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CN-122005855-A - Double mRNA (messenger ribonucleic acid) combined targeted delivery system for treating peripheral arterial disease of lower limb and application thereof

CN122005855ACN 122005855 ACN122005855 ACN 122005855ACN-122005855-A

Abstract

The invention discloses a double mRNA combined targeting delivery system for treating Peripheral Arterial Disease (PAD) of lower limbs and application thereof. The system includes a microcrystalline nanocarbon targeted drug delivery matrix having a messenger ribonucleic acid (mRNA) of the IL-4 gene and/or VEGF gene supported thereon. The matrix comprises matrix microcrystalline nanocarbon, folic Acid (FA), dextran Sulfate (DS), polyethyleneimine (PEI) and polyethylene glycol (PEG) which are modified on the surface of the matrix. The system realizes high-efficiency endocytosis by double targeting of folic acid and dextran sulfate on macrophages, utilizes mRNA to rapidly express protein in cytoplasm, avoids the risk of genome integration, and cooperatively promotes functional angiogenesis and tissue repair by IL-4 and VEGF co-delivery. Preferably, the system may be entrapped in an injectable hydrogel to form a gel complex system that allows for slow sustained release of the drug. The invention also provides a preparation method of the system and application of the system in preparation of medicines for treating ischemic diseases. The system has the advantages of high targeting efficiency, rapid expression, high vascular maturity, lasting effect and the like, and provides a new strategy for PAD treatment.

Inventors

  • HAO YIZHOU
  • ZHANG PENG
  • Huang Lehang

Assignees

  • 广州墨羲科技有限公司

Dates

Publication Date
20260512
Application Date
20260408

Claims (14)

