human IL-2 [200 U / ml] was added to each well. Cells were then cultured again (37°C, 5% CO2) for 96 h. Figure 44A and Figure 44B The image shows cell type-specific RNA transfection (Thy1.1-RNA expression) and DNA transfection (Venus nanoparticle DNA expression) analyzed by flow cytometry.

[0527] For both LNPs, low RNA and DNA transfection was observed in B cells and monocytes. LNPs containing novel lipids showed higher RNA and DNA transfection in T cells. Both novel lipids tested are potent candidates for targeted transfection of T cells.

[0528] Figure 35A and Figure 35B The in vitro evaluation in hPBMCs is shown using the reported thiolipin for targeted transfection of T cells. A comparison of two different LNPs containing two different ionizable lipids demonstrates the high RNA and DNA transfection efficiency of the reported thiolipin formulated in LNP1 and LNP2. Figure 44A Plot the percentage (y-axis) of Thy1.1-expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD14+ monocytes) in all single cells and live cells. Figure 44BDescribe the percentage of CD4+ T cells and CD8+ T cells expressing Venus-nanoplasmid.

[0529] Example 12: Manufacturability and in vitro studies of LNP formulations composed of novel ionizable lipid BNT-72 This embodiment evaluates the manufacturability and in vitro biological effects of an LNP formulation composed of the provided lipid (with a sulfone branched tail and a hydroxyl head) BNT-72. For this purpose, BNT-72 was formulated against a baseline (BM) formulation, and the LNPs were characterized and tested in vitro. To determine the effect of the N / P ratio on the BNT-72 formulation, particles were prepared at three different N / P ratios. The LNPs were prepared by microfluidic mixing of the lipid mixture in ethanol and an RNA solution in an acidic aqueous buffer. Details of the formulations are given in the table below.

[0530] The size of LNP was evaluated using DLS. Figure 36A The hydrodynamic diameter of the baseline formulation was found to be 70 nm, as expected. The size of the BNT-72 formulation varied with the N / P ratio. Formulations with diameters of 148 nm and 191 nm were obtained at N / P ratios of 4 and 8, respectively. At an N / P ratio of 12, the size of the BNT-72 formulation decreased significantly (114 nm). The PDI value was found to be ≤0.2, indicating a narrow size distribution. The zeta potential of the formulation was assessed by electrophoretic light scattering. Figure 36B The formulation exhibits a neutral zeta potential.

[0531] The mRNA encapsulation efficiency of the formulation was evaluated using Ribogreen assays. Figure 37A The BNT-72 formulation showed high accessibility at N / P ratios of 4 and 8. The mRNA integrity of the formulation was assessed by capillary electrophoresis and fragment analysis. Figure 37B Generally, no fragmentation was observed in the formulation.

[0532] The cytotoxicity and transfection efficiency of the formulation were evaluated on three different cell lines (C2C12, HepG2, and RAW 264) at three different mRNA concentrations (12.5 ng, 25 ng, and 50 ng). Figures 38A-38C Generally, the formulation did not show significant cytotoxicity under the test conditions. The formulation was able to effectively transfect the tested cell lines. Figures 39A-39C The transfection efficiency of the BNT-72 formulation increased significantly with increasing N / P ratio. At all N / P ratios, the BNT-72 formulation was superior to the BM formulation, except for HepG2 cells (the transfection efficiency was comparable at N / P ratios of 4 and 8).

[0533] Example 13: Comparative Analysis of Tail Bases This example considers the differences in transfection efficiency, size, and physical characteristics of complexes containing thiolipin (BNT-76) and conventional lipid (BNT-90) in different cell lines:

[0534] Methods for characterizing complexes are provided in the various embodiments herein.

[0535] Figure 40 Bar graphs showing the size (nm) and polydispersity index (PDI) of complexes containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0536] Figure 41 A bar graph showing the Z-potential (mV) of a complex containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0537] Figure 42 Bar graph showing RNA integrity (%) of complexes containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0538] Figure 43 Bar graphs showing the permeability and pH of complexes containing BNT-76, BNT-90, or the baseline lipid CM12_BM.

[0539] Figures 44A-44D To demonstrate C2C12 cells ( Figure 44A ), HepG2 cells ( Figure 44B ), RAW cells ( Figure 44C ) and Hek293 cells ( Figure 44D A series of bar graphs showing the transfection efficiency of ).

[0540] Figures 45A-45D To demonstrate C2C12 cells ( Figure 45A ), HepG2 cells ( Figure 45B ), RAW cells ( Figure 45C ) and Hek293 cells ( Figure 45D A series of bar graphs showing cell viability.

[0541] Example 14: Synthesis of Thiolipids BL-199 and BL-200 Synthesis Sodium 6-hydroxyhexane-1-sulfonate, sodium bromide (4) 6-Bromohexane-1-ol (2.0 g, 1 equivalent, 11 mmol) was dissolved in water (10 mL) and ethanol (10 mL), then sodium sulfite (2.1 g, 1.5 equivalent, 17 mmol) was added, and the mixture was heated to 100 °C and stirred overnight. The mixture was evaporated and co-evaporated with 2 × 20 mL toluene, 2 × 20 mL EtOH, and 2 × 20 mL MeCN to obtain crude sodium 6-hydroxyhexane-1-sulfonate·sodium bromide (4.1 g, 11 mmol, quantified).

[0542] 6-Chlorohexane-1-sulfonyl chloride (5) Method A: A solution of sodium 6-hydroxyhexane-1-sulfonate (1.5 g, 65% Wt, 1 equivalent, 4.8 mmol) in DCM (40 mL) and DMF (0.1 mL) was mixed with thionyl chloride (2.3 g, 1.4 mL, 4 equivalent, 19 mmol), and the mixture was heated to reflux and stirred for 4 hours. The reactants were then concentrated under vacuum, dissolved in DCM, and filtered to obtain a crude product (1.5 g, 4.7 mmol, 98%), which was used without further purification.

[0543] Method B: Sodium 6-hydroxyhexane-1-sulfonate bromide (6.7 g, 1 equivalent, 22 mmol) and POCl3 (33 g, 20 mL, 10 equivalent, 0.22 mol) were charged into a 50 mL round-bottom flask. The resulting white suspension was heated to 110 °C and stirred for 30 hours, after which heating was stopped and the reaction mixture was stirred at room temperature over the weekend. The reaction was stopped and concentrated under reduced pressure. The crude product was dissolved in 15 mL of DCM, filtered, and back-extracted 3× with 20 mL of DCM to obtain 6-chlorohexane-1-sulfonyl chloride (4.7 g, 21 mmol, 98%), which was used without further purification.

