Cationic polymer, drug delivery system containing cationic polymer and application

Abstract

The invention relates to the technical field of medicines, in particular to a cationic polymer, a drug delivery system containing the same and application. The cationic polymer is a cationic polymer PEI modified by a cyclic disulfide group, the cyclic disulfide group is shown as a formula (I)-a formula (III), the polymer provided by the invention can be used as a drug carrier, especially a carrier of a nucleic acid drug, and targeted delivery of extrahepatic organ tissues is realized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to cationic polymers and drug delivery systems and applications comprising the same. Background Technology

[0002] In the field of medical treatment, gene therapy is not only an important means of treating genetic defects, but also a crucial strategy for treating tumors and neurological diseases. Gene vectors are key to the success of gene delivery, especially in delivering active ingredients to intracellular targets. Appropriate delivery carriers are needed to provide sufficient protection and allow the active ingredients to be effectively delivered to specific tissues within the body. However, delivery carriers must overcome various extracellular and intracellular barriers to reach their intracellular target sites. Although viral vectors are effective for gene delivery compared to non-viral vectors, their use comes with several risks, including toxicity, immunogenicity, and limitations on the size of the genetic material cargo. Non-viral vectors are generally safer and easier to mass-produce, but their transfection efficiency is relatively low.

[0003] The use of cationic polymers as nonviral synthetic carriers for delivering active ingredients, more specifically nucleic acids such as mRNA, to target cells has attracted considerable attention.

[0004] As a negatively charged nucleic acid macromolecule, the inherent instability of mRNA limits its direct application in vivo. How to efficiently deliver mRNA drugs to target tissues and cells is a key problem that urgently needs to be solved in the field of mRNA drug development. Nanomaterial-based delivery systems have been developed to enhance cytoplasmic delivery of mRNA. This method can not only effectively protect mRNA from ribozyme degradation and prolong its metabolic cycle in vivo, but also effectively increase drug accumulation in target tissues and organs, enhancing therapeutic efficacy. In particular, delivery systems represented by lipid nanoparticles (LNPs) have achieved great commercial success. However, LNPs still have many limitations, such as complex formulation processes, demanding storage conditions, and a tendency to accumulate in the liver. These characteristics greatly limit the treatment and application of mRNA drugs for non-hepatic related diseases.

[0005] Therefore, developing novel delivery systems for targeted delivery to extrahepatic organs is a pressing issue that needs to be addressed.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] This invention provides a novel cationic polymer, namely, a cationic polymer, polyethyleneimine (PEI), containing cyclic disulfide groups. By utilizing the sulfur-sulfide interaction between the cyclic disulfide groups and free thiol groups, and by appropriately adjusting the hydrophilicity / hydrophobicity of the polymer and the spacing between functional groups, stable encapsulation of nucleic acid drugs (e.g., mRNA) and thiol-mediated cellular uptake are achieved, thereby effectively enhancing the delivery efficiency of nucleic acid drugs (e.g., mRNA) and realizing targeted delivery to extrahepatic organs and tissues.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a polymer or a pharmaceutically acceptable salt, isomer, or stable isotopic derivative thereof, wherein the polymer is a cationic polymer PEI modified with an RO group, wherein the RO group is selected from one of formulas (I)-(III). , , ; Wherein, R1 is selected from , where a is 0 or 1, and b is 0, 1, 2 or 3; R3 is a direct key or c is an integer from 1 to 5; R2 is selected from H, -CH3, -CH2CH3, , .

[0009] Preferably, the PEI is a dendritic PEI or a linear PEI.

[0010] Preferably, the molecular weight (MW) of the PEI is 600~25000, more preferably 600~10000, and even more preferably 600~6000.

[0011] Preferably, the polymer has the structural formula shown in formula (IV) or (V). , ; in, R is selected from H or R0; In equation (IV), R is selected from H or R0; R may be the same or different, provided that at least one R is R0; n is an integer from 1 to 10, preferably an integer from 2 to 8, more preferably an integer from 3 to 6, and most preferably 3; m is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50; p is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50.

[0012] Preferably, the polymer has the following structural formula:

[0013] Wherein, m is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50; q is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50; p is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50. R is selected from H or R0; R4 is a direct key or c is an integer from 1 to 5; or, R4 is selected from... Where a is 0 or 1, b is an integer from 1 to 20, preferably an integer from 1 to 10; or R4 is selected from... , where n is an integer from 1 to 20, preferably an integer from 1 to 10.

[0014] Preferably, the polymer has the structural formula shown in formula (VI) or (VII). , .

[0015] In a second aspect, the present invention provides a method for preparing the polymer described in any one of the above claims or its pharmaceutically acceptable salts, isomers, or stable isotope derivatives, the method comprising: reacting polyethyleneimine with a compound containing a cyclic disulfide group (such as dithiopentane-trimethylene carbonate) in the presence of a solvent and a catalyst to obtain the polymer.

[0016] Thirdly, the present invention provides the use of the polymer described in any of the above claims, or its pharmaceutically acceptable salts, isomers, or stable isotope derivatives, in the preparation of drug delivery carriers; Preferably, the target organ or tissue of the drug delivery carrier includes at least one of the following: spleen, pancreas, lymph nodes, spinal cord, brain, or placenta; and / or, The target organ or tissue of the drug delivery vehicle includes tumor tissue and / or immune cells.

[0017] Fourthly, the present invention provides a polymer complex comprising any of the polymers described above or a pharmaceutically acceptable salt, isomer, or stable isotope derivative thereof, optionally further comprising a therapeutic agent and / or a preventive agent; Preferably, the therapeutic agent and / or preventive agent is selected from one or more of nucleic acids, peptides, and negatively charged small molecule drugs; Preferably, the nucleic acid is selected from one or more of voluntary RNA, small interfering RNA, antisense oligonucleotides, and short hairpin RNA.

[0018] Preferably, the method includes: reacting the polymer, the therapeutic agent, and / or the preventive agent under acidic conditions to prepare the polymer complex.

[0019] Fifthly, the present invention provides a drug delivery system comprising the polymer complex described above.

[0020] In a sixth aspect, the present invention provides the use of the polymer described in any of the above claims or its pharmaceutically acceptable salts, isomers, stable isotope derivatives, or polymer complexes described in the above claims, or the drug delivery system described in the above claims, in the preparation of drugs that precisely deliver functional mRNA to specific organs or tissues; Preferably, the specific organ or tissue includes at least one of the following: spleen, pancreas, lymph nodes, spinal cord, brain, or placenta; and / or, The specific organ or tissue includes tumor tissue and / or immune cells.

[0021] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: The cyclic disulfide functionalized cationic polymer disclosed in this invention possesses highly efficient cellular uptake and cytoplasmic delivery capabilities. Nanoparticles encapsulating functional nucleic acid (mRNA) drugs achieve rapid intracellular internalization efficiency through sulfur-sulfur exchange, thereby enabling highly efficient cytoplasmic delivery of nucleic acid (mRNA) in various cell lines. The cyclic disulfide functionalized cationic polymer disclosed in this invention features a simple, efficient, and controllable preparation method, while also exhibiting good protection and loading properties for nucleic acid drugs. The cyclic disulfide functionalized cationic polymer prepared in this invention enables precise targeted delivery to extrahepatic organs and tissues in vivo, providing a universal and effective platform for the development of nucleic acid (mRNA) drugs targeting extrahepatic diseases, and possesses enormous application potential. Attached Figure Description

[0022] Figure 1 This is a characterization of the polymer prepared in Example 1, where A is the DTC of this invention. 17 -The chemical synthesis reaction formula for PEI polymer synthesis; B is DTC. 17 -PEI polymer 1 1H NMR (600 MHz, DMSO-d6) characterization spectrum.

