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CN-121971634-A - Blood brain barrier nucleic acid drug delivery system based on glycosyl functional nucleic acid, and preparation method and application thereof

CN121971634ACN 121971634 ACN121971634 ACN 121971634ACN-121971634-A

Abstract

The invention discloses a blood-brain barrier-crossing nucleic acid drug delivery system based on glycosyl functional nucleic acid, and a preparation method and application thereof, and belongs to the technical field of biomedicine. The core of the system is glycosylated transferrin receptor aptamer (GTA), which is formed by covalently linking a glycosyl modification module to a transferrin receptor binding sequence, which can bind to a therapeutic nucleic acid to form a complex. The invention remarkably improves the blood-brain barrier crossing efficiency and brain targeting of nucleic acid drugs through a double-channel synergistic mechanism formed by transferrin receptor mediated transcytosis and glucose transporter assisted ingestion, and the GTA can be automatically prepared through a DNA solid phase synthesis platform, has controllable structure, stable batch, high biocompatibility and low immunogenicity. The delivery system can be compatible with a plurality of nucleic acid medicaments such as siRNA, ASO, cpG ODN and the like, and has wide application prospect in the treatment of central nervous system diseases such as glioblastoma and the like.

Inventors

  • JI DINGKUN
  • TAN WEIHONG
  • HAN YONGQI
  • WANG MINGHUA

Assignees

  • 上海交通大学医学院附属仁济医院

Dates

Publication Date
20260505
Application Date
20260403

Claims (10)

