CN-121971650-A - Protein delivery method for targeted reversal of nucleus pulposus cell aging

CN121971650ACN 121971650 ACN121971650 ACN 121971650ACN-121971650-A

Abstract

The invention discloses a protein delivery method for targeted reversal of nucleus pulposus cell aging, which relates to the technical field of biomedical engineering and comprises the steps of preparing a recombinant OSK reprogramming protein mixed solution of OCT4 protein, SOX2 protein and KLF4 protein, using the recombinant OSK reprogramming protein mixed solution, loading on carboxymethyl chitosan nanoparticles functionalized by nuclear localization signal peptide and endosome escape peptide L17E through electrostatic adsorption and hydrophobic effect to form a nucleus targeted protein drug-carrying core, coating the nucleus targeted protein drug-carrying core and membrane vesicles extracted from M1 polarized macrophages through ultrasonic fusion and sequential extrusion technology to prepare a bionic delivery unit, and applying the bionic delivery unit into a pathological change intervertebral disc of an intervertebral disc degeneration model animal in an intra-intervertebral disc injection mode to targeted delivery of nucleus pulposus cells. The bionic delivery unit has the dual functions of actively regulating pathological change microenvironment and efficiently starting intracellular reprogramming, and constitutes a multi-target and synergistic advanced treatment strategy.

Inventors

ZHAN JIAWEN
ZHAO KE
CUI YONGZHI
DU YUXUAN
LIU LI

Assignees

中国中医科学院望京医院(中国中医科学院骨伤科研究所)

Dates

Publication Date: 20260505
Application Date: 20260203

Claims (10)