[0023] Figure 2This section describes the physicochemical characterization of different polymer nanoparticles in Example 2, where A represents the particle size and zeta potential of different DTC-PEI nanoparticles; B represents the size characterization using TEM; C represents the encapsulation efficiency of different polymers for mRNA; and D represents the DTC... 17 - Storage stability diagram of PEI drug-loaded nanoparticles at 4°C; E is the transfection efficiency of nanoparticles in Example 2 for efficiently delivering mRNA to multiple cell lines.

[0024] Figure 3 Example 3 shows the enhancement of thiol-mediated cellular uptake by the dithiopentane modification of nanoparticles. A is a schematic diagram of the thiol-mediated cellular uptake mechanism; B is a laser scanning confocal microscope image after the thiol inhibitor DTNB blocks thiol-mediated cellular uptake; and C is a flow cytometry analysis of thiol-mediated cellular uptake.

[0025] Figure 4 This is the endosome escape of DTC-PEI nanoparticles in Example 4. Among them, A is a confocal microscope image of endosome escape at 2, 4 and 8 h after nanoparticle uptake; B is the Pearson correlation coefficient between nanoparticles and lysosomes at three different time points; C is the verification of the endosome escape mechanism.

[0026] Figure 5 This describes the spleen-specific targeted delivery of different DTC-PEI nanoparticles in vivo 6 hours after intravenous injection in Example 5. A represents in vivo imaging of mice and bioluminescence imaging of the heart, liver, spleen, lung, and kidney in vitro; B represents the bioluminescence intensity of the spleen region; C represents the proportional relationship of bioluminescence intensity of the heart, liver, spleen, lung, and kidney in vitro; D and E) show the trends of protein expression changes in mice at 6, 24, and 48 hours.

[0027] Figure 6 This refers to the pancreas-specific targeted delivery of different DTC-PEI nanoparticles in vivo 6 hours after intraperitoneal injection in Example 6. A represents in vivo imaging of mice and bioluminescence imaging results of the heart, liver, spleen, lung, kidney, and pancreas in vitro; B represents the bioluminescence intensity of the pancreas region; C represents the relative proportions of bioluminescence intensity of the heart, liver, spleen, lung, kidney, and pancreas in vitro; D and E represent in vivo transfection imaging results after intraperitoneal injection of the commercial transfection reagent SM-102 LNP and the distribution ratio of mRNA expression in each major organ.

[0028] Figure 7This describes the lymph node-specific targeted delivery achieved in vivo 6 hours after subcutaneous injection of different DTC-PEI nanoparticle tails in Example 7. A shows the bioluminescence imaging results of in vivo mouse imaging; bioluminescence intensity of the isolated heart, liver, spleen, lung, and kidney; B shows the bioluminescence imaging results; C shows the comparison of bioluminescence intensity; the distribution of nanoparticles in organs such as the heart, liver, spleen, lung, and kidney; D shows the imaging of Cy5 fluorescence signal; and E shows the comparison of fluorescence signal intensity of naked mRNA and polymer-encapsulated mRNA enriched in different organs.

[0029] Figure 8 This refers to the targeted delivery of different DTC-PEI nanoparticles to the spinal cord and brain in vivo after intrathecal injection for 6 hours in Example 8. In this example, A represents the bioluminescence imaging results of mouse in vivo imaging; B represents the bioluminescence intensity; C represents the relative proportion of bioluminescence intensity in the heart, liver, spleen, lung, kidney, spinal cord, and brain; and D represents the bioluminescence imaging results in the heart, liver, spleen, lung, kidney, spinal cord, and brain.

[0030] Figure 9 In Example 9, different DTC-PEI nanoparticles were intravenously injected for 6 hours to achieve placental-targeted delivery in vivo. In this example, A is the bioluminescence imaging result of mouse in vivo imaging; B is the bioluminescence intensity; C and D are the (C) bioluminescence imaging and (D) intensity of the heart, liver, spleen, lung, kidney, placenta and embryo, respectively.

[0031] Figure 10 This describes the synthesis and characterization of the LPEI-En polymer in Example 10; where A is the chemical reaction formula for the synthesis of LPEI-En in this invention; and B is the chemical reaction formula for LPEI-E14. 1 1H NMR (600 MHz, DMSO-d6) spectrum.

[0032] Figure 11 This describes the preparation and physicochemical characterization of the LPEI-En@mRNA complex in Example 11; it includes the particle size and Zeta potential of the nanoparticles, as well as the results of gel electrophoresis experiments.

[0033] Figure 12 This is a characterization of the efficient mRNA delivery in vitro to cells from the LPEI-En@mRNA polymer complex in Example 12, including the results of inverted fluorescence microscopy.

[0034] Figure 13 This is the physicochemical characterization of the LPEI-EnDm@mRNA complex in Example Fourteen, including the particle size and transfection percentage of the polymer and mRNA after assembly under different DTC and alkane chain modifications.

[0035] Figure 14This is the result of inverted fluorescence microscopy of HeLa cells transfected with LPEI-EnDm@mRNA in Example 14. Detailed Implementation

[0036] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0037] Through long-term and in-depth research, the inventors have creatively proposed a cationic polymer that can effectively encapsulate nucleic acids, such as mRNA, as a carrier and has good stability. Experiments have shown that the cationic polymer of this invention can effectively deliver nucleic acid drugs to extrahepatic organs or tissues, achieving precise targeted delivery to extrahepatic organs and tissues in vivo. Based on this, the present invention was completed.

[0038] On one hand, the present invention provides a polymer or a pharmaceutically acceptable salt, isomer, or stable isotopic derivative thereof, wherein the polymer is a cationic polymer PEI modified with an RO group, wherein the RO group is selected from one of formulas (I)-(III). , , ; Wherein, R1 is selected from , where a is 0 or 1, and b is 0, 1, 2 or 3; R3 is a direct key or c is an integer from 1 to 5 (e.g., 1, 2, 3, 4, 5). R2 is selected from H, -CH3, -CH2CH3, , .

[0039] In some embodiments, the PEI is a dendritic PEI or a linear PEI.

[0040] In some embodiments, the molecular weight (MW) of the PEI is 600 to 25,000.

[0041] In some embodiments, the molecular weight (MW) of the PEI is 600 to 10,000.

[0042] In some embodiments, the molecular weight (MW) of the PEI is 600 to 6000.

[0043] In a specific and preferred embodiment, the molecular weight (MW) of the PEI is 1800.

[0044] In some embodiments, the polymer has the structural formula shown in formula (IV) or (V). , ; in, R is selected from H and R0; In equation (IV), R is selected from H or R0; R may be the same or different, provided that at least one R is R0; n is an integer from 1 to 10; m is an integer from 1 to 60; p is an integer from 1 to 60.

[0045] In some implementations, n is an integer from 2 to 8, such as 2, 3, 4, 5, 6, 7, 8.

[0046] In some implementations, n is an integer from 3 to 6.

[0047] In some implementations, n is 3.

[0048] In some implementations, m is an integer from 1 to 60, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc.

[0049] In some implementations, m is an integer from 10 to 50.

[0050] In some implementations, m is an integer from 20 to 50.

[0051] In some implementations, m is an integer from 20 to 50.

[0052] In some implementations, p is an integer from 10 to 50, such as 10, 15, 20, 25, 30, 35, 40, 45, 50; In some implementations, p is an integer from 20 to 50.