[0544] 6-Chloro-N-decylhexane-1-sulfonamide (6) Method A: A solution of decyl-1-amine (280 mg, 0.8 equivalent, 1.78 mmol) and triethylamine (451 mg, 621 μL, 2 equivalent, 4.45 mmol) in DCM (5.0 mL) was cooled to 0 °C, and then 6-chlorohexane-1-sulfonyl chloride (488 mg, 1 equivalent, 2.23 mmol) in 3 mL of DCM was slowly added. The mixture was stirred overnight at room temperature. The mixture was then evaporated to dryness and purified by FCC using E:H from 0% EtOAc to 50% to provide 6-chloro-N-decylhexane-1-sulfonamide (215 mg, 632 μmol, 28.4%).

[0545] Method B: A solution of decyl-1-amine (280 mg, 0.8 equivalent, 1.78 mmol) and K₂CO₃ (616 mg, 2 equivalent, 4.45 mmol) in MeCN (5.0 mL) was cooled to 0 °C, and then 6-chlorohexane-1-sulfonyl chloride (488 mg, 1 equivalent, 2.23 mmol) in 3 mL of DCM was slowly added. The mixture was stirred overnight at room temperature. The mixture was then evaporated to dryness and purified by FCC using E:H from 0% EtOAc to 50% to provide 6-chloro-N-decylhexane-1-sulfonamide (205 mg, 603 μmol, 27.1%).

[0546] BL-199 A suspension of 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester (315 mg, 1 equivalent, 713 μmol), 6-chloro-N-decylhexane-1-sulfonamide (267 mg, 1.1 equivalent, 784 μmol), K₂CO₃ (98.5 mg, 1 equivalent, 713 μmol), and KI (118 mg, 1 equivalent, 713 μmol) in a mixture of MeCN (3.57 mL) and CPME (3.57 mL) was heated to reflux overnight. The mixture was evaporated to dryness, water was added, and the product was extracted with EtOAc (2×). The organic layer was washed with brine, dried, and evaporated under vacuum. The crude product was purified by FCC using EtOAc:EtOH (3:1) in heptane to provide 8-((6-(N-decylaminosulfonyl)hexyl)(2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester (450 mg, 604 μmol, 70.0%).

[0547] 6-Chloro-N-decyl-N-octylhexane-1-sulfonamide (7) A solution of N-octyldec-1-amine (2.0 g, 1 equivalent, 7.4 mmol) and triethylamine (2.3 g, 3.1 mL, 3 equivalent, 22 mmol) in DCM (70 mL) was cooled to 0 °C, and then 6-chlorohexane-1-sulfonyl chloride (2.2 g, 1.33 equivalent, 9.9 mmol) in 8 mL of DCM was slowly added and stirred overnight. The reaction mixture was concentrated under reduced pressure to obtain a crude product. This crude product was coated onto a hydromatrix and purified using FCC with 0%–25% EtOAc in heptane to obtain 6-chloro-N-decyl-N-octylhexane-1-sulfonamide (657 mg, 1.45 mmol, 20%).

[0548] BL-200 In a 50 mL round-bottom flask, 391 mg (1 equivalent, 885 μmol) of heptadecanoyl octanoate and 400 mg (1 equivalent, 885 μmol) of 6-chloro-N-decyl-N-octylhexane-1-sulfonamide were mixed in CPME (4.0 mL) / MeCN (4.0 mL), and potassium iodide (294 mg, 2 equivalent, 1.77 mmol) and potassium carbonate (489 mg, 4 equivalent, 3.54 mmol) were added. The mixture was heated at 90 °C overnight. The reaction mixture was cooled to room temperature, filtered through a diatomaceous earth pad, and evaporated under vacuum. The crude material was dissolved in a small amount of heptane / EtOAc and purified on a silica column to provide 8-((6-(N-decyl-N-octylaminosulfonyl)hexyl)(2-hydroxyethyl)amino)octanoic acid heptadecanyl ester (254 mg, 296 μmol, 33.5%).

[0549] BL-201 and BL-202 Synthesis Sodium 7-ethoxy-7-oxoheptane-1-sulfonate (9) A solution / suspension of sodium sulfite (15.7 g, 1.9 equivalent, 124 mmol) in water (49.7 mL) was added to a solution of ethyl 7-bromoheptanate (15.5 g, 1 equivalent, 65.4 mmol) in EtOH (39.8 mL), and the reaction mixture was stirred overnight at 80 °C. The reaction mixture was concentrated under vacuum and co-evaporated with EtOH (1×) and toluene (3×) to produce crude sodium 7-ethoxy-7-oxohepane-1-sulfonate (31.2 g, 66 mmol) as a waxy solid.

[0550] Ethyl 7-(chlorosulfonyl)heptanoate (10) DMF (239 mg, 253 μL, .05 equivalent, 3.27 mmol) was added to a suspension of sodium 7-ethoxy-7-oxoheptane-1-sulfonate (30.9 g, 55% Wt, 1 equivalent, 65.4 mmol) in toluene (262 mL). Thionyl chloride (77.8 g, 47.7 mL, 10 equivalent, 654 mmol) was added dropwise, and the mixture was stirred under reflux for 3 days. The mixture was evaporated to semi-dryness and co-evaporated twice with toluene. Water was added, and the product was extracted with EtOAc (2×), washed with brine, dried, and evaporated to dryness to provide ethyl 7-(chlorosulfonyl)heptaate (24.2 g, 66 mmol).

[0551] Ethyl 7-(N-hexyl-N-octylaminosulfonyl)heptanoate (11) At 0 °C, a cold (cooled to 0 °C) solution of ethyl 7-(chlorosulfonyl)heptanate (17.2 g, 70% Wt, 1 equivalent, 46.9 mmol) in DCM (176 mL) was added dropwise to a solution of N-hexyloctyl-1-amine (15.0 g, 0.02 L, 1.5 equivalent, 70.3 mmol) in DCM (176 mL). The mixture was stirred for 20 min, and then evaporated to dryness. Ethyl 7-(N-hexyl-N-octylaminosulfonyl)heptanate (7.09 g, 16.3 mmol, 34.9%) was obtained by FCC purification.