  1. 1. A dual-mRNA combined targeting delivery system for treating peripheral arterial disease of lower limb is characterized by comprising a microcrystalline nano-carbon targeting drug delivery matrix, wherein the matrix is a nano-particle for loading IL-4 and/or VEGF mRNA, the IL-4 mRNA is functionally designed according to an original sequence with the GenBank accession number of [ NM_000589.4 ]; The microcrystalline nano-carbon targeted drug delivery matrix comprises matrix microcrystalline nano-carbon, folic acid, dextran sulfate, polyethyleneimine and polyethylene glycol which are modified on the surface of the matrix microcrystalline nano-carbon, wherein the modification density of FA is 20-50 mu g/m < 2 >, the modification density of DS is 30-80 mu g/m < 2 >, the modification density of PEG is 100-200 mu g/m < 2 >, and the modification density of PEI is 150-300 mu g/m < 2 >, and the modification densities of FA and DS are optimized and screened. It was found that targeting binding capacity to folate receptor positive macrophages is significantly reduced when the FA modification density is below 25 μg/m2, whereas binding of DS to scavenger receptors is likely to be affected by steric hindrance and costs are increased when above 50 μg/m 2. Similarly, the DS density is in the range of 45-70 mug/m < 2 >, which not only ensures the efficient scavenger receptor mediated endocytosis, but also does not cause the increase of nonspecific uptake. Therefore, the modification densities of FA and DS are respectively controlled to be 25-50 mug/m < 2 > and 45-70 mug/m < 2 >, which are key technical parameters for realizing the maximization of the dual-targeting synergistic effect; the microcrystalline nano carbon is characterized in that 1) the basic unit (carbon microchip) has higher crystallinity (70 percent), 2) the stacking mode is disordered, rich most probable mesopores mainly comprising 1.8-2.5 nanometers are formed, 3) the whole particle has high specific surface area (700-1100 m < 2 >/g) and pore volume (2.5-3.5 cm < 3 >/g), and the structure is different from completely amorphous carbon, and also different from graphite, carbon nanotube or graphene with long-range ordered lattice or regular/two-dimensional structure, but is a unique carbon nanomaterial with highly functionalized mesoporous structure; The Zeta potential of the matrix microcrystalline nano carbon is-20 to-10 mV (pH=7.4 phosphate buffer solution is detected), the cytotoxicity grade is 1 (ISO 10993-5 standard), the highly developed and interconnected mesoporous network formed by disordered stacking of carbon micro-crystalline sheets provides two major core functional advantages for the invention, 1) the ultra-high loading capacity is that the pore volume of up to 2.5-3.5 cm < 3 >/g and the specific surface area of 700-1100 m < 2 >/g provide sufficient space for physical adsorption of mRNA and electrostatic binding anchor points of subsequent PEI, which is a structural basis for realizing >90% loading rate, 2) the control release and protection function is that abundant 2-3 nm mesopores can effectively limit the entry of nuclease, protect mRNA from degradation and delay the diffusion release of mRNA, and the unique structure of the microcrystalline nano carbon can realize the physical structure guarantee of continuous expression for 7-14 days, and the unique load and protection of IL-4 and/or mRNA are obviously superior to the carbon nano-carbon tubes with the same loading rate and protection effect or the same size as the wall of the amorphous carbon nano-tubes; After the gene delivery system is injected into the lower limb muscle, the targeted enrichment rate of macrophages through folic acid receptor and scavenger receptor dual paths at the ischemic part of the lower limb is more than or equal to 70 percent, the retention time of the vector is more than or equal to 7 days, and the continuous expression of IL-4 and/or VEGF mRNA at the focus part can be realized for 7-14 days.
  2. 2. The gene delivery system according to claim 1, wherein the mesoporous size distribution of the matrix microcrystalline nanocarbon is 1-10 nm Kong Zhanbi-80% in the area-aperture distribution of the differential integral pores measured by BJH method (adsorption), 2-3 nm Kong Zhanbi-40%, 10-30 nm Kong Zhanbi-30%, more than 30nm Kong Zhanbi-10%, and 1-10 nm Kong Zhanbi-50% in the aperture distribution of the differential integral pores measured by BJH method (adsorption), 2-3 nm Kong Zhanbi-15%, 10-30 nm Kong Zhanbi-20%, more than 30nm Kong Zhanbi-40%.
  3. 3. The gene delivery system according to claim 1, wherein the microcrystalline nanocarbon is synthesized by pulse modulation radio frequency plasma enhanced chemical vapor deposition, the method comprising ① introducing precursor gas comprising methane (purity not less than 99.9%), hydrogen (purity not less than 99.9%), argon (purity not less than 99.9%) and oxygen (purity not less than 99.9%) into an inductively coupled plasma reactor, wherein the flow rate of methane is 3-10 sccm, the flow rate of hydrogen is 150-250 sccm, the flow rate of argon is 250-350 sccm, the flow rate of oxygen is 0.1-0.15 sccm, and the gas is introduced into the reaction zone after being uniformly mixed; ② in the pulse radio frequency plasma condition, the reaction pressure is 0.01-1 Pa, the radio frequency band is 70MHz, the pulse frequency is 400-600 Hz, the pulse duty ratio is 18% -23%, the average power is 1000-2100W, the reaction zone temperature is 1800-2200 ℃, the reaction time is 90 min, the carbon microchip is formed rapidly in the air flow, ③ the air flow containing the carbon microchip generated in the step ② is quenched rapidly by a spiral cooling sleeve quenching device at the speed of >1000 ℃ per second under the condition of <100 ℃, the carbon microchip is self-assembled and stacked to form primary particles of the matrix microcrystalline nano carbon in the quenching process, ④ collects the primary particles, and uses 30% -50% hydrogen peroxide solution to oxidize for 3-6 hours at 50-70 ℃ to enable the oxygen content to reach 8% -12% (detected by an X-ray photoelectron spectrometer), wherein the oxygen-containing functional groups mainly comprise hydroxyl groups (accounting for 40% -60%) and carboxyl groups (accounting for 30% -50%).