[0053] In some embodiments, the polymer has the following structural formula:

[0054] Wherein, m is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50; q is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50; p is an integer from 1 to 60, preferably an integer from 10 to 50, and more preferably an integer from 20 to 50. R is selected from H or R0; R4 is a direct key or c is an integer from 1 to 5; or, R4 is selected from... Where a is 0 or 1, b is an integer from 1 to 20, preferably an integer from 1 to 10; or R4 is selected from... , where n is an integer from 1 to 20, preferably an integer from 1 to 10.

[0055] In some embodiments, the polymer has the structural formula shown in formula (VI) or (VII). , .

[0056] On one hand, the present invention provides a method for preparing the polymer described in any of the above claims or its pharmaceutically acceptable salts, isomers, or stable isotope derivatives, the method comprising: reacting polyethyleneimine with a compound containing a cyclic disulfide group (such as dithiopentane-trimethylene carbonate) in the presence of a solvent and a catalyst to obtain the polymer.

[0057] In some embodiments, the compound containing a cyclic disulfide group is dithiopentane-trimethylene carbonate.

[0058] In some embodiments, the solvent is selected from DMSO.

[0059] In some embodiments, the catalyst is selected from one or more of triazabicyclo[4.4.0]dodec-5-ene, 1,8-diazabicyclo[5.4.0]undec-7-ene, and triethylamine.

[0060] In some embodiments, the reaction is carried out at a temperature of 35-40°C for 15-36 hours.

[0061] In some embodiments, the molar ratio of the polyethyleneimine and dithiopentane-trimethylene carbonate is 1:(1~30), for example 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, etc.

[0062] In some embodiments, the molar ratio of the catalyst to the polyethyleneimine is 1:(0.2~10), for example 1:0.3, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.

[0063] In some embodiments, the reaction further includes a post-processing step comprising dialyzing the reaction product in DMSO using a dialysis bag with a molecular weight cutoff of 1000-100000 Da, followed by dialyzing in a gradient acidic buffer to obtain the polymer. After freeze-drying, it can be stored for a long period.

[0064] On the one hand, the present invention also provides a polymer complex comprising the polymer described in any one of the preceding claims or a pharmaceutically acceptable salt, isomer, or stable isotope derivative thereof, optionally further comprising a therapeutic agent and / or a preventive agent.

[0065] In some embodiments, the therapeutic and / or preventive agents are selected from one or more of nucleic acids, peptides, and negatively charged small molecule drugs.

[0066] In some embodiments, the therapeutic and / or preventative agents are selected from nucleic acids.

[0067] In some embodiments, the nucleic acid is DNA or RNA.

[0068] In some embodiments, the nucleic acid is selected from one or more of spontaneous RNA, small interfering RNA, antisense oligonucleotides, and short hairpin RNA.

[0069] In some implementations, the nucleic acid is selected as spontaneous RNA (mRNA).

[0070] In some embodiments, the mRNA expresses a fluorescent reporter protein, a gene-editing protein, or a protein with therapeutic functions.

[0071] On the one hand, the present invention also provides a method for preparing the polymer complex described above, the method comprising: reacting the polymer, the therapeutic agent and / or the preventive agent under acidic conditions to prepare the polymer complex.

[0072] In some embodiments, the acidic condition is a pH of 4.0.

[0073] In some embodiments, the acidic condition is a sodium acetate buffer solution with a pH of 4.0.

[0074] In some embodiments, the therapeutic and / or preventive agents are selected from one or more of nucleic acids and negatively charged small molecule drugs.

[0075] In some embodiments, the therapeutic and / or preventative agents are selected from nucleic acids.

[0076] In some embodiments, the nucleic acid is selected from one or more of spontaneous RNA, small interfering RNA, antisense oligonucleotides, and short hairpin RNA.

[0077] In some implementations, the nucleic acid is selected as spontaneous RNA (mRNA).

[0078] In some embodiments, the mass ratio of mRNA to polymer is 1:(2~100), for example: 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, etc. When the mass of both is controlled within this range, a good overall balance can be achieved in terms of the stability and drug loading capacity of the formed nanoparticles.

[0079] In one aspect, the present invention also provides a drug delivery system comprising the polymer complex described above.

[0080] The drug delivery system of the present invention delivers a polymer complex (DTC) x -PEI 1800 @mRNA combines the targeting of tissues and organs with the selective killing specificity of functional mRNAs, achieving dual-specific delivery and therapeutic effects to tissues, organs, and cells.

[0081] On the one hand, the present invention provides the use of the polymer described in any of the above claims or its pharmaceutically acceptable salts, isomers, or stable isotope derivatives in the preparation of drug delivery carriers.

[0082] In some embodiments, the target organ or tissue of the drug delivery carrier includes at least one of the spleen, pancreas, lymph nodes, spinal cord, brain, or placenta.

[0083] On the one hand, the present invention provides the use of the polymer described in any of the above claims or its pharmaceutically acceptable salts, isomers, stable isotope derivatives, or polymer complexes described above, or the drug delivery system described above, in the preparation of drugs for the precise delivery of mRNA to specific tissues and cells; In some embodiments, the specific tissues and cells include at least one of the spleen, pancreas, lymph nodes, spinal cord, brain, or placenta.

[0084] On the one hand, the present invention provides the use of the polymer described in any of the preceding claims or its pharmaceutically acceptable salts, isomers, stable isotope derivatives, or polymer complexes described above, or the drug delivery system described above, for transfecting cells.

[0085] In some embodiments, the cells are selected from tumor cells and / or immune cells.

[0086] In some embodiments, the tumor cells are selected from human pancreatic cancer cells PANC-1, human ovarian cancer cells SK-OV-3, human astrocytoma U87, human ovarian cancer cells SK-OV-3, mouse melanoma cells B16-F10, mouse prostate cancer cells RM1, mouse lung cancer cells LLC, mouse colon cancer cells MC38, mouse breast cancer cells 4T1, mouse mononuclear macrophage leukemia cells RAW264.7, and human T lymphocyte leukemia cells Jurkat.

[0087] In some embodiments, the immune cells include bone marrow-derived dendritic cells DC2.4.

[0088] Terminology Explanation: As used herein, the term "PEI" refers to polyethyleneimine (PEI), also known as polyazidepropane, a water-soluble polymer. Polyethyleneimine exists in two forms: linear and branched. Linear polyethyleneimine contains only secondary amines, while branched polyethyleneimine contains primary, secondary, and tertiary amine groups. Polyethyleneimine can condense RNA into nanoparticles, which can adhere to negatively charged cell surface residues and enter the cell via endocytosis. Those skilled in the art can select appropriate PEIs based on the specific circumstances, and these appropriate PEIs are all within the scope of this invention. Furthermore, the cationic polymers of this invention are not limited to PEI.

[0089] The compounds of this invention comprise isotopic derivatives thereof. The term "isotopic derivative" refers to a compound whose structure differs only in the presence of one or more isotopically enriched atoms. For example, compounds having the structure disclosed herein use "deuterium" or "tritium" instead of hydrogen, or use... 18 F-fluorine labeling ( 18 F isotopes) can be used instead of fluorine, or... 11 C-, 13 C-, or 14 C-enriched carbon ( 11 C-, 13 C-, or 14 C-carbon labeling; 11 C-, 13 C-, or 14Compounds in which carbon atoms are replaced by C-isotopes are within the scope of this disclosure. Such compounds can be used as analytical tools or probes in, for example, biological assays, or as in vivo diagnostic imaging tracers for diseases, or as tracers for pharmacodynamic, pharmacokinetic, or receptor studies. The various deuterated forms of compounds disclosed herein refer to compounds in which each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds by referring to relevant literature. Commercially available deuteration starting materials can be used in the preparation of deuterated forms of compounds, or they can be synthesized using conventional techniques with deuteration reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane. Deuterated compounds generally retain activity comparable to undeuterated compounds, and better metabolic stability can be achieved when deuterated at certain specific sites, resulting in certain therapeutic advantages.