[0552] N-hexyl-N-octyl-7-oxoheptane-1-sulfonamide (12) Ethyl 7-(N-hexyl-N-octylaminosulfonyl)heptanate (3.1 g, 1 equivalent, 7.1 mmol) was dissolved in toluene (74 mL) under an argon atmosphere and cooled to -78 °C. A solution of DIBAL-H iM (1.1 g, 7.5 mL, 1.0 mol concentration, 1.05 equivalent, 7.5 mmol) in toluene (15 mL) was then added dropwise while maintaining the temperature at -78 °C, and the mixture was stirred for 1 hour. The reaction was quenched, and then 1.5 mL of methanol was added at -78 °C, and the mixture was stirred for 20 minutes. Water was then added, and the crude product was extracted with EtOAc to provide N-hexyl-N-octyl-7-oxoheptane-1-sulfonamide (2.6 g, 6.7 mmol, 93%).

[0553] BL-201 2-Aminoethanol-1-ol (65.32 mg, 1 equivalent, 1.069 mmol) and N-hexyl-N-octyl-7-oxohepane-1-sulfonamide (1000 mg, 2.4 equivalent, 2.566 mmol) were dissolved in DCM (12.83 mL) and stirred for 5 minutes. Sodium triacetoxyborohydride (679.9 mg, 3 equivalent, 3.208 mmol) was then added and the mixture was stirred overnight. The mixture was then quenched with saturated NaHCO3 and the crude product was extracted with DCM (2×). The combined DCM layers were washed with brine, dried (Na2SO4), and evaporated to dryness. The crude product was coated onto a hydromatrix and purified by FCC using 0%–50% EtOAc / EtOH (3 / 1) in heptane to yield 7,7'-((2-hydroxyethyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (282 mg, 349 μmol, 32.6%).

[0554] BL-202 3-Aminoprop-1-ol (80.32 mg, 1 equivalent, 1.069 mmol) and N-hexyl-N-octyl-7-oxohepane-1-sulfonamide (1000 mg, 2.4 equivalent, 2.566 mmol) were dissolved in DCM (12.83 mL) and stirred for 5 minutes. Then, sodium triacetoxyborohydride (679.9 mg, 3 equivalent, 3.208 mmol) was added and the mixture was stirred overnight. The mixture was quenched with saturated NaHCO3 and the crude product was extracted with DCM (2×). The combined DCM layers were washed with brine, dried (Na2SO4), and evaporated under vacuum. The crude product was coated onto a hydromatrix and purified by FCC using 0%–50% EtOAc / EtOH (3 / 1) in heptane to produce 7,7'-((3-hydroxypropyl)azanidinediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (259 mg, 315 μmol, 29.5%).

[0555] BL-203 and BL-204 Synthesis 7-Hydroxyheptan-1-sulfonate bromide (14) 6-Bromohexyl acetate (750 mg, 1 equivalent, 3.36 mmol) was dissolved in water (6.72 mL), then sodium sulfite (636 mg, 1.5 equivalent, 5.04 mmol) was added, and the mixture was heated to 100 °C and maintained for 17 hours. The mixture was then evaporated under vacuum and used without further purification.

[0556] 7-Chloro-heptane-1-sulfonyl chloride / 7-bromo-heptane-1-sulfonyl chloride (15) Sodium 7-hydroxyheptane-1-sulfonate sodium bromide (5.0 g, 1 equivalent, 16 mmol) and POCl3 (24 g, 15 mL, 10 equivalent, 0.16 mol) were packed into a 50 mL round-bottom flask. The resulting white suspension was heated to 110 °C and stirred for 3 days. The reaction mixture was cooled to room temperature and filtered through a diatomaceous earth mat. The dark brown filtrate was evaporated on a rotary evaporator and the black residue was back-extracted with toluene (3×) to obtain a crude mixture of 7-chloroheptane-1-sulfonyl chloride / 7-bromo-heptane-1-sulfonyl chloride (3.7 g, 14–16 mmol), which was used without further purification.

[0557] 7-Chloro-N-hexyl-N-octylheptane-1-sulfonamide / 7-bromo-N-hexyl-N-octylheptane-1-sulfonamide (16) A solution of dioctylamine (2.9 g, 1 equivalent, 12 mmol) and triethylamine (2.4 g, 3.3 mL, 2 equivalent, 24 mmol) in DCM (0.11 L) was cooled to 0 °C. A crude mixture of 7-chloro-heptane-1-sulfonyl chloride and 7-bromo-heptane-1-sulfonyl chloride (16–18 mmol) in DCM (3 mL) was then slowly added. The reaction mixture was evaporated under vacuum, and the residue was coated onto a hydromatrix and purified by silica column chromatography using 5%–40%–100% EtOAc in heptane to provide a mixture of 7-chloro-N,N-dioctylheptane-1-sulfonamide and 7-bromo-N,N-dioctylheptane-1-sulfonamide (300 mg, 6.2–6.8 mmol) for use without further purification.

[0558] BL-203 A mixture of 4-(4-methylpiperazin-1-yl)but-1-amine (300 mg, 1 equivalent, 1.75 mmol), 7-chloro-N-hexyl-N-octylheptane-1-sulfonamide (1.58 g, 2.2 equivalent, 3.85 mmol), K₂CO₃ (533 mg, 2.2 equivalent, 3.85 mmol), and KI (640 mg, 2.2 equivalent, 3.85 mmol) in MeCN (4.38 mL) / CPME (4.38 mL) was heated to 100 °C and stirred overnight. The solvent was then evaporated under vacuum, and the crude product was coated onto a hydromatrix. Purification was performed by FCC using 0%–70% MeOH in 7M NH₃ / DCM (1:9), yielding three impurity fractions. The purest batch was further purified using 5%–45% of DCM (7M NH3 in 10% MeOH: 90% DCM) to produce 7,7'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (131 mg, 143 μmol, 8.14%).

[0559] BL-204 A mixture of 2-(4-methylpiperazin-1-yl)ethyl-1-amine (250 mg, 262 μL, 1 equivalent, 1.75 mmol), 7-chloro-N-hexyl-N-octylheptane-1-sulfonamide (1.57 g, 2.2 equivalent, 3.84 mmol), K₂CO₃ (531 mg, 2.2 equivalent, 3.84 mmol), and KI (637 mg, 2.2 equivalent, 3.84 mmol) in MeCN (4.36 mL) / CPME (4.36 mL) was heated to 100 °C and stirred overnight. The solvent was then evaporated under vacuum. The crude extract was dissolved in a minimum amount of DCM and the resulting solution was purified by FCC using 0%–8% MeOH in the DCM to provide 7,7'-((2-(4-methylpiperazin-1-yl)ethyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (306 mg, 344 μmol, 19.7%).

