CN-122005832-A - SiTNF-alpha delivery vehicle based on sulfated hyaluronic acid-polyethyleneimine-polyphenol conjugate and application thereof

Abstract

The present invention relates to a siTNF-alpha delivery vehicle based on a sulfated hyaluronic acid-polyethyleneimine-polyphenol conjugate and its use. Considering that the existing siRNA carrier is difficult to be applied to the prevention and substitution treatment of inflammatory diseases, the invention synthesizes the siRNA carrier based on the S-HA-PEI-PP conjugate, which obviously reduces the original cytotoxicity of PEI, HAs good biocompatibility, can control the particle size of the carrier within the range which is favorable for macrophage ingestion, is easy to be identified and phagocytized by macrophages, and improves the delivery efficiency of siRNA. In particular, the S-HA-PEI-CA carrier can obviously improve the anti-inflammatory effect by means of a multi-target regulation mechanism, realizes the synergistic effect of siTNF-alpha high-efficiency delivery and anti-inflammatory function, breaks the limitation of the traditional siRNA carrier in treating inflammatory diseases, and provides a new thought for the development of an siRNA delivery system.

Inventors

• Yan Dieshu
• LI RUITING
• HAN ZIYAN
• Bu Linghan
• DING FANGFANG

Assignees

• 江南大学

Dates

Publication Date

20260512

Application Date

20260324

Claims (10)

1. 1. Use of a sulfated hyaluronic acid-polyethyleneimine-polyphenol conjugate in the preparation of an siRNA delivery vehicle, characterized in that the sulfated hyaluronic acid-polyethyleneimine-polyphenol conjugate is obtained by a cross-linking reaction of a conjugate formed from polyethyleneimine and a polyphenol compound, which is a carboxyl-containing polyphenol or a carboxylated polyphenol, with sulfated hyaluronic acid.
2. 2. Use according to claim 1, characterized in that it comprises at least one of the following features: (1) The polyphenol compound comprises caffeic acid, chlorogenic acid, gallic acid, glycyrrhizic acid, baicalin or carboxylated rutin; (2) The siRNA comprises siRNA for knocking out or knocking down a tumor necrosis factor-alpha encoding gene; (3) The mass ratio of the polyethylenimine, the polyphenol compound and the sulfated hyaluronic acid is (1-100): 1-100; (4) The structural formula of the conjugate formed by the polyethyleneimine and the polyphenol compound is shown as follows: Wherein R 1 is independently selected from the group consisting of polyphenols or H and at least one R 1 is a polyphenol and n is an integer between 1 and 100; (5) The structure of the sulfated hyaluronic acid is shown as the following formula: Wherein R ́ is independently selected from SO 3 - or H.
3. 3. A composition for preparing an siRNA delivery vehicle, comprising: A conjugate of a polyethylenimine and a polyphenol compound of the formula: Wherein R 1 is independently selected from the group consisting of polyphenols or H and at least one R 1 is a polyphenol and n is an integer between 1 and 100; sulfated hyaluronic acid.
4. 4. The composition of claim 3, wherein the composition further comprises an siRNA; The siRNA comprises siRNA for knocking out or knocking down a tumor necrosis factor-alpha encoding gene.
5. 5. Use of a composition according to claim 3 or 4 for the preparation of a genetic medicament.
6. 6. A method for preparing an siRNA drug, comprising the steps of: Mixing a structure shown in the following formula with siRNA, then mixing with sulfated hyaluronic acid, and performing amide condensation under proper conditions to prepare the siRNA drug; Wherein R 1 is independently selected from a polyphenol or H and at least one R 1 is a polyphenol and n is an integer between 1 and 100.
7. 7. The siRNA medicine prepared by the preparation method of claim 6.
8. 8. A sulfated hyaluronic acid-polyethyleneimine-caffeic acid conjugate, characterized in that the conjugate formed by polyethyleneimine and caffeic acid is obtained by a crosslinking reaction with sulfated hyaluronic acid.
9. 9. The sulfated hyaluronic acid-polyethyleneimine-caffeic acid conjugate according to claim 8, wherein the sulfated hyaluronic acid-polyethyleneimine-caffeic acid conjugate has the structural formula: Wherein R ́ is independently selected from SO 3 - or H, R 2 is independently selected from caffeic acid or H, m is an integer between 10 and 100, and n is an integer between 10 and 100.
10. 10. Use of the composition of claim 3 or 4, the gene drug of claim 5, the siRNA drug of claim 7, the sulfated hyaluronic acid-polyethyleneimine-caffeic acid conjugate of claim 8 or 9 for the preparation of an anti-inflammatory product or for the preparation of a macrophage phenotype polarization modulator.