[0090] As used herein, the term “pharmaceutical acceptable” means that such compounds, materials, compositions, and / or dosage forms are suitable for contact with patient tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within reasonable medical judgment, have a reasonable benefit / risk ratio, and are effective for their intended use.

[0091] "Pharmaceutically acceptable salt" refers to a salt of the compounds of this invention that is safe and effective in mammalian use and possesses the expected biological activity. The salt can be prepared separately during the final isolation and purification of the compound, or by reacting a suitable group with a suitable base or acid. Bases commonly used to form pharmaceutically acceptable salts include inorganic bases as well as organic bases. Acids commonly used to form pharmaceutically acceptable salts include inorganic acids as well as organic acids.

[0092] As used herein, the term "isomer" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may include one or more chiral centers and / or double bonds and thus exist as a stereoisomer, such as a double-bonded isomer (i.e., a geometric E / Z isomer) or a diastereomer (e.g., an enantiomer (i.e., (+) or (-))) or cis / trans isomer. This disclosure covers any and all isomers of the compounds described herein, including stereoisomeric pure forms (e.g., geometrically pure, enantiomeric pure, or diastereomeric pure) and mixtures of enantiomers and stereoisomers, such as racemates. Mixtures of enantiomers and stereoisomers of compounds, and the ways in which they are resolved into their constituent enantiomers or stereoisomers, are well known.

[0093] As used in this article, the singular forms of “a,” “an,” and “the” include plural references, and vice versa, unless the context clearly indicates otherwise.

[0094] The terms involved in this invention have been defined above. Those skilled in the art can also understand the above terms in conjunction with the prior art. The following is a further description based on the content of this invention and the definition of the terms.

[0095] The "room temperature" mentioned is not a specific temperature value, but refers to a temperature range of 10-30℃.

[0096] In this invention, the term "about" when used to refer to a measurable value, such as mass, time, temperature, etc., means that it can fluctuate within a certain range around a specific value, such as ±10%, ±5%, ±1%, ±0.5%, or ±0.1%.

[0097] In this embodiment of the invention, the particle size and distribution of polymer micelles, as well as the surface Zeta potential, were determined by dynamic laser light scattering (DLS) and electrophoresis using a Zetasizer Nano-Z (Malvern). The DTC content in the polymer was tested using a dual-beam UV-Vis spectrophotometer (UH5300 Hitachi). Nuclear magnetic resonance (NMR) measurements were performed using deuterated dimethyl sulfoxide (DMSO-d6) as the solvent on an Agilent 600 MHz DD2 (DirectDrive2) spectrometer. 1 1H NMR spectrum, where chemical shifts are based on residual solvent peaks (DMSO: 1 ¹H NMR: δ 2.50. The CCK-8 assay used a ThermoMultiskan FC microplate reader to measure the UV absorbance of the samples at 450 nm. Flow cytometry (BD FACSCalibur) was used to characterize the cellular uptake of polymer nanoparticles and mRNA transfection. The in vivo mRNA delivery efficiency of the polymer nanoparticles was assessed using a small animal imaging system (Caliper IVIS II system, Perkin Elmer).

[0098] In the embodiments of this invention, all reagents involved are commercially available or synthesized according to known methods. For example, dithiopentane-trimethylene carbonate (DTC) is not limited to the method disclosed in reference PMID: 35009324. The cells used in the embodiments of this application include, but are not limited to, human fibroblasts L929, human pancreatic cancer cells PANC-1, human ovarian cancer cells SK-OV-3, human astrocytoma U87, human ovarian cancer cells SK-OV-3, mouse melanoma cells B16-F10, mouse prostate cancer cells RM1, mouse lung cancer cells LLC, mouse colon cancer cells MC38, mouse breast cancer cells 4T1, mouse bone marrow-derived dendritic cells DC2.4, mouse mononuclear macrophage leukemia cells RAW264.7, and human T lymphocyte leukemia cells Jurkat, all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. They were prepared using a combination of 10% fetal bovine serum (FBS, Gibco) and 1% penicillin (100 IU / mL penicillin and 100 IU / mL penicillin). Cells were cultured under standard conditions in DMEM medium (HyClone, USA) or RPMI 1640 cell medium (Gibco) containing μg / mL streptomycin.

[0099] The design of reporter gene and therapeutic mRNA sequences is based on the following: The mRNA sequences used in this invention are designed from reference sequences of commonly used reporter genes and cytokines in publicly available databases, including GFP, Luciferase, tdTomato, OVA, interleukin-10 (IL-10), and interleukin-12 (IL-12). The coding sequences (CDS) of these genes are derived from publicly available sequences in the corresponding NCBI GenBank or RefSeq databases, and codon optimization can be performed as needed to improve their translation efficiency in mammalian cells. For example, the reference sequences include GFP (GenBank accession number L29345), tdTomato (GenBank accession number KJ081243), and mouse IL-10 (RefSeq accession number NM_010548). The codon optimization process follows principles such as mammalian preferred codon usage, GC content control, removal of potentially repressive sequences, and adaptation to in vitro transcription systems, without altering the protein amino acid sequence. The mRNA sequences used in the experiments of this invention were all designed using these reference sequences as templates, which does not affect the understanding of the technical effects of this invention by those skilled in the art.

[0100] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sam brook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0101] Example 1: Synthesis and Characterization of DTC-PEI Polymer

[0102] DTC 17 - Take the synthesis process of PEI polymer as an example ( Figure 1 A), take the dendritic PEI 1800 DTC (180 mg, 0.1 mM) and DTC (326.4 mg, 1.7 mM) were dissolved in 5 mL of DMSO solvent, and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD, 41.7 mg, 0.3 mM) were added as a catalyst. The reaction was carried out at 37 °C with stirring for 24 h. After the reaction, the mixture was dialyzed twice in DMSO solvent for 4 h each time using a dialysis bag with a molecular weight cutoff of 1000 Da to remove unreacted reactants and catalyst. Then, the mixture was dialyzed in 100 mM sodium acetate buffer (pH 4.0), and gradually replaced with neutral deionized water. Finally, DTC was obtained by freeze-drying. 17 - Lyophilized powder of DTC-PEI polymer. The DTC-PEI polymer was dissolved in deuterated DMSO solvent, and the ring-opening reaction of DTC was studied using nuclear magnetic resonance spectroscopy. Yield: 85%. 1 H NMR (600 MHz, DMSO-) d 6 (ppm): δ 4.0 (-COC-C) H 2 -C), δ3.4 (-CC) H 2 -OH) and δ3.02 (-C(C H 2 SSC H 2 )-).

[0103] DTC 17Taking PEI polymer as an example, 17 DTC units are modified onto the primary amine and some secondary amines of polyethyleneimine, respectively, where the DTC ring-opening generates a hydroxycarbamate-like structure. 17 -The 1H NMR spectrum of the PEI polymer shows ( Figure 1 (B) The open-ring DTC exhibits three distinct chemical shifts at 4.0, 3.4, and 3.0 ppm. Integrating the peak areas at these shifts yields a peak area ratio of 1:1:2, similar to the predicted result, confirming that DTC... 17 -PEI was successfully synthesized.