[0560] BL-205 and BL-206 Synthesis 9-Hydroxynonane-1-sulfonate.NaBr (20) 9-Bromonon-1-ol (9.6 g, 1 equivalent, 43 mmol) was suspended in water (0.11 L), sodium sulfite (6.5 g, 1.2 equivalent, 52 mmol) was added, and the mixture was stirred overnight at 95 °C. The mixture was cooled, washed with MTBE, and the aqueous layer was evaporated to dryness to provide sodium 9-hydroxynonane-1-sulfonate sodium bromide·NaBr (18.3 g, 52.4 mmol). The product was used without further purification.

[0561] 9-Chlorononane-1-sulfonyl chloride (21) 9-Hydroxynonane-1-sulfonate·NaBr salt (6.18 g, 1 equivalent, 17.7 mmol) was suspended in POCl3 (30 g, 18 mL, 11 equivalent, 0.19 mol) and the reaction mixture was stirred at (initial) 110 °C. After 70 hours, the reaction mixture was concentrated and co-evaporated with toluene and DCM to produce crude 9-chlorononane-1-sulfonyl chloride. The Cl:Br ratio was 1:0.35. The product was used without further purification.

[0562] N-Butyl-9-chloro-N-hexylnonane-1-sulfonamide (22) A solution of N-butylhexane-1-amine (2.09 g, 0.75 equivalents, 13.3 mmol) and triethylamine (5.37 g, 7.38 mL, 3 equivalents, 53.1 mmol) in DCM (32 mL) was cooled to 0 °C, and then 9-chlorononane-1-sulfonyl chloride / bromine (4.62 g, 1 equivalent, approximately 17.7 mmol) (crude mixture) in DCM (22 mL) was slowly added, and the reaction mixture was stirred at room temperature for 4 days. The reaction mixture was concentrated under vacuum, resuspended in DCM / MeOH, coated onto hydromatrix, and purified by FCC using 1%–25% EtOAc in heptane to obtain a Cl / Br mixture of N-butyl-9-halogen-N-hexylnonane-1-sulfonamide (1.5 g), with a Cl / Br ratio of 1:1 according to NMR.

[0563] BL-205 2-Aminoethanol (42.1 mg, 1 equivalent, 689 μmol), N-butyl-9-chloro-N-hexylnonane-1-sulfonamide (500 mg, 1.9 equivalent, 1.31 mmol), K₂CO₃ (95.2 mg, 1 equivalent, 689 μmol), and KI (114 mg, 1 equivalent, 689 μmol) were suspended in a mixture of MeCN (3.27 mL) and CPME (3.27 mL) in a sealed tube and heated to 120 °C overnight. The mixture was evaporated, absorbed into EtOAc / water, and extracted with EtOAc (2×). The extract was washed with brine, dried (Na₂SO₄), and evaporated to dryness.

[0564] 9,9'-((2-hydroxyethyl)azinediyl)bis(N-butyl-N-hexylnonane-1-sulfonamide) (244 mg, 324 μmol, 47.1%) was obtained by purification with 0%-100% EtOAc in heptane via FCC.

[0565] (4-(bis(9-(N-butyl-N-hexylaminosulfonyl)nonyl)amino)butyl)tert-butyl carbamate (23) To a solution of N-butyl-9-chloro-N-hexylnonane-1-sulfonamide (1.14 g, 3 equivalents, 2.98 mmol) and (4-aminobutyl)carbamate tert-butyl ester (187 mg, 190 μL, 1 equivalent, 993 μmol) in a mixture of CPME (4.97 mL) and MeCN (4.97 mL), KI (660 mg, 4 equivalents, 3.97 mmol) and K2CO3 (686 mg, 5 equivalents, 4.97 mmol) were added and the mixture was heated to 90 °C and stirred overnight. The reaction was stopped, filtered through diatomaceous earth, washed twice with heptane, and concentrated under reduced pressure to obtain the crude product. This crude product was coated onto a hydromatrix and purified using FCC with 5%–45% EtOAc:EtOH (3:1) in heptane to provide (4-(bis(9-(N-butyl-N-hexylaminosulfonyl)nonyl)amino)butyl)carbamate tert-butyl ester (550 mg, 625 μmol, 63.0%).

[0566] 9,9'-((4-aminobutyl)azanidinediyl)bis(N-butyl-N-hexylnonane-1-sulfonamide) hydrochloride (24) HCl (456 mg, 3.13 mL, 4.0 mol concentration, 20 equivalences, 1 mmol) was added to a solution of (4-(bis(9-(N-butyl-N-hexylaminosulfonyl)nonyl)amino)butyl)carbamate (550 mg, 1 equivalent, 625 μmol) in DCM (1.0 mL), and this mixture was stirred for 2 hours and then evaporated under vacuum to obtain 9,9'-((4-aminobutyl)azanidinediyl)bis(N-butyl-N-hexylnonane-1-sulfonamide) hydrochloride (495 mg, 607 μmol, 97.0%). The product was used without further purification.

[0567] BL-206 At 0 °C, thiophosgene (69.6 mg, 46.4 μL, 85% Wt, 1.2 Equivalent, 515 μmol) was added dropwise to a solution of 9,9'-((4-aminobutyl)azanidinediyl)bis(N-butyl-N-hexylnonane-1-sulfonamide) hydrochloride (350 mg, 1 equivalent, 429 μmol) and Et3N (130 mg, 179 μL, 3 equivalent, 1.29 mmol) in DCM (5.0 mL), and the reaction mixture was stirred at room temperature for 5 h. Then, thiophosgene (29.6 mg, 19.7 μL, 0.6 equivalent, 257 μmol) was added, and the reaction mixture was stirred for 3 h. The reaction mixture was cooled to 0 °C, and a solution of dimethylamine in THF (193 mg, 2.15 mL, 2.0 mol concentration, 10 equivalent, 4.29 mmol) was added. The mixture was heated to 40 °C and stirred for 1 h. The solvent was evaporated under vacuum and the crude product was coated onto hydromatrix. Purification was performed by FCC using 5%–50% EtOAc:EtOH (3:1) in heptane to provide 9,9'-((4-(3,3-dimethylthiourea)butyl)azanediyl)bis(N-butyl-N-hexylnonane-1-sulfonamide) (340 mg, 392 μmol, 80.1%).

[0568] BL-207 and BL-208 Synthesis Ethyl 7-(N,N-dioctylaminosulfonyl)heptanoate (25) At 0 °C, ethyl 7-(chlorosulfonyl)heptaate (1400 mg, 87% Wt, 1 equivalent, 4.744 mmol) in 10 mL of DCM was added dropwise to a solution of dioctylamine (3.437 g, 4.30 mL, 3 equivalents, 14.23 mmol) in DCM (13.0 mL). The mixture was stirred for 20 minutes. The reaction mixture was coated onto Hydromatrix and purified by FCC.