  1. 1. A blood brain barrier nucleic acid drug delivery system based on a glycosyl functional nucleic acid, characterized in that the delivery system comprises a glycosylated transferrin receptor aptamer, the glycosylated transferrin receptor aptamer comprises a transferrin receptor aptamer, and a glycosyl modification module linked to the transferrin receptor aptamer through a linking sequence, the glycosyl modification module is a glycosyl modified nucleotide; The glycosyl in the glycosyl modification module is selected from any one or more of glucose, mannose, galactose or derivatives thereof, the connecting sequence is connected to the 5' end of the transferrin receptor aptamer, and the length of the connecting sequence is 5-30 nt.
  2. 2. The blood brain barrier nucleic acid drug delivery system of claim 1, wherein the glycosylated transferrin receptor aptamer is formed by site-directed integration of a glycosylphosphoramidite monomer with a transferrin receptor aptamer with a linker sequence on a DNA solid phase synthesis platform.
  3. 3. The blood brain barrier nucleic acid drug delivery system of claim 1, further comprising a therapeutic nucleic acid with which the glycosylated transferrin receptor aptamer can bind to form a complex, the therapeutic nucleic acid comprising siRNA, antisense oligonucleotides (ASOs), and CpG oligodeoxynucleotides (CpG ODNs).
  4. 4. The blood brain barrier nucleic acid drug delivery system of claim 1, wherein the glycosyl in the glycosyl modification module is glucose.
  5. 5. The blood brain barrier nucleic acid drug delivery system of claim 1, wherein the glycosyl modification module comprises at least three glycosyl modified nucleotides.
  6. 6. The blood brain barrier nucleic acid drug delivery system of claim 1, wherein the linker sequence is 10nt in length.
  7. 7. A method of preparing a blood brain barrier nucleic acid drug delivery system based on a glycosyl functional nucleic acid according to any one of claims 1 to 6, comprising the steps of: S1, preparing raw materials, namely selecting a plurality of glycosyl phosphoramidite monomers containing protecting groups, and connecting transferrin receptor aptamer with a connecting sequence to a solid phase carrier; s2, deprotection, namely placing the solid phase carrier in a dichloromethane solution containing 2% -5% (v/v) trichloroacetic acid, reacting for 30-60S at 20-25 ℃, removing protecting groups, and washing 3-5 times by using anhydrous acetonitrile after the reaction; s3, activating and condensing, namely adding glycosyl phosphoramidite monomer and 5-ethylthio-1H-tetrazole activator solution into the system, and reacting for 30-60S at 20-25 ℃ to form a phosphotriester bond; s4, capping, namely adding capping reagent A solution and capping reagent B solution, reacting for 10-20 seconds, and sealing free 5' -hydroxyl which does not participate in condensation, wherein the solution A is a composition of acetic anhydride, pyridine and tetrahydrofuran, and the solution B is a composition of N-methylimidazole and tetrahydrofuran; S5, oxidizing, namely adding an iodine/water/pyridine/tetrahydrofuran mixed oxidizing reagent, reacting for 10-30S, and oxidizing the phosphotriester bond into a phosphotriester bond; S6, circularly extending, namely repeating the steps S2-S5, and sequentially adding corresponding glycosyl phosphoramidite monomers according to a target sequence to obtain a precursor of the glycosylated transferrin receptor aptamer; S7, post-treatment, namely transferring the solid phase carrier into concentrated ammonia water, treating for 12-20 hours at room temperature, removing all protecting groups, cooling, centrifuging, collecting supernatant, and purifying by high performance liquid chromatography to obtain the glycosylated transferrin receptor aptamer.
  8. 8. The method of manufacturing as claimed in claim 7, comprising the steps of: S1, preparing raw materials, namely selecting a plurality of glycosyl phosphoramidite monomers containing protecting groups, and connecting transferrin receptor aptamer with a connecting sequence to a solid phase carrier; S2, deprotection, namely placing the solid phase carrier in a dichloromethane solution containing 3% (v/v) trichloroacetic acid, reacting for 35S at 25 ℃, removing protecting groups, and flushing with anhydrous acetonitrile for 3 times after the reaction; s3, activating and condensing, namely adding 0.1M glycosyl phosphoramidite monomer anhydrous acetonitrile solution and 0.25M 5-ethylthio-1H-tetrazole activator solution into a system, and reacting for 45S at 25 ℃ to form a phosphotriester bond; S4, capping, namely adding capping reagent A solution and B solution, reacting for 15 seconds, and closing free 5' -hydroxyl which does not participate in condensation, wherein the A solution is a composition of acetic anhydride, pyridine and tetrahydrofuran with the volume ratio of (1-3) (15-17), and the B solution is a composition of N-methylimidazole and tetrahydrofuran with the volume ratio of (2) (17-19); s5, oxidizing, namely adding a mixed oxidizing reagent consisting of iodine/water/pyridine/tetrahydrofuran, reacting for 20 seconds, and oxidizing a phosphotriester bond into a phosphotriester bond, wherein the concentration of iodine in the mixed oxidizing reagent is 0.02M, and the volume ratio of water, pyridine and tetrahydrofuran is (1-2): (10-30): (75-85); S6, circularly extending, namely repeating the steps S2-S5, and sequentially adding corresponding glycosyl phosphoramidite monomers according to a target sequence to obtain a precursor of the glycosylated transferrin receptor aptamer; S7, post-treatment, namely transferring the solid phase carrier into concentrated ammonia water, treating for 16 hours at room temperature, removing all protecting groups, cooling to room temperature, centrifuging, collecting supernatant, concentrating, purifying by high performance liquid chromatography, performing gradient elution by using a mobile phase A and a mobile phase B, monitoring 260 nm ultraviolet absorption, collecting fractions corresponding to a main product peak, and performing freeze drying to obtain the glycosylated transferrin receptor aptamer, wherein the A phase is 0.1M TEAA buffer solution, and the B phase is acetonitrile.
  9. 9. The method of manufacturing of claim 7, further comprising: S8, mixing the glycosylated transferrin receptor aptamer with a complementary nucleic acid drug, adding PBS containing 10 mM MgCl 2 , and stirring at 37 ℃ overnight to obtain the nucleic acid drug delivery system loaded with the nucleic acid drug.
  10. 10. Use of a blood brain barrier nucleic acid drug delivery system based on glycosyl functional nucleic acids according to any one of claims 1-6 or prepared by the method of preparation according to any one of claims 7-9 for the preparation of a medicament for the treatment of brain diseases, including brain tumors, central nervous system inflammatory diseases or gene-defective neurological diseases.