[0104] Following the above synthesis method, DTCx-PEI polymers with different DTC graft numbers were prepared, where x = 10, 14, 20, 23.

[0105] Example 2: Synthesis and Physicochemical Characterization of the DTC-PEI@mRNA Complex First, the mRNA was diluted to 20 μg / mL using HEPES buffer (10 mM, pH 5.2) to obtain an aqueous mRNA solution, which was then reconstituted with 100 mM sodium acetate solution to DTC. x -PEI polymers (x=10, 14, 17, 20, 23) were dissolved to form a polymer aqueous solution with a concentration of 10 mg / mL. Next, an appropriate amount of the polymer aqueous solution was placed at the bottom of a 1.5 mL EP tube, and the mRNA aqueous solution was quickly mixed with the polymer using a pipette. After standing for 5 min, DTC was obtained. x -PEI 1800 @mRNA: By adjusting the mixing ratio of polymer and mRNA, DTC-PEI@mRNA nanoparticles with different nitrogen-to-phosphorus ratios were obtained. The particle size and zeta potential of the prepared nanoparticles with a nitrogen-to-phosphorus ratio of 10 were determined by dynamic light scattering (DLS). Ribogreen experiments were used to characterize the encapsulation of mRNA by the polymer.

[0106] The particle size of the prepared nanoparticles was detected using DLS. Five different DTCx-PEI@mRNAs (x=10, 14, 17, 20, 23) had similar particle sizes. Figure 2 The hydrated particle size of DTC (dichlorodimethylamine) is approximately 65 nm, the PDI is about 0.15, and the zeta potential is about +15 mV. Transmission electron microscopy (TEM) was used to examine the DTC. 17 Morphological analysis was performed on PEI@mRNA, and TEM results showed ( Figure 2(B) Nanoparticles prepared using a simple mixing method exhibited a uniform particle size distribution, with a size of approximately 35 nm. To more accurately quantify the encapsulation efficiency of DTCx-PEI polymer for mRNA, the small molecule dye Ribogreen was used to detect the content of unencapsulated free mRNA. Ribogreen is a highly efficient and specific RNA-binding dye that generates a fluorescent signal upon binding to RNA and does not interfere with DNA or other molecules. The fluorescence signal intensity is strongly correlated with the RNA content. Ribogreen experimental results showed ( Figure 2 In the study (C), with a drug loading rate of 16%, the encapsulation efficiency of the five DTCx-PEI polymers for mRNA was all above 95%, indicating that DTCx-PEI polymers have a strong encapsulation ability for mRNA and can efficiently form complexes with mRNA. Meanwhile, the prepared DTC... 17 -PEI nanoformulations were stored at 4°C to monitor their storage stability and the protective ability of the polymer carrier for mRNA. Samples were taken weekly over a four-week monitoring period, and transfection efficiency was validated in 293T cells. Results showed that ( Figure 2 DTC 17 -PEI@mRNA exhibits good storage stability, and the transfection efficiency of the nano-formulation did not decrease significantly over four weeks, demonstrating that the polymer carrier can effectively protect mRNA from nuclease degradation.

[0107] To further investigate the ability of polymeric carriers to deliver mRNA in other cells, a variety of tumor cells were used, including human pancreatic cancer cells PANC-1, human ovarian cancer cells SK-OV-3, human brain astrocytoma U87, human ovarian cancer cells SK-OV-3, mouse melanoma cells B16-F10, mouse prostate cancer cells RM1, mouse lung cancer cells LLC, mouse colon cancer cells MC38, mouse breast cancer cells 4T1, and various immortalized immune cells DC2.4, RAW264.7, and Jurkat cells, for DTC. 17 The mRNA transfection capability of the PEI polymer vector was screened. The specific steps are as follows: 1 × 10⁻⁶ mRNA transfection vectors were used for each cell type. 5 Cells were seeded at a density of 100 cells per well in a 24-well plate and incubated at 37°C until 80% confluence was achieved. Samples were prepared according to the method described above for preparing polymer nanoparticles and the manual for the commercial transfection reagent Lipo 2000. After co-incubation with cells for 4 h, the medium was changed with fresh medium and incubated for another 16 h. Transfection efficiency was then assessed using an inverted fluorescence microscope and flow cytometry.

[0108] The experimental results are as follows ( Figure 2 E), for various tumor cells (human pancreatic cancer cells PANC-1, human ovarian cancer cells SK-OV-3, mouse melanoma cells B16-F10, human brain astrocytoma U87, mouse prostate cancer cells RM1, mouse lung cancer cells LLC), DTC 17 -PEI polymers can all achieve highly efficient transfection, with an eGFP positivity rate greater than 75%, and some cells (U87, RM1, LLC) even reaching nearly 100% transfection efficiency. In contrast, the commercial transfection reagent Lipo 2000 only achieves a transfection positivity rate of less than 40% under the same conditions. Analysis of mRNA protein translation levels was performed using DTC. 17 The PEI polymer group generally exhibited a stronger green fluorescence signal, with nearly two-fold increased eGFP protein expression levels compared to the Lipo group, indicating that DTC... 17 -PEI polymers exhibit stronger mRNA delivery efficiency compared to the commercial transfection reagent Lipo 2000.

[0109] Example 3: Dithiopentane modification enhances thiol-mediated cellular uptake.

[0110] To investigate the role of dithiopentane rings on the surface of polymer nanoparticles in cellular uptake, this example synthesized TMC using trimethylene carbonate (TMC). 20 -PEI, as DTC 17 The negative control group for -PEI polymers, TMC and DTC have similar cyclic carbonate structures, but TMC does not contain a dithiopentane structure. Meanwhile, the thiol-reactive reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used to remove free thiol groups from the cell surface, and the interaction between cell surface thiol groups and the dithiopentane rings on the nanoparticle surface was studied using flow cytometry and confocal imaging. Figure 3 (A)

[0111] The specific experimental steps are as follows: In the confocal microscopy experiment, a laser scanning confocal microscope was used to more directly observe the interaction between dithiopentane rings and thiol groups on the cell membrane surface. DC2.4 cells were seeded in confocal culture dishes (1×10⁻⁶ cells / year). 5 Cells / well), incubated overnight at 37°C. Treatment groups were pretreated with 20 mM DTNB for 2 h, then incubated with DTC at 4°C. 17Cells were co-incubated with PEI for 1 h. Cells were washed three times with PBS to remove free Cy5 NPs from the culture medium. Then, cells were stained with FITC-WGA (green, 5 μg / mL) for 30 min, fixed with 4% paraformaldehyde for 10 min, and stained with DAPI (blue, 1 μg / mL) for 3 min. After each staining, the cells were washed three times with PBS to remove excess dye. Finally, the prepared samples were photographed using a confocal laser microscope to observe the co-localization of the cell membrane and nanoparticles.

[0112] In flow cytometry experiments, DC2.4 cells were seeded in 24-well plates (1×10⁻⁶ cells / well). 5 Cells per well were incubated overnight at 37 °C. Treatment and untreated groups were set up, with the treatment group receiving DTNB (20 mM) 2 h pretreatment, followed by incubation at 4 °C with DTC. 17 -PEI or TMC 20 -PEI co-incubation continued for 1 h. DC2.4 cells were then digested and collected, tested using a BD flow cytometer, and the flow cytometry data were analyzed using FlowJo software.