[0569] N,N-Dioctyl-7-oxoheptane-1-sulfonamide (26) Ethyl 7-(N,N-dioctylaminosulfonyl)heptanate (1100 mg, 1 equivalent, 2.382 mmol) was back-extracted twice with toluene, dissolved in toluene (25 mL) under argon atmosphere, and cooled to -78 °C. A solution of DIBAL-H 1M (372.7 mg, 2.620 mL, 1.0 mol concentration, 1.1 equivalent, 2.620 mmol) in toluene (5.0 mL) was then added dropwise while maintaining the temperature at -78 °C. The mixture was stirred for 1 hour, the reaction was quenched with methanol (1.5 mL) at -78 °C, and the mixture was stirred for 20 minutes. Water and 2 M NaOH were then added, and the crude product was extracted with EtOAc to provide N,N-dioctyl-7-oxoheptane-1-sulfonamide (1.35 g, 2.8 mmol, 120%), which was used without further purification.

[0570] BL-207 In a 20 mL screw-cap vial, N,N-dioctyl-7-oxoheptane-1-sulfonamide (400 mg, 2.5 equivalents, 958 μmol) and 4-aminobut-1-ol (34.1 mg, 1 equivalent, 383 μmol) were dissolved in DCM (3.83 mL). Sodium triacetoxyborohydride (203 mg, 2.5 equivalents, 958 μmol) was then added, and the mixture was stirred for 30 minutes. The mixture was quenched with saturated NaHCO3, and the crude product was extracted with DCM (2×). The combined DCM layers were washed with brine, dried, and evaporated to dryness. The crude product was coated onto a hydromatrix and purified by FCC using 0%–50% EtOAc:EtOH (3:1) in heptane to provide 7,7'-((4-hydroxybutyl)azanidinediyl)bis(N,N-dioctylheptane-1-sulfonamide) (198 mg, 222 μmol, 57.9%).

[0571] (4-(bis(7-(N,N-dioctylaminosulfonyl)heptyl)amino)butyl)tert-butyl carbamate (27) N,N-dioctyl-7-oxoheptane-1-sulfonamide (600 mg, 1 equivalent, 1.26 mmol) and (4-aminobutyl)carbamate tert-butyl ester (238 mg, 1 equivalent, 1.26 mmol) were dissolved in DCM (5.06 mL), and sodium triacetoxyborohydride (670 mg, 2.5 equivalent, 3.16 mmol) was added after 3 minutes. The mixture was stirred overnight at room temperature. The solvent was evaporated under vacuum and the crude product was purified by FCC using 0%–25% EtOAc in heptane to provide (4-(bis(7-(N,N-dioctylaminosulfonyl)heptyl)amino)butyl)carbamate tert-butyl ester (440 mg, 444 μmol, 35.1%, CAD purity: 98.4%).

[0572] 7,7'-((4-aminobutyl)azonidinediyl)bis(N,N-dioctylheptane-1-sulfonamide)hydrochloride (28) A solution of tert-butyl carbamate (440 mg, 1 equivalent, 444 μmol) in DCM (2.22 mL) was mixed with 4M HCl in dioxane (162 mg, 1.11 mL, 4.0 mol concentration, 10 equivalent, 4.44 mmol) and stirred overnight. The solvent was evaporated under vacuum to obtain 7,7'-((4-aminobutyl)azanidinediyl)bis(N,N-dioctylheptane-1-sulfonamide) hydrochloride (418 mg, 450 μmol, 102%). The product was used without further purification.

[0573] BL-208 At 0 °C, phosgene (72.1 mg, 48.0 μL, 85% Wt, 1.2 equivalent, 533 μmol) was added dropwise to a solution of 7,7'-((4-aminobutyl)azanidinediyl)bis(N,N-dioctylheptane-1-sulfonamide) hydrochloride (412 mg, 1 equivalent, 444 μmol) and Et3N (135 mg, 186 μL, 3 equivalent, 1.33 mmol) in DCM (6.0 mL), and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was cooled to 0 °C, and a solution of dimethylamine in THF (20.0 mg, 222 μL, 2.0 mol concentration, 1 equivalent, 444 μmol) was added, after which the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum, and the crude product was coated onto hydromatrix. Purified by FCC using 20%–70% EtOAc:EtOH (3:1) in heptane, 7,7'-((4-(3,3-dimethylthiourea)butyl)azanediyl)bis(N,N-dioctylheptane-1-sulfonamide) (281 mg, 287 μmol, 64.7%) was provided.

[0574] Synthesis of BL-209 Sodium 6-hydroxyhexane-1-sulfonate, sodium bromide 6-Bromohexane-1-ol (2.0 g, 1 equivalent, 11 mmol) was dissolved in water (10 mL) and EtOH (10 mL), then sodium sulfite (2.1 g, 1.5 equivalent, 17 mmol) was added, and the mixture was heated to 100 °C and stirred overnight. The mixture was evaporated and co-evaporated with 2 × 20 mL toluene, 2 × 20 mL EtOH, and 2 × 20 mL MeCN to obtain crude sodium 6-hydroxyhexane-1-sulfonate·sodium bromide (4.1 g, 11 mmol, 100%).

[0575] 6-Chlorohexane-1-sulfonyl chloride Method A: A solution of sodium 6-hydroxyhexane-1-sulfonate sodium bromide (3.85 g, 1 equivalent, 12.5 mmol) in DCM (40 mL) and DMF (0.1 mL) was added to a solution of thionyl chloride (5.97 g, 3.66 mL, 4 equivalents, 50.1 mmol), and the mixture was heated to reflux and stirred for 4 hours. The reaction was then stopped by concentration under reduced pressure to obtain a crude product. This product was dissolved in DCM, filtered, back-extracted 3× with DCM, and used as a crude product in the next reaction.

[0576] Method B: Sodium 6-hydroxyhexane-1-sulfonate sodium bromide (6.7 g, 1 equivalent, 22 mmol) and POCl3 (33 g, 20 mL, 10 equivalent, 0.22 mol) were packed into a 50 mL round-bottom flask. The resulting white suspension was heated to 110 °C and stirred for 30 hours, after which heating was stopped and the reaction mixture was stirred at room temperature over the weekend. The reaction was stopped and concentrated under reduced pressure. The crude product was dissolved in 15 mL of DCM, filtered, and back-extracted 3× with 20 mL of DCM to obtain 6-chlorohexane-1-sulfonyl chloride (4.7 g, 21 mmol, 98%), which was used without further purification.