Description

SiTNF-alpha delivery vehicle based on sulfated hyaluronic acid-polyethyleneimine-polyphenol conjugate and application thereof Technical Field The invention relates to the technical field of gene drug delivery, in particular to a siTNF-alpha delivery carrier based on sulfated hyaluronic acid-polyethyleneimine-polyphenol conjugate and application thereof. Background Tumor necrosis factor-alpha (Tumor necrosis factor-alpha, TNF-alpha) is a key inflammatory mediator produced by the body after trauma or infection. The factor induces the release of pro-inflammatory factors such as Interleukin-6 (Interleukin-6, IL-6) and Interleukin-1 beta (Interleukin-1 beta, IL-1 beta) by activating classical nuclear factor- κB signaling pathway, thereby amplifying inflammatory cascade reaction and accelerating pathological process. Based on its core regulatory role in inflammatory response, TNF- α has become an important molecular target for the treatment of acute inflammation. In recent years, gene silencing techniques based on small interfering RNAs (SMALL INTERFERING RNA, SIRNA) have provided new directions for the regulation of inflammation. By specifically inhibiting TNF-alpha gene expression, the technology can effectively block the activation of downstream inflammatory signal channels, and has the potential of treating acute inflammatory diseases. However, the only current lot of siRNA delivery carrier lipid nanoparticles (Lipid nanoparticles, LNPs) may elicit non-specific immune responses due to their inherent immunogenicity, and in vivo accumulation toxicity may exacerbate tissue damage, making it difficult to meet the safety requirements of preventive interventions and alternative therapies for inflammatory diseases. In addition to lipids, polyethyleneimine (Polyethylenimine, PEI) as a cationic polymer representation has been of great interest in siRNA delivery system research. The action mechanism is mainly based on the characteristic of high-density positive charges on a molecular chain, on one hand, the high-efficiency endocytosis of the siRNA is mediated through electrostatic adsorption and the combination of the molecular chain and a negatively charged cytoplasmic membrane, and on the other hand, the unique proton sponge effect of the compound can obviously enhance the escape efficiency of endosomes and ensure the effective release [7] of the siRNA in cytoplasm. More notably, PEI has the immune regulation property, can inhibit the expression of proinflammatory cytokines, can exert anti-inflammatory effect by regulating mechanisms such as macrophage polarization and the like, and shows the advantage of dual functions of carrier-therapy. However, excessive cationic charge of PEI molecules can disrupt cell membrane integrity and cause damage to organelles, severely limiting its use in clinical transformations. Therefore, how to optimize the charge distribution of PEI by molecular engineering means so as to realize the safe and efficient siTNF-alpha delivery system construction becomes a technical problem to be broken through in the field. Hyaluronic acid has become an ideal choice for novel drug delivery vehicles by virtue of the characteristics of high hydrophilicity, viscoelasticity, biodegradability, low sensitization, excellent biocompatibility and the like, and researches show that compared with PEI, the hyaluronic acid component in the hyaluronic acid-PEI conjugate can effectively neutralize excessive positive charges on the surface of PEI molecule through charge shielding effect, remarkably reduce nonspecific damage to cell membranes and maintain anti-inflammatory activity of PEI. Compared with hyaluronic acid, the sulfated hyaluronic acid has certain macrophage targeting and anti-inflammatory activity, and the sulfated hyaluronic acid can also remarkably prolong the half life of the hyaluronic acid by enhancing the steric hindrance effect with hyaluronidase, thereby realizing remarkable expansion of the treatment window. The present invention therefore proposes a strategy for constructing a sulfated hyaluronic acid-PEI complex as siTNF- α delivery vehicle, possibly achieving both the objective of enhanced anti-inflammatory activity and reduced frequency of administration. However, under physiological conditions, the sulfated hyaluronic acid carries strong negative charges and generates severe electrostatic complexation with PEI cations, so that the critical aggregation concentration of the sulfated hyaluronic acid is greatly reduced under the physiological conditions, the stability of the preparation is obviously affected, and secondly, the capacity of loading siTNF-alpha of PEI is severely reduced due to excessive neutralization of charges of the sulfated hyaluronic acid. The contradictory conformational roles at the molecular level suggest how molecular reconstitution of the sulfated hyaluronic acid-PEI complex is critical to successful construction of the siTNF-alpha vector, while maintaining the affinity