[0113] Confocal microscopy was used to observe the interaction between the cell membrane and the polymer carrier. Cell membrane structures were labeled with the dye FITC-WGA, and it can be seen that (…). Figure 3 (B) The nanoparticles with red signals are mostly located on the outer side of the cell membrane, ruling out the possibility of cellular uptake, indicating that the nanoparticles with dithiopental rings have undergone chemical cross-linking with the thiol groups on the cell membrane surface. After pretreatment with DTNB, the number of carriers adsorbed on the membrane surface decreased significantly. The dithiopental rings on the carrier surface can interact strongly with the thiols on the cell membrane surface, and this interaction further promotes the internalization of nanoparticles into the cell.

[0114] Among them, TMC-PEI NPs exhibited similar physicochemical properties as DTC-PEI NPs, including particle size and zeta surface charge. The thiol inhibitor DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) was used to pre-consume free thiols on the cell membrane surface, blocking thiol-mediated cellular uptake. With the use of DTNB, DTC... 17 Cellular uptake in the PEI polymer group decreased from 12605 to 7620, a reduction of approximately 40%, while that in the TMC group, which served as a negative control, decreased by 40%. 20 - No significant change was observed in the uptake of PEI polymers. Figure 4 C). This indicates that DTC 17The high cellular uptake of PEI polymers is related to thiol-mediated endocytosis. The sulfur-sulfur exchange between the dithiopentane ring on the nanoparticle surface and free mercaptoethanol on the cell membrane surface mediates cell surface adsorption, enhancing cellular uptake. Meanwhile, TMC... 20 -PEI nanoparticles do not exhibit this force.

[0115] Example 4: Study on endosomal escape and its mechanism The study investigated the endosome escape behavior of polymer nanoparticles at different time points. The specific steps were as follows: DC2.4 cells were seeded in confocal culture dishes (1×10⁻⁶ cells / year). 5 (1 cell / well), incubate overnight at 37°C. Then DTC 17 - PEI was added to cells and incubated for 1 h, followed by medium changes. Treatments were performed at 0, 1, 3, and 7 h. The treatment process was as follows: cells were washed three times with PBS to remove Cy5-doped nanoparticles from the culture medium; lysosomes were stained with Lyso-Tracker DND-26 (green, 50 nM) for 30 min; cells were fixed with 4% paraformaldehyde for 10 min; and nuclei were stained with DAPI (blue, 1 μg / mL) for 3 min. After each staining, cells were washed three times with PBS to remove excess dye. Finally, the prepared samples were photographed using a confocal laser microscope. Data were processed using ImageJ software, and the Pearson coefficient was calculated using the JACOP plugin.

[0116] To investigate the mechanism of polymer nanoparticle incorporation escape, the specific steps are as follows: DC2.4 cells were seeded in 24-well plates (1×10⁻⁶ cells / well). 5 Cells were incubated overnight at 37°C (cells / well). Cells were pretreated 0.5 hours beforehand with the endosome acidification inhibitor chloroquine (10 μM) and the proton pump inhibitor bafilomycin A1 (25 nM), respectively, followed by DTC treatment. 17 Cells were transfected with PEI@mGFP and incubated for 2 h, then cultured in fresh medium for another 16 h. DC2.4 cells were then digested and collected, tested using a BD flow cytometer, and the flow cytometry data were analyzed using FlowJo software.

[0117] Confocal microscopy was used to observe the co-localization of nanoparticles and endosomals. First, the polymer carrier was labeled with red Cy5, and lysosomes were labeled with green Lysotracker. The co-localization of the polymer carrier and endosomals was observed at 1, 2, 4, and 8 h, and analyzed using ImageJ software. The experimental results are as follows (…). Figure 4In cell A, at 1 h, numerous red dotted fluorescence was clearly observed around the cell nucleus, indicating that a large number of nanoparticles were taken up by the cell. However, most of these nanoparticles overlapped with the endosomes, appearing as yellow dotted fluorescence, with only a few showing red fluorescence. This indicates that most nanoparticles were still trapped within the endosomes. From 2 h to 4 h, the red dotted fluorescence gradually increased, indicating that nanoparticles were continuously escaping from the endosomes. However, due to ongoing endocytosis, the yellow dotted fluorescence also increased. After 4 h, the uptake of nanoparticles by the cell began to decrease, and the yellow dotted fluorescence began to decrease, gradually being replaced by red fluorescence. The change in the Pearson coefficient also reflects the process of endosome escape. Figure 4 In the first two hours (0-2 h), the internalization of cells was greater than the escape efficiency of endosomes, and the colocalization coefficient between nanoparticles and lysosomes increased continuously. However, between 2 and 8 h, the colocalization coefficient between the two began to decrease continuously as the internalization weakened and the number of endosomes escaped increased.

[0118] Two small molecule drugs associated with endosome escape, chloroquine and bafloxacin, were used to investigate DTC-modified DTC. 17 -The endosome escape mechanism of PEI polymers. Chloroquine, with its protonable multi-level amine structure, effectively inhibits endosome acidification and enhances the carrier's ability to escape from the endosome. Bafloxacin, a proton pump inhibitor, inhibits endosome acidification by hindering hydrogen ion transport. Results showed ( Figure 4 (C), after treatment with chloroquine, DTC 17 The transfection positivity rate of the PEI polymer group did not increase significantly, remaining at around 60%. In contrast, the transfection efficiency of the Lipo 2000 group increased from 20% to 37%, indicating that DTC... 17 -PEI polymer groups exhibit sufficiently high integrity escape efficiency. Furthermore, after treatment with bafloxacin, DTC... 17 The transfection positivity rate of the PEI polymer group decreased significantly, from 60% to 14%, reflecting the role of the proton pump in the treatment of DTC. 17 -PEI polymers are crucial for mRNA delivery, indicating that their endosome escape is mainly achieved through the proton sponge effect. The transfection efficiency of the Lipo 2000 group was almost not reduced, which is consistent with the literature reports that Lipo 2000 is mainly composed of liposomes and achieves endosome escape mainly through membrane fusion, without relying on the presence of proton pumps.

[0119] Example 5: Spleen-specific mRNA targeted delivery Following the method described in Example 2, five DTCx-PEI@mLuc (x=10, 14, 17, 20, 23) polymer nanoparticle formulations encapsulating Luc-mRNA were prepared, with an mRNA concentration of 37.5 μg / mL and a polymer-to-mRNA mass ratio of 6. The nanoparticle formulations were injected intravenously into C57BL / 6 mice (n=3, mRNA injection dose: 0.375 mg / kg). Six hours after injection, D-fluorescein potassium salt (150 mg / kg) was injected intraperitoneally into the mice. Five minutes later, in vivo imaging of the mice was performed using the Caliper IVIS II system (Perkin Elmer). The mice were then euthanized, and their major organs (heart, liver, spleen, lungs, and kidneys) were dissected. Bioluminescence imaging of the isolated organs was then performed again using the IVIS imaging system.

[0120] Experimental results show that ( Figure 5 (A) All five different DTCx-PEI polymers can effectively deliver mRNA and exhibit excellent spleen-targeted transfection capabilities. Among them, DTC... 17 -PEI polymers exhibit the best in vivo transfection performance, with bioluminescence values ​​reaching 3 × 10⁻⁶. 7 p / s, approximately 2 to 3 times that of other groups ( Figure 5 (B). To accurately quantify the specificity of polymer carrier transfection in vivo, mice were euthanized 6 hours after transfection, and their major organs, including the heart, liver, spleen, lungs, and kidneys, were dissected. Their bioluminescence values ​​were then quantified. The results showed ( Figure 5 (C), all five polymer vectors showed the ability to specifically target and transfect the spleen, among which DTC 17 -PEI polymers exhibited the strongest spleen-specific transfection capability, with spleen transfection fluorescence intensity accounting for as high as 93.7%. The duration of expression after in vivo transfection with the polymer vector was investigated. At three time points—6 h, 24 h, and 48 h post-transfection (Figures 5D-E)—the highest protein expression level was observed at 6 h post-transfection, while at 48 h it decreased to less than one-tenth of the original level, indicating rapid transient mRNA expression in the liver with high safety. In contrast, the commercially available transfection reagent SM-102 LNP showed that over 95% of protein expression occurred in the liver, which is not conducive to transfection of different organs.