[0577] 6-Chloro-N-hexyl-N-octylhexane-1-sulfonamide To a solution of N-hexyloctyl-1-amine (1.5 g, 1.0 equivalent, 6.8 mmol) and triethylamine (1.4 g, 1.9 mL, 2 equivalent, 14 mmol) in DCM (34 mL), 6-chlorohexane-1-sulfonyl chloride (1.5 g, 1 equivalent, 6.8 mmol) was added. The resulting mixture was stirred overnight. The reactants were diluted with 25 mL of DCM, washed with water (3 × 25 mL) and brine, dried over Na₂SO₄, filtered, and concentrated under vacuum to provide the crude product. Purification was performed by FCC using 0%–20% EtOAc in heptane to provide 6-chloro-N-hexyl-N-octylhexane-1-sulfonamide (110 mg, 278 μmol, 4.1%).

[0578] (4-(bis(6-(N-hexyl-N-octylaminosulfonyl)hexyl)amino)butyl)tert-butyl carbamate A solution of 6-chloro-N-hexyl-N-octylhexane-1-sulfonamide (631 mg, 3 equivalents, 1.59 mmol) and (4-aminobutyl)carbamate tert-butyl ester (100 mg, 102 μL, 1 equivalent, 531 μmol) in a mixture of CPME (2.66 mL) and MeCN (2.66 mL) was added with KI (353 mg, 4 equivalents, 2.12 mmol) and K₂CO₃ (367 mg, 5 equivalents, 2.66 mmol) and heated to 90 °C and stirred for 48 h. The mixture was concentrated under vacuum and purified by FCC using 5%–50% EtOAc:EtOH (3:1) in heptane to provide (4-(bis(6-(N-hexyl-N-octylaminosulfonyl)hexyl)amino)butyl)carbamate tert-butyl ester (300 mg, 331 μmol, 62.2%).

[0579] 6,6'-((4-aminobutyl)azanidinediyl)bis(N-hexyl-N-octylhexane-1-sulfonamide) hydrochloride A solution of (4-(bis(6-(N-hexyl-N-octylaminosulfonyl)hexyl)amino)butyl)carbamate tert-butyl (610 mg, 1 equivalent, 672 μmol) in DCM (4.0 mL) was added with 4 M HCl in dioxane (245 mg, 1.68 mL, 4.0 mol concentration, 10 equivalent, 6.72 mmol) and stirred overnight. The solution was then concentrated under vacuum to obtain crude 6,6'-((4-aminobutyl)azanidinediyl)bis(N-hexyl-N-octylhexane-1-sulfonamide) hydrochloride (507 mg, 601 μmol, 89.4%), which was used without further purification.

[0580] BL-209 At 0 °C, phosgene (97.5 mg, 65.0 μL, 85% Wt, 1.2 equivalent, 721 μmol) was added dropwise to a solution of 6,6'-((4-aminobutyl)azanidinediyl)bis(N-hexyl-N-octylhexane-1-sulfonamide) hydrochloride (507 mg, 1 equivalent, 601 μmol) and TEA (182 mg, 251 μL, 3 equivalent, 1.80 mmol) in DCM (5.0 mL), and the reaction mixture was stirred overnight at room temperature. The reaction mixture was cooled to 0 °C, and a solution of dimethylamine in THF (271 mg, 3.00 mL, 2.0 mol concentration, 10 equivalent, 6.01 mmol) was added and stirred for 3.5 h. The reaction mixture was concentrated under vacuum to obtain the crude product. Purified by FCC using 5%–75% EtOAc:EtOH (3:1) in heptane, 6,6'-((4-(3,3-dimethylthiourea)butyl)azanediyl)bis(N-hexyl-N-octylhexane-1-sulfonamide) (99 mg, 0.10 mmol, 17%).

[0581] BL-210 Synthesis 6-Hydroxyhexane-1-sulfonate bromide 6-Bromohexane-1-ol (2.0 g, 1 equivalent, 11 mmol) was dissolved in water (10 mL) and EtOH (10 mL), then sodium sulfite (2.1 g, 1.5 equivalent, 17 mmol) was added, and the mixture was heated to 100 °C and stirred overnight. The mixture was evaporated and co-evaporated with 2 × 20 mL toluene, 2 × 20 mL EtOH, and 2 × 20 mL MeCN to obtain crude 6-hydroxyhexane-1-sulfonic acid (4.1 g, 11 mmol, 100%).

[0582] 6-Chlorohexane-1-sulfonyl chloride Method A: A solution of sodium 6-hydroxyhexane-1-sulfonate sodium bromide (3.85 g, 1 equivalent, 12.5 mmol) in DCM (40 mL) and DMF (0.1 mL) was mixed with thionyl chloride (5.97 g, 3.66 mL, 4 equivalents, 50.1 mmol) and heated to reflux with stirring for 4 hours. The reaction was then stopped by concentration under reduced pressure to obtain the crude product. This was dissolved in DCM, filtered, back-extracted 3× with DCM, and used as a crude product in the next reaction.

[0583] Method B: Sodium 6-hydroxyhexane-1-sulfonate sodium bromide (6.7 g, 1 equivalent, 22 mmol) and POCl3 (33 g, 20 mL, 10 equivalent, 0.22 mol) were packed into a 50 mL round-bottom flask. The resulting white suspension was heated to 110 °C and stirred for 30 hours, after which heating was stopped and the reaction mixture was stirred at room temperature over the weekend. The reaction was stopped and concentrated under reduced pressure. The crude product was dissolved in 15 mL of DCM, filtered, and back-extracted 3× with 20 mL of DCM to obtain 6-chlorohexane-1-sulfonyl chloride (4.7 g, 21 mmol, 98%), which was used without further purification.

[0584] 6-Chloro-N-decyl-N-octylhexane-1-sulfonamide A solution of N-octyldec-1-amine (2.0 g, 1 equivalent, 7.4 mmol) and triethylamine (2.3 g, 3.1 mL, 3 equivalent, 22 mmol) in DCM (70 mL) was cooled to 0 °C, and then 6-chlorohexane-1-sulfonyl chloride (2.2 g, 1.33 equivalent, 9.9 mmol) in 8 mL of DCM was slowly added and stirred overnight. The reaction mixture was concentrated under reduced pressure to obtain a crude product. This crude product was coated onto a hydromatrix and purified using FCC heptane / EtOAc 0=>25%. Fractions containing the product were combined and concentrated under reduced pressure to obtain 6-chloro-N-decyl-N-octylhexane-1-sulfonamide (657 mg, 1.45 mmol, 20%).