1. A protein delivery method for targeted reversal of nucleus pulposus cell aging is characterized by comprising the steps of preparing a recombinant OSK reprogramming protein mixed solution of OCT4 protein, SOX2 protein and KLF4 protein; The recombinant OSK reprogramming protein mixed solution is used for loading on carboxymethyl chitosan nano-particles functionalized by nuclear localization signal peptide and endosome escape peptide L17E through electrostatic adsorption and hydrophobic action, so as to form a nuclear targeting protein drug-carrying core; coating the core targeting protein drug-carrying core and membrane vesicles extracted from M1 polarized macrophages by ultrasonic fusion and sequential extrusion technology to prepare a bionic delivery unit; Applying the bionic delivery unit into a pathological change intervertebral disc of an intervertebral disc degeneration model animal in an intra-intervertebral-disc injection mode, and carrying out targeted delivery on nucleus pulposus cells; the bionic delivery unit neutralizes inflammatory factors and weakens inflammatory microenvironment in the pathological change intervertebral disc through M1 polarized macrophage membrane vesicles, and realizes endosome escape through endosome escape peptide L17E after being internalized by nucleus pulposus cells; the nuclear localization signal peptide enters the nucleus, the recombinant OSK reprogramming protein mixed solution is interpreted and released in the environment of high concentration glutathione in the nucleus, and the partial reprogramming program is started to reverse the aging of the nucleus cell.
2. The method for delivering protein targeted to reverse aging of nucleus pulposus cells according to claim 1, wherein preparing the recombinant OSK reprogramming protein mixture of OCT4 protein, SOX2 protein and KLF4 protein comprises the steps of: respectively transforming plasmids encoding OCT4 protein, SOX2 protein and KLF4 protein genes into expression hosts; Culturing and inducing expression of the transformed escherichia coli competent cells of the gene plasmids of the OCT4 protein, the SOX2 protein and the KLF4 protein to obtain bacteria expressing the OCT4 protein, bacteria expressing the SOX2 protein and bacteria expressing the KLF4 protein; Respectively lysing the thalli expressing OCT4 protein, thalli expressing SOX2 protein and thalli expressing KLF4 protein, and purifying to obtain OCT4 protein solution, SOX2 protein solution and KLF4 protein solution; and mixing the OCT4 protein solution, the SOX2 protein solution and the KLF4 protein solution according to a preset molar ratio to obtain the recombinant OSK reprogramming protein mixed solution.
3. The method for delivering protein for targeting reversal of aging of nucleus pulposus cells according to claim 2, wherein the recombinant OSK reprogramming protein mixture is loaded on carboxymethyl chitosan nanoparticle functionalized by nuclear localization signal peptide and endosomal escape peptide L17E through electrostatic adsorption and hydrophobic interaction to form a nuclear targeting protein drug-loaded core, comprising the following steps: Dissolving carboxymethyl chitosan in a buffer solution, and reacting with a cross-linking agent to prepare carboxymethyl chitosan nano particles; Covalently connecting the nuclear localization signal peptide and the endosome escape peptide L17E to the carboxymethyl chitosan nanoparticle through chemical reaction to form the carboxymethyl chitosan nanoparticle functionalized by the nuclear localization signal peptide and the endosome escape peptide L17E; Mixing and incubating the recombinant OSK reprogramming protein mixed solution with carboxymethyl chitosan nanoparticles functionalized by nuclear localization signal peptide and endosome escape peptide L17E; In the mixed incubation process of the recombinant OSK reprogramming protein mixed solution and the carboxymethyl chitosan nanoparticle functionalized by the nuclear localization signal peptide and the endosome escape peptide L17E, the recombinant OSK reprogramming protein mixed solution is loaded on the carboxymethyl chitosan nanoparticle functionalized by the nuclear localization signal peptide and the endosome escape peptide L17E through electrostatic adsorption and hydrophobic action to form a nuclear targeting protein drug-carrying core.
4. The method for delivering protein for targeted reversal of aging of nucleus pulposus cells according to claim 3, wherein the biomimetic delivery unit is prepared by coating a nuclear targeted protein drug-carrying core and membrane vesicles extracted from M1 polarized macrophages by ultrasonic fusion and sequential extrusion technology, and comprises the following steps: Resuspending membrane vesicles extracted from M1 polarized macrophages in phosphate buffer to obtain membrane vesicle suspension, and mixing the core aqueous solution of the core targeting protein drug carrier with the membrane vesicle suspension according to equal volume to obtain a mixed solution of the core targeting protein drug carrier core and the membrane vesicle suspension; carrying out ultrasonic treatment on the mixed solution of the nuclear targeting protein drug-carrying core and the membrane vesicle suspension, and inducing the primary fusion of the membrane vesicle and the nuclear targeting protein drug-carrying core; Sequentially extruding the mixed solution of the core of the nuclear targeting protein drug-carrying core after ultrasonic treatment and the membrane vesicle suspension through a polycarbonate membrane with a decreasing aperture; in the sequential extrusion process, membrane vesicles are coated on the surface of a core-targeted protein drug-carrying core to form a bionic delivery unit with a core-shell structure.
5. The method for delivering protein for targeted reversal of aging of nucleus pulposus cells according to claim 4, wherein the targeted delivery of nucleus pulposus cells is performed by administering the bionic delivery unit into a diseased intervertebral disc of an intervertebral disc degeneration model animal by means of intra-discal injection, comprising the steps of: Resuspending the bionic delivery unit in phosphate buffer solution to prepare bionic delivery unit injection suspension, Enabling the intervertebral disc degeneration model animal to enter an anesthetic state in an anesthetic mode, and positioning a pathological change intervertebral disc under the anesthetic state of the intervertebral disc degeneration model animal; sucking the bionic delivery unit injection suspension by using a microinjector, penetrating a needle head of the microinjector into a nucleus pulposus region of a pathological change intervertebral disc, pushing a piston of the microinjector, and injecting the bionic delivery unit injection suspension into the pathological change intervertebral disc in an intra-intervertebral-disc injection mode; The biomimetic delivery unit injection suspension is distributed in the diseased intervertebral disc, and the biomimetic delivery unit contacts with the nucleus pulposus cells and initiates the targeted delivery process to the nucleus pulposus cells.
6. The method for delivering protein targeted to reverse aging of nucleus pulposus cells according to claim 5, wherein the biomimetic delivery unit neutralizes inflammatory factors and attenuates inflammatory microenvironments within the diseased disc through M1 polarized macrophage membrane vesicles, comprising the steps of: The bionic delivery unit releases M1 polarized macrophage membrane vesicle in the pathological change intervertebral disc, and the receptor on the surface of the M1 polarized macrophage membrane vesicle is combined with pro-inflammatory cytokines in the pathological change intervertebral disc; The combination process of M1 polarized macrophage membrane vesicle and pro-inflammatory cytokine neutralizes the bioactivity of the pro-inflammatory cytokine, and the concentration of the pro-inflammatory cytokine in the pathological change intervertebral disc is reduced after the pro-inflammatory cytokine is neutralized; The decrease in the concentration of pro-inflammatory cytokines in the diseased disc weakens the inflammatory microenvironment driven by the pro-inflammatory cytokines.
7. The method for delivering a protein targeted to reverse aging of a nucleus pulposus cell according to claim 6, wherein the endosome escape is achieved through the endosome escape peptide L17E after internalization by the nucleus pulposus cell, comprising the steps of: The nucleus pulposus cell intakes the bionic delivery unit into the cell through the internal memory effect, and the internal memory foam containing the bionic delivery unit is fused with the early endosome to form the early endosome; early endosomes mature gradually into late endosomes, the internal environment is acidified, and in the acidic environment, endosome escape peptide L17E is activated; The activated endosome escape peptide L17E interacts with the endosome membrane to damage the integrity of the endosome membrane, and after the integrity of the endosome membrane is damaged, the nuclear targeting protein drug-carrying core is released from the endosome into cytoplasm of the nucleus pulposus cell to realize the escape of the endosome.
8. The protein delivery method for targeted reversal of nuclear cell senescence according to claim 7, characterized in that the partial reprogramming is initiated to reverse nuclear cell senescence by means of nuclear localization signal peptide into nucleus, interpreting recombinant OSK reprogramming protein mixture in high concentration glutathione environment in nucleus, comprising the steps of: The nuclear localization signal peptide positioned on the surface of the nuclear targeting protein drug-carrying core is identified and combined by the input protein in cytoplasm, and the combination of the nuclear localization signal peptide and the input protein guides the nuclear targeting protein drug-carrying core to pass through the nuclear pore complex to enter the nucleus, and the nuclear targeting protein drug-carrying core entering the nucleus is exposed to a high concentration glutathione environment; The disulfide bond of the nuclear targeting protein medicine carrying core is triggered to be broken by the high-concentration glutathione environment, so that the nuclear targeting protein medicine carrying core is degraded, and after the nuclear targeting protein medicine carrying core is degraded, the loaded recombinant OSK reprogramming protein mixed solution is released into the nucleus; The released recombinant OSK reprogramming protein mixed solution combines OCT4 protein, SOX2 protein and KLF4 protein with a specific gene promoter region in the cell nucleus, and the combination of OCT4 protein, SOX2 protein and KLF4 protein with the gene promoter region activates a transcription program related to cell rejuvenation; The activated transcription process down-regulates senescence-associated markers, and initiates a partial reprogramming process to reverse nuclear cell senescence.
9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, wherein the processor, when executing the computer program, performs the steps of the protein delivery method of any one of claims 1-8 for targeted reversal of nuclear cell senescence.
10. A computer-readable storage medium, on which a computer program is stored, characterized in that the computer program, when executed by a processor, implements the steps of the protein delivery method of any one of claims 1 to 8 for targeted reversal of nuclear cell senescence.