  4. 4. The gene delivery system according to claim 1, wherein folic acid and dextran sulfate are covalently modified on the surface of the microcrystalline carbon substrate through a connecting arm PEG, the number average molecular weight of the PEG is 2000-10000 Da, the molar ratio of folic acid to PEG is 0.8-1.2:1, the mass ratio of dextran sulfate to PEG is 0.5-1.5:1, the PEG is connected with hydroxyl on the surface of the microcrystalline carbon substrate through an ester bond formed by carboxyl at one end, the surface of the microcrystalline carbon substrate is cationized through linear PEI, the mass ratio of PEI to microcrystalline carbon substrate is 0.1-0.5:1, the Zeta potential of the microcrystalline carbon substrate is +30 to +50 mV after PEI modification, mRNA is loaded on the surface of the microcrystalline carbon substrate through electrostatic effect, and the mRNA loading rate is more than or equal to 90% (detected through gel electrophoresis).
  5. 5. The gene delivery system of claim 1, wherein the gene delivery system is entrapped in a biodegradable hydrogel selected from one or more of hyaluronic acid, gelatin, alginate, chitosan, poly (lactic-co-glycolic acid) (PLGA) or derivatives thereof to form a gel complex system, wherein the gel complex system achieves sustained release of mRNA by slow degradation of the hydrogel after injection for a duration of up to 14-28 days.
  6. 6. The gene delivery system of claim 1, wherein the surface of the matrix microcrystalline carbon is further doped with nitrogen in an amount of 1% -5% (atomic percent), the nitrogen being in the form of pyrrole nitrogen, pyridine nitrogen and graphite nitrogen, wherein the ratio of pyrrole nitrogen is greater than or equal to 50%.
  7. 7. The gene delivery system of claim 2, further comprising a particle size control step after step ④, wherein the oxidized primary particles are crushed in a dispersion medium by a strong ball mill, further crushed and refined by ultrasonic, and then subjected to centrifugal classification and membrane filtration, so that the particle size of the final microcrystalline nanocarbon targeted drug delivery matrix is 90-160 nm.
  8. 8. The gene delivery system of any one of claims 1-5, wherein the system achieves a cumulative release rate of IL-4 mRNA of 60% -90% over 48 hours in a simulated lower limb ischemic microenvironment (pH 6.5-7.0, 10 mM H 2 O 2 in phosphate buffer).
  9. 9. A method of preparing the gene delivery system of any one of claims 1-5, comprising the steps of: (1) Providing a matrix microcrystalline nanocarbon of the microcrystalline nanocarbon targeted drug delivery matrix; (2) Dispersing the substrate microcrystalline nano carbon in a buffer solution, adding carbodiimide and N-hydroxysuccinimide to activate carboxyl on the surface of the substrate microcrystalline nano carbon, and then mixing the substrate microcrystalline nano carbon with a polyethyleneimine solution for reaction to obtain PEI modified microcrystalline nano carbon; covalently connecting the folic acid-polyethylene glycol derivative and the dextran sulfate-polyethylene glycol derivative with activated PEI modified microcrystalline nanocarbon to obtain a folic acid and dextran sulfate dual-targeting modified microcrystalline nanocarbon targeted drug delivery matrix; (3) mRNA loading, namely mixing and incubating mRNA containing IL-4 genes and/or VEGF genes with the microcrystalline nano-carbon targeted drug delivery matrix obtained in the step (2) in phosphate buffer solution, and forming a compound through electrostatic action to obtain the gene delivery system.
  10. 10. The gene delivery system of any one of claims 1-6, wherein the gene delivery system is formulated as a lyophilized gel formulation comprising 5% trehalose lyoprotectant, and is stable for at least 12 months at 2-8 ℃ and 6 months at 25 ℃.
  11. 11. A pharmaceutical composition comprising a therapeutically effective amount of the gene delivery system of any one of claims 1-6, and a pharmaceutically acceptable carrier or adjuvant, preferably the pharmaceutical composition is an injectable gel formulation, the gene delivery system being dispersed in a hydrogel matrix.
  12. 12. Use of the gene delivery system of any one of claims 1-6 or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the treatment of ischemic disease.
  13. 13. The use according to claim 10, wherein the ischemic disease is selected from peripheral arterial disease, severe limb ischemia, myocardial infarction, diabetic foot ulcer or chronic wound healing disorder.
  14. 14. The use according to claim 10 or 11, wherein the gene delivery system is administered by intramuscular injection for targeting macrophages at the focal site, inducing polarization of macrophages to the reparative M2 phenotype by IL-4, promoting vascular endothelial cell proliferation and migration by VEGF, and improving ischemic microenvironment, promoting pericytes and fibroblasts to participate in angiogenesis, improving vascular maturation and tissue repair, and ultimately promoting functional angiogenesis and recovery of muscle function at the ischemic site by the synergistic effect of IL-4 and VEGF.