[0585] (4-(bis(6-(N-decyl-N-octylaminosulfonyl)hexyl)amino)butyl)tert-butyl carbamate To a solution of 6-chloro-N-decyl-N-octylhexane-1-sulfonamide (681 mg, 2.7 equivalents, 1.51 mmol) and (4-aminobutyl)carbamate tert-butyl ester (105 mg, 1 equivalent, 558 μmol) in a mixture of CPME (2.79 mL) and MeCN (2.79 mL), KI (370 mg, 4 equivalents, 2.23 mmol) and K₂CO₃ (385 mg, 5 equivalents, 2.79 mmol) were added and the mixture was heated to 90 °C and stirred overnight. The reactants were concentrated under vacuum and purified by FCC using 5%–50% EtOAc:EtOH (3:1) in heptane to provide (4-((6-(N-decyl-N-octylaminosulfonyl)hexyl)amino)butyl)carbamate tert-butyl ester (115 mg, 190 μmol, 34.1%).

[0586] 6,6'-((4-aminobutyl)azanidinediyl)bis(N-decyl-N-octylhexane-1-sulfonamide) hydrochloride A solution of tert-butyl carbamate (265 mg, 1 equivalent, 260 μmol) in DCM (3.0 mL) was mixed with 4 M HCl in dioxane (190 mg, 1.30 mL, 4.0 mol concentration, 20 equivalent, 5.20 mmol) and stirred overnight. The solution was then concentrated under vacuum to obtain crude 6,6'-((4-aminobutyl)azanidinediyl)bis(N-decyl-N-octylhexane-1-sulfonamide) hydrochloride (230 mg, 241 μmol, 92.6%), which was used without further purification.

[0587] BL-210 At 0 °C, phosgene (39.0 mg, 26.0 μL, 85% Wt, 1.2 equivalent, 289 μmol) was added dropwise to a solution of 6,6'-((4-aminobutyl)azanidinediyl)bis(N-decyl-N-octylhexane-1-sulfonamide) hydrochloride (230 mg, 1 equivalent, 241 μmol) and TEA (73.0 mg, 101 μL, 3 equivalent, 722 μmol) in DCM (5.0 mL), and the mixture was stirred overnight at room temperature. Dimethylamine (108 mg, 1.20 mL, 2.0 mol concentration, 10 equivalent, 2.41 mmol) in THF was added to the reaction mixture, and the mixture was stirred for 1.5 h. The reaction mixture was concentrated under vacuum to obtain the crude product. Purified by FCC using 5%–75% EtOAc:EtOH (3:1) in heptane, 6,6'-((4-(3,3-dimethylthiourea)butyl)azanediyl)bis(N-decyl-N-octylhexane-1-sulfonamide) (149.8 mg, 139.9 μmol, 58.2%) was provided.

[0588] Example 15: Preparation of functi...

Claims

1. A compound represented by Formula I: Formula I or a pharmaceutically acceptable salt thereof, wherein: I 21. The compound of any one of claims 1-17, wherein G is -OH. L 1 and L 2 each independently is an optionally substituted C1-C 30 aliphatic group; L 3 is a bond, an optionally substituted C1-C 10 aliphatic group or an optionally substituted 2 to 10 membered heteroaliphatic group comprising 1 to 4 heteroatoms selected from the group consisting of N, O, and S; X 1 and X 2 are each independently selected from the group consisting of a bond, -OC(O)-, -C(O)O-, -S(O)2N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-, -OC(S)C(R 1 )2-, -C(R 1 )2C(S)O-, and -S-; wherein one or both of X 1 or X 2 is selected from the group consisting of -S(O)2N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-, -OC(S)C(R 1 )2-, -C(R 1 )2C(S)O-, and -S-. Each R 1 Independently for each case the optionally substituted C1-C 20 Aliphatic or H; T 1 and T 2 each independently is optionally substituted C3-C 30 aliphatic; G is -N(R 2 )C(S)N(R 2 )2, -OH, -N(R 2 )2, -N + (R 3 )3, -N(R 5 )C(O)R 3 , -N(R 5 )S(O)2R 3 , -N(R 5 )C(O)N(R 3 )2, -CH(N-R 2 ), -R 4 or -S(O)2R 3 ; each R is independently selected from the group consisting of H, optionally substituted C1-C6 aliphatic, and OR 2 independently selected in each instance from the group consisting of H, optionally substituted C1-C6 aliphatic, and OR 3 independently selected in each instance from the group consisting of H, optionally substituted C1-C6 aliphatic, and OR 2 two instances of R, together with the atoms to which they are attached, form an optionally substituted 4- to 12-membered heterocyclic or optionally substituted 4- to 12-membered heteroaryl ring comprising 1 to 4 heteroatoms selected from N, O, and S; each R is independently selected from the group consisting of H and an optionally substituted C1-C6alkyl group; 3 independently selected from the group consisting of H and an optionally substituted C1-C6alkyl group; 10 an aliphatic group; and R 4 It is a 4- to 12-membered heterocycle with 1 to 4 heteroatoms selected from N, O, and S, optionally substituted, and a 4- to 12-membered heteroaryl group with 1 to 4 heteroatoms selected from N, O, and S, substituted with -(CH2). 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C6-C in )2 are replaced 12 Aryl group, or with an oxy group, -(CH2) 0-6 -OH or -(CH2) 0-6 -N(R 5 One or more of the C3-C in )2 are substituted 12 Cycloaliphatic; Each R 5 Independently selected from H and optionally substituted C1-C6 aliphatic compounds.

2. The compound of claim 1, wherein L 1 and L 2 each is C1-C 30 alkylene.

3. The compound of claim 1 or 2, wherein L 1 and L 2 each independently is -(CH2) 6-10 -.

4. The compound of any one of claims 1-3, wherein L 1 and L 2 are the same.

5. The compound of any one of claims 1-3, wherein L 1 and L 2 are different.

6. The compound of any one of claims 1-5, wherein X 1 and X 2 are each independently selected from -S(O)2N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-, -OC(S)C(R 1 )2-, -C(R 1 )2C(S)O-, and -S-.

7. The compound of any one of claims 1-5, wherein X 1 and one of X 2 is a bond, -OC(O)-, or -C(O)O-, and the other of X 1 and X 2 is -S(O)2N(R 1 )-, -N(R 1 )S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R 1 )2-, -OC(S)C(R 1 )2-, -C(R 1 )2C(S)O-, or -S-.

8. The compound of any one of claims 1-5, wherein X is -S(O)2N(R 1 )-, wherein R 1 is C1-C 1 aliphatic, and X is -C(O)O-. 10 2 8. The compound of any one of claims 1-5, wherein X is -S(O)2N(R 1 )-, wherein R 1 is C1-C 1 aliphatic, and X is -C(O)O-. 10 2 8. The compound of any one of claims 9. The compound of any one of claims 1-5, wherein X 1 and X 2 are each -S(O)2N(R 1 )-, wherein each R 1 is independently C1-C 10 aliphatic.