[0121] Example 6: Pancreas-Specific mRNA Targeted Delivery Intraperitoneal injection can effectively avoid the first-pass effect in the liver, which may help to alter the in vivo distribution of nanoparticles. To investigate the effect of intraperitoneal injection on in vivo mRNA expression, five different DTCx-PEI polymers (x=10, 14, 17, 20, 23) were used to load mRNA encoding firefly luciferase and were intraperitoneally injected into mice at a dose of 0.375 mg / kg. In vivo mRNA transfection was characterized using an in vivo imaging system 6 h later.

[0122] Experimental results show that ( Figure 6 In group A), different levels of bioluminescent signals were expressed in the abdomen of each group of mice, indicating that the polymer carrier can also achieve effective mRNA expression via intraperitoneal injection. Furthermore, the in vivo expression of these five different DTC-PEI polymers showed a gradually increasing effect. Figure 6 (B), of which DTC 23 The total bioluminescence value of the PEI polymer group even reached 7.6 × 10⁻⁶. 8 p / s. Subsequently, the protein expression and distribution in various organs of the mouse were quantified, and surprisingly ( Figure 6 In mice, common organs such as the heart, liver, spleen, lungs, and kidneys did not show significant transfection, while significant protein expression was observed in the pancreatic tissue, accounting for over 95% of the total protein expression in all organs. These experimental results indicate that intraperitoneal injection of mRNA protein expression exhibits a clear pancreatic tissue-targeting nature. Weak expression in other organs may help reduce off-target side effects of related drugs, potentially providing new treatment options for pancreatic-related diseases. Commercially available lipid nanoparticles SM102 LNP (… Figure 6 After intraperitoneal injection, the liver region showed significant protein expression, accounting for as much as 52%, while pancreatic tissue transfection accounted for only 45%. The non-specificity of transfection made it more likely to cause systemic and hepatotoxicity, which reflects some of the advantages of dithiopentane-modified polymer carriers.

[0123] Example 7: Lymph Node-Specific mRNA Targeted Delivery mRNA nanoparticles were prepared according to the method described in Example 2. 0.2 mg / kg of the mRNA drug was injected subcutaneously into both sides of the base of the mouse tail. Six hours later, in vivo mRNA transfection was characterized using an in vivo imaging system. The results showed ( Figure 7 (A) Significant bioluminescent signals were observed in the inguinal lymph nodes of mice, indicating that the polymer carrier can achieve lymph node targeting via subcutaneous injection. Surprisingly, the signal was primarily observed in the lymph node region, with a small amount appearing at the injection site, and it did not accumulate or express in organs such as the heart, liver, spleen, lungs, or kidneys. Figure 7(Cy5-doped nanoparticles were used to study the biodistribution after subcutaneous injection. The results showed that the nanoparticles did not enter the bloodstream to other organs; Cy5 signaling was only observed in lymph nodes and the injection site.) Figure 7 (Drug-mediated desorption / retention), this characteristic of local residence will help reduce systemic toxicity and other side effects. The above experimental results show that, through subcutaneous injection at the tail base, the protein expression of mRNA exhibits obvious lymph node targeting properties.

[0124] Example 8: Targeted delivery of spinal cord and brain tissue-specific mRNAs mRNA nanoparticles were prepared according to the method described in Example 2. Intrathecal injection was administered into the cerebrospinal fluid of mice via a needle inserted into the L5-L6 intervertebral space. In vivo mRNA transfection was characterized using an in vivo imaging system 6 hours later. Experimental results showed ( Figure 8 In the spinal cord and brain regions of mice, significant bioluminescent signals were observed (in the middle A region). It is noteworthy that ( Figure 8 Even at extremely low mRNA doses (approximately 0.04 mg / kg), this injection method (B) achieved excellent mRNA delivery to the central nervous system, with a total bioluminescence value reaching 2 × 10⁷ p / s, indicating extremely high mRNA utilization efficiency. Subsequently, the distribution of protein expression in various organs of mice was quantified, and the results showed (…). Figure 8 The majority of the mRNA (mRNA) proteins are expressed in the spinal cord of the spine and the medulla oblongata of the brain, demonstrating that intrathecal injection can achieve highly efficient targeting of the central nervous system.

[0125] Example 9: Placenta-specific mRNA targeted delivery The mRNA nanoparticles were prepared according to the method described in Example 2 and administered intravenously to pregnant mice. Six hours later, the in vivo mRNA transfection status was characterized using an in vivo imaging system. The experimental results showed ( Figure 9 In contrast to healthy mice, pregnant mice showed a significant bioluminescent signal in their abdomens, with the bioluminescence intensity increasing by nearly an order of magnitude. Figure 9 (B). Subsequently, the distribution of protein expression in various organs of the mouse was quantified, and the results showed ( Figure 9 In the mouse placenta, apart from a small amount of protein expression observed in the spleen, most of the protein expression was distributed in the placental structure, and no protein expression was observed in the fetus. This indicates that the nanoparticles cannot penetrate the blood barrier of the fetus and will not affect the normal development of the fetus.

[0126] Example 10: Synthesis and Characterization of LPEI-En Polymer See Figure 10In section A, taking the synthesis process of LPEI-E14 polymer as an example, we take linear PEI... 2500 Tetradecyl acrylate was dissolved in 5 mL of propanol, and triethylamine (TEA) was added as a catalyst. The reaction was carried out at 37 °C with stirring for 24 h. After the reaction, the mixture was dialyzed in deionized water for 4 h using a dialysis bag with a molecular weight cutoff of 1000 Da to remove unreacted raw materials and catalyst. Finally, the mixture was freeze-dried to obtain the lyophilized powder of LPEI-E14 polymer. The LPEI-E14 polymer was dissolved in deuterated chloroform solvent for NMR testing.

[0127] Taking the LPEI-80E14 polymer with an 80% E14 modification ratio as an example, 45 E14 units were modified onto the secondary amine of polyethyleneimine, resulting in a Michael addition reaction between the PEI amino groups and the acrylate. The 1H NMR spectrum of the LPEI-80E14 polymer showed different chemical shifts at 3.7, 4.2, and 6.0 ppm. By integrating the peak areas at these shifts, the ratio of modified to unmodified amino group peak areas was calculated to be approximately 4:1, similar to the predicted result, proving the successful modification of LPEI-E14. Figure 10 (B)

[0128] LPEI-En polymers (n=12, 16, 18) were prepared according to the above method.

[0129] Example 11: Preparation and Physicochemical Characterization of LPEI-En@mRNA Polymer Complex First, mRNA was diluted to 20 μg / mL using HEPES buffer (10 mM, pH 5.2) to obtain an aqueous mRNA solution. Then, LPEI-En polymers (n=12, 14, 16, 18) were reconstituted with ethanol to obtain a polymer solution with a concentration of 10 mg / mL. Next, an appropriate amount of polymer solution was placed at the bottom of a 1.5 mL EP tube, and the mRNA aqueous solution was quickly mixed with the polymer using a pipette. After standing for 5 min, an LPEI-En@mRNA polymer complex was obtained. By adjusting the En modification ratio in the polymer and mixing it with mRNA, different LPEI-En@mRNA nanoparticles were obtained. The particle size and Zeta potential of the prepared nanoparticles with a nitrogen-to-phosphorus ratio of 10 were determined using dynamic light scattering (DLS).