Description

Blood brain barrier nucleic acid drug delivery system based on glycosyl functional nucleic acid, and preparation method and application thereof Technical Field The invention relates to the technical field of biomedicine, in particular to a blood-brain barrier-crossing nucleic acid drug delivery system based on glycosyl functional nucleic acid, and a preparation method and application thereof. Background The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art. The nucleic acid medicine (including small interfering RNA, antisense oligonucleotide, cpG oligodeoxynucleotide, etc.) has unique advantages in the fields of gene silencing, immune regulation and accurate treatment, so that the medicine has wide clinical application prospect. However, nucleic acid drugs themselves have large molecular weight, negatively charged physicochemical properties, are easily degraded by nucleases in vivo, and are difficult to penetrate cell membranes into target cells, and the presence of the Blood-Brain Barrier (BBB) further limits their clinical transformation and use in the treatment of central nervous system diseases. The blood brain barrier is a highly selective physiological barrier formed by brain microvascular endothelial cells, can effectively block exogenous drug molecules from entering brain tissues, and is a major obstacle in drug development of the central nervous system. The currently reported nucleic acid drug brain delivery strategies mainly include Lipid Nanoparticles (LNP), polymer vectors, viral vectors, and receptor-mediated transport systems, etc., but all suffer from significant drawbacks: 1) Although the lipid nano particles and the polymer carrier (such as polyethyleneimine and PAMAM dendritic polymer) have good encapsulation performance and cell uptake efficiency, potential toxicity and in-vivo nonspecific aggregation problems exist, and the biosafety needs to be improved; 2) The virus vector has high transfection efficiency, but has strong immunogenicity, limited loading capacity and insertion mutation risk, and the safety is difficult to meet clinical requirements; 3) Although a receptor-mediated transmembrane transport system (such as transferrin receptor or insulin receptor-mediated transport system) can realize a certain degree of blood-brain barrier crossing, the receptor-mediated transmembrane transport system is limited by the problems of receptor saturation effect, competitive binding, reduced targeting accuracy in the brain and the like, and the delivery efficiency needs to be improved; 4) The nucleic acid aptamer serving as a novel targeting molecule has the advantages of high affinity, low immunogenicity, easiness in modification and the like, but the mediated cross-blood brain barrier system still has the defects of low delivery efficiency and insufficient targeting in brain, for example, the system mediated by the transferrin receptor aptamer has difficulty in considering the crossing efficiency and focus positioning accuracy. The prior art generally has the problems of single transfer path limitation, insufficient targeting precision, poor structure controllability, complicated preparation process, potential biotoxicity and the like, and the development of a nucleic acid drug delivery system which has a structure precision controllability, a dual transmembrane mechanism and can maintain the targeting characteristic in the brain is needed to improve the utilization efficiency and the treatment effect of nucleic acid drugs in central nervous system diseases. Disclosure of Invention Aiming at the defects in the prior art, the invention aims to provide a nucleic acid drug delivery system based on glycosylated transferrin receptor aptamer (GTA), which improves the brain delivery efficiency and targeting property through a double-channel collaborative transmembrane mechanism so as to overcome the problems of low delivery efficiency, insufficient targeting precision, complex structure, potential toxicity and the like of nucleic acid drugs crossing blood brain barriers in the prior art, and has the advantages of chemical synthesis, large-scale production and high biocompatibility. In order to achieve the above purpose, the invention adopts the following technical scheme: A blood brain barrier nucleic acid drug delivery system based on a glycosyl functional nucleic acid, characterized in that the delivery system comprises a glycosylated transferrin receptor aptamer, the glycosylated transferrin receptor aptamer comprises a transferrin receptor aptamer, and a glycosyl modification module connected with the transferrin receptor aptamer through a connecting sequence, the glycosyl modification module is a glycosyl modified nucleotide; wherein