Description

Double mRNA (messenger ribonucleic acid) combined targeted delivery system for treating peripheral arterial disease of lower limb and application thereof Technical Field The invention belongs to the technical field of biological medicines and nano materials, and particularly relates to a gene delivery system for treating peripheral arterial diseases of lower limbs. More specifically, the invention relates to an IL-4 and/or VEGF mRNA delivery system which takes nitrogen-doped medium Kong Weijing-state nano carbon as a matrix and is subjected to double targeting modification by folic acid and dextran sulfate, a preparation method thereof and application thereof in promoting functional angiogenesis of a lower limb ischemia part and improving lower limb blood supply deficiency. Background Peripheral Arterial Disease (PAD) and its end-stage severe limb ischemia (CLI) are serious global health problems characterized by limb hypoperfusion leading to pain, ulcers, gangrene and high amputation rates. Current revascularization therapy and drug therapies have limited effectiveness. Gene therapy, particularly delivery of pro-angiogenic factors (such as VEGF), has demonstrated potential, but has often failed in clinical trials due to immature angiogenesis, poor stability and lack of targeting. Interleukin-4 (IL-4) is a key immunoregulatory cytokine capable of inducing macrophage polarization to M2 phenotype with repair and angiogenesis promoting functions, and provides a new strategy for remodelling ischemic microenvironment through immunoregulation. However, IL-4 proteins have a very short half-life in vivo, requiring repeated high doses of administration, and are prone to side effects. In recent years, mRNA therapies have received attention for their unique advantages. Compared with plasmid DNA, mRNA does not need to enter the nucleus, can be translated into functional protein in cytoplasm, has quicker expression, does not have genome integration risk, and has higher safety. However, mRNA molecules are poorly stable and are susceptible to nuclease degradation, requiring efficient delivery systems for protection. Non-viral vectors are of interest for better safety and regulatory concerns, mainly including cationic polymers, liposomes, inorganic nanocarriers, and the like. The gene delivery process of non-viral vectors encompasses key steps of gene loading, cellular uptake, endosome/lysosomal escape, and gene expression. Each step plays a key role in successful translation of the gene within the target cell and ultimately in achieving biological function. Lipid nanoparticles (Lipid nanoparticles, LNPs) and liposomes that mimic cell membrane structures are widely used for nucleic acid delivery. LNPs and liposome complexes are capable of effectively encapsulating nucleic acids, preventing degradation of nucleic acids, promoting cellular internalization and endosomal escape. LNPs is prepared by precisely mixing cationic or ionizable lipid, polyethylene glycol-lipid, cholesterol and phospholipid according to a specific molar ratio. Wherein positively charged cations or ionizable lipids bind to negatively charged nucleic acids, cholesterol increases stability, promotes membrane fusion, polyethylene glycol-lipids control particle size and stability to avoid aggregation, and phospholipids mediate nucleic acid encapsulation. However, the liposome still has the problems of low transfection efficiency, poor targeting, unstable serum, easy rapid clearance by a mononuclear phagocyte system and the like. Therefore, it is important to develop a novel gene vector that combines high-efficiency load, stable delivery, active targeting and good biocompatibility. Particularly in the lower limb ischemic pathology microenvironment, high levels of Reactive Oxygen Species (ROS), acidic metabolites and inflammatory factors can further exacerbate the degradation and instability of conventional liposome vectors, resulting in substantial leakage or inactivation of their entrapped therapeutic genes before they reach the target cells. In addition, the dense interstitial pressure of ischemic tissue also impedes the effective penetration of traditional nanoparticles. Therefore, the development of a novel intelligent response type carrier which can not only overcome the systematic defects, but also adapt to local special microenvironments (such as oxidative stress and acidic pH) of ischemia is very important for realizing the efficient gene therapy of PAD. Inorganic nanomaterials, particularly graphene and its derivatives, are receiving a great deal of attention in the field of drug delivery due to their large specific surface area, ease of functionalization and good biocompatibility. However, the conventional graphene material has the problems of easy agglomeration of sheets, nonuniform size, poor structural stability and the like, and limits the application of the conventional graphene material as an intravenous injection carrier. In the invention, t