10. The compound of any one of claims 1-5, wherein X 1 and X 2 are the same.

11. The compound of any one of claims 1-5, wherein X 1 and X 2 are different.

12. The compound of any one of claims 1-11, wherein T 1 and T 2 are each independently selected from the group consisting of optionally substituted C3-C 20 alkyl.

13. The compound of any one of claims 1-11, wherein T 1 and T 2 are each independently selected from: 、 、 、 、 、 、 、 、 、 ； 、 、 、 ； , , and .

14. The compound of claim 1, wherein the moiety -L 1 -X 1 -T 1 is selected from the group consisting of: 、 、 、 、 、 、 、 and .

15. The compound of claim 1 or claim 14, wherein the moiety -L 2 -X 2 -T 2 is selected from the group consisting of: , , , , , , , and .

16. The compound of claim 1, wherein moiety -L 1 -X 1 -T 1 and moiety -L 2 -X 2 -T 2 are each independently selected from the group consisting of: 。 17. The compound of any one of claims 1-16, wherein L 3 is optionally substituted C1-C 10 aliphatic.

18. The compound of any one of claims 1-17, wherein G is -N(R 2 )C(S)N(R 2 )2 or -N(R 5 )S(O)2R 3 .

19. The compound of claim 18, wherein G is -N(R 2 )C(S)N(R 2 )2.

20. The compound of claim 19, wherein G is -N(H)C(S)N(R 2 )2, wherein each R 2 is selected from optionally substituted C1-C6 aliphatic and OH.

22. The compound of any one of claims 1-17, wherein G is an optionally substituted 4- to 12-membered heterocyclic ring.

23. The compound of any one of claims 1-17, wherein G is selected from:

26. The compound of claim 1, wherein the compound is represented by Formula Ila: 、 、 、 、 、 、 、 、 、 、 and .

24. The compound of claim 1, wherein the moiety -L 3 - G is selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , and .

25. The compound of claim 1, wherein -L 3 - G is selected from: 、 、 、 、 、 、 、 、 、 and . Ila or a pharmaceutically acceptable salt thereof.

27. The compound of claim 1, wherein the compound is represented by Formula lib: Lib or a pharmaceutically acceptable salt thereof.

28. The compound of claim 1, wherein the compound is represented by Formula lie: lie or a pharmaceutically acceptable salt thereof.

29. The compound of claim 1, wherein the compound is represented by Formula Ilia: Ilia or a pharmaceutically acceptable salt thereof.

30. The compound of claim 1, wherein the compound is represented by Formula Illb: Illb or a pharmaceutically acceptable salt thereof.

31. The compound of claim 1, wherein the compound is represented by Formula IIIc: IIIc or a pharmaceutically acceptable salt thereof.

32. The compound of claim 1, wherein the compound is represented by Formula IIId: IIId or a pharmaceutically acceptable salt thereof.

33. The compound of claim 1, wherein the compound is represented by Formula IIIe: IIIe or a pharmaceutically acceptable salt thereof.

34. The compound of claim 1, wherein the compound is represented by Formula IIIf: IIIf or a pharmaceutically acceptable salt thereof.

35. The compound of claim 1, wherein the compound is represented by Formula IIIg: IIIg or a pharmaceutically acceptable salt thereof.

36. The compound of claim 1, wherein the compound is selected from Table 1.

37. A particle comprising the compound of any one of claims 1-36 and a nucleic acid.

38. The particle of claim 37, wherein the nucleic acid is RNA, DNA, or a mixture thereof.

39. The particle of claim 38, wherein the RNA is mRNA.

40. The particle of claim 39, wherein the RNA is modRNA, circRNA, saRNA, taRNA, or uRNA.

41. The particle of claim 37, wherein the DNA is linear DNA, plasmid DNA, minicircle DNA, nanoplasmid DNA, dogbone DNA, or transposon.

42. The particle of any one of claims 37-41, wherein the particle further comprises one or more of a helper lipid, a polymer-conjugated lipid, or a sterol.

43. The particle of claim 42, wherein the helper lipid is a phospholipid. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 44. The particle of claim 43, wherein the phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, more preferably selected from the group consisting of: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), didocosanoylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (CI 6 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanyl-phosphatidylethanolamine (DPyPE), and combinations thereof.

45. The particle of any one of claims 42-44, wherein the polymer-conjugated lipid is selected from the group consisting of: poly(ethylene glycol) (PEG)-bound lipids, poly(sarcosine) (pSar)-bound lipids, poly(aminoethoxyethoxyacetic acid) (pAEEA)-bound lipids; and poly(2-methylaminoethoxyethoxyacetic acid) (pMAEEA)-bound lipids.

46. The particle of any one of claims 42-45, wherein the polymer-conjugated lipid is a PEG-lipid selected from the group consisting of: PEG-DAG, PEG-PE, PEG-S-DAG, PEG2000-DMG, PEG-cer, PEG dialkyloxypropyl carbamates, and combinations thereof.

47. The particle of any one of claims 42-46, wherein the solid sterol is selected from beta-sitosterol, stigmasterol, cholesterol, cholecalciferol, ergocalciferol, calcipotriol, botulinum toxin, lupeol, ursolic acid, oleanolic acid, cycloartenol, lanosterol, or alpha-tocopherol.

48. The particle of any one of claims 42-47, wherein the particle is characterized by an N / P ratio of about 4 to about 16.

49. A method of increasing or causing an increase in RNA expression in a target in a subject, the method comprising administering to the subject a composition comprising the particle of any one of claims 42-48.

50. The method of claim 49, wherein the target is selected from the group consisting of lung, liver, spleen, heart, brain, lymph node, bladder, kidney, and pancreas.

51. A method of treating a disease, disorder, or condition in a subject, the method comprising administering to the subject a composition comprising the particle of any one of claims 42-48.

52. The method of claim 51, wherein the disease, disorder, or condition is an infectious disease, a cancer, a genetic disorder, an autoimmune disease, or a rare disease.

53. The method of any one of claims 48-52, wherein the particle is administered parenterally or intranasally.

54. The method of claim 53, wherein the particle is administered intramuscularly, subcutaneously, intradermally, or intravenously.

55. The particle of any one of claims 42-48 for use as a medicament.

56. The particle of any one of claims 42-48 for use in the treatment and / or prevention of a disease or disorder, wherein the disease or disorder is an infectious disease, a cancer, a genetic disorder, an autoimmune disease, or a rare disease.

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