[0130] The particle size of the prepared nanoparticles was detected using DLS. Different LPEI-En @mRNAs showed similar particle sizes. (See [link to DLS analysis]). Figure 11The hydrated particle size was 100-200 nm, the PDI was approximately 0.05-0.3, and the zeta potential was around +10 to +30 mV. The nanoparticles prepared using a simple mixing method exhibited a uniform particle size distribution. To more accurately quantify the encapsulation efficiency of the LPEI-En polymer for mRNA, gel electrophoresis was used to detect the content of unencapsulated free mRNA. The gel electrophoresis results showed that, with a drug loading rate of 10%, the LPEI-En polymer effectively encapsulated mRNA, with no significant mRNA leakage. Furthermore, no significant fluorescence of nucleic acid and dye was observed in the wells as the alkane chain length increased, indicating that LPEI-En has a strong encapsulation ability for mRNA and can efficiently form complexes with it.

[0131] Example 12: High-efficiency delivery of mRNA to cells in vitro using the LPEI-En@mRNA polymer complex. To further investigate the ability of the polymer vector to deliver mRNA in cells, HeLa cells were used to screen the mRNA transfection capability of LPEI-En@mRNA. The specific steps are as follows: each cell type was transfected at 1×10⁻⁶ mRNAs. 5 Cells were seeded at a density of [number] cells per well in a 24-well plate and incubated at 37°C until 80% confluence was achieved. Samples were prepared using the method described above for preparing polymer nanoparticles. After co-incubating with cells for 4 h, the medium was changed with fresh culture medium and incubated for another 16 h. Transfection efficiency was then assessed using an inverted fluorescence microscope. See [link to documentation]. Figure 12 The experimental results are as follows: Analysis of the mRNA protein translation level showed that the polymer group generally exhibited a stronger green fluorescence signal, indicating that the LPEI-En polymer phase exhibits a stronger mRNA delivery efficiency.

[0132] Example 13: Synthesis of LPEI-EnDm Polymer

[0133] Linear polyethyleneimine (PEI, Mn = 2.5 kDa) and acrylate monomers were first dissolved in anhydrous methanol / tetrahydrofuran (1:1, v / v) mixed solvent and transferred to a sealed round-bottom flask under nitrogen protection. The mixture was stirred at 50 °C for 24 h. After the reaction was completed, 1,2-dithiopentanetrimethylene carbonate (DTC) and tetrabutylammonium bromide (TBAB, 5 mol%) were added dropwise at room temperature with stirring as phase transfer catalysts. The reaction was continued at 37 °C for 24 h to trigger the ring-opening polymerization modification of DTC through nucleophilic addition. After the reaction was completed, the product was dried at 40 °C under a vacuum of 1 mbar to remove the solvent, yielding the final product.

[0134] Example 14: Preparation and Physicochemical Characterization of the LPEI-EnDm@mRNA Complex First, mRNA was diluted to 20 μg / mL using sodium acetate buffer (10 mM, pH 4.0) to obtain an aqueous mRNA solution. Then, the LPEI-EnDm polymer (m=10, 15, 20; n=10, 15, 20) was reconstituted with ethanol to obtain a polymer solution with a concentration of 10 mg / mL. Next, an appropriate amount of polymer solution was placed at the bottom of a 1.5 mL EP tube, and the mRNA aqueous solution was quickly mixed with the polymer using a pipette. After standing for 5 min, the LPEI-EnDm@mRNA polymer complex was obtained. By adjusting the modification ratio of En and Dm in the polymer, different LPEI-EnDm@mRNA nanoparticles were obtained by mixing with mRNA. The particle size and Zeta potential of the nanoparticles with a nitrogen-to-phosphorus ratio of 10 were determined by DLS. The results are shown in [link to DLS diagram]. Figure 13 .

[0135] To further investigate the ability of the polymer vector to deliver mRNA in cells, HeLa cells were used to screen the mRNA transfection capability of LPEI-EnDm@mRNA. The specific steps are as follows: 1 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 100 μg / well in a 24-well plate and incubated at 37°C until 80% confluence was achieved. Samples were prepared using the method described above for preparing polymer nanoparticles. After co-incubation with cells for 4 h, the medium was changed with fresh culture and incubated for another 16 h. Transfection efficiency was then assessed using an inverted fluorescence microscope. Results are shown below. Figure 14 .

[0136] The transfection efficiencies of representative alkane-chain and DTC-modified polymers were compared. Taking 2EA-D10E20 as an example, the representative modified acrylate is 2EA, the modification ratio is LPEI:DTC:2EA molar ratio of 1:10:20, and the molecular weight is 8100. The transfection efficiencies of polymers with different molecular weights were compared. Among them, IDA, with an appropriate ratio of DTC, can have a smaller particle size distribution and higher transfection efficiency. This indicates that the LPEI-DmEn polymer phase exhibits strong mRNA delivery efficiency under appropriate combination.

[0137] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A polymer or a pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof, characterized in that, The polymer is a cationic polymer PEI modified by a R0 group, wherein the R0 group is selected from one of formula (I) to (III), 、 、 ； wherein R1 is selected from R3 is a direct bond or is R2 is selected from H, -CH3, -CH2CH3, 2. The polymer or pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof of claim 1, wherein, The PEI is a dendritic PEI or a linear PEI.

3. The polymer or pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof of claim 1 or 2, wherein, The polymer has a structural formula as shown in formula (IV), wherein R is the same or different, R is selected from H or R0 and at least one R is R0; n is an integer from 1 to 10; Alternatively, the polymer has a structural formula as shown in formula (V), wherein R is selected from H or R0; m is an integer from 1 to 60; p is an integer from 1 to 60; Alternatively, the polymer has a structural formula as shown below: #imgpt9# wherein m is an integer from 1 to 60, q is an integer from 1 to 60, p is an integer from 1 to 60, R is selected from H or R0, and R4 is a direct bond or is 4. The polymer or pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof of claim 3, wherein, The polymer has a structural formula as shown in formula (VI) or (VII), 5. A method of preparing the polymer or pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof according to any one of claims 1 to 4, characterized in that, The preparation method comprises: reacting polyethyleneimine and a compound containing a cyclic disulfide group in the presence of a solvent and a catalyst to obtain the polymer.

6. Use of the polymer or pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof of any one of claims 1 to 4 in the preparation of a drug delivery carrier.

7. A polymer composite, characterized by The polymer complex comprises the polymer or pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof of any one of claims 1 to 4, and optionally further comprises a therapeutic agent and / or a prophylactic agent.

8. The method of producing a polymer composite according to claim 7, characterized in that, The method comprises: reacting the polymer, the therapeutic agent and / or the prophylactic agent under acidic conditions to prepare the polymer complex.

9. A drug delivery system, characterized by The drug delivery system comprises the polymer complex of claim 7.

10. Use of the polymer or pharmaceutically acceptable salt, isomer, stable isotopic derivative thereof of any one of claims 1 to 4, or the polymer complex of claim 7, or the drug delivery system of claim 9 in the preparation of a medicament for precisely delivering functional mRNA to a specific organ or tissue.

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