Description

Protein delivery method for targeted reversal of nucleus pulposus cell aging Technical Field The invention relates to the technical field of biomedical engineering, in particular to a protein delivery method for targeted reversal of nucleus pulposus cell aging. Background The key pathological link of disc degeneration is the senescence of nucleus pulposus cells and the inflammation-senescence vicious circle initiated by the same. In order to reverse cell senescence, a partial reprogramming technology shows great potential, can younger senescent cells on the premise of not inducing tumor risk, and clinical transformation of the technology faces major obstacle that the existing gene delivery method has safety risks such as insertion mutation, and direct delivery of reprogramming proteins is limited by multiple biological barriers such as poor in-vivo stability, difficulty in crossing cell membranes and nuclear membranes, so that nuclear delivery efficiency is extremely low and cannot effectively play a role. Existing partial reprogramming strategies based on gene delivery face significant challenges in clinical transformations. Firstly, the introduction of exogenous genes has the safety problems of insertion mutation, difficult precise control of continuous expression, potential immunogenicity and the like, although the direct delivery of reprogramming proteins can avoid genetic modification risks, protein drugs are easy to be enzymatically hydrolyzed in complex in vivo environments, have poor stability, and have functions of overcoming multiple biological barriers such as cell membranes, endosomal barriers, nuclear membranes and the like, so that the nuclear delivery efficiency is extremely low, and effective treatment concentration is difficult to achieve, and most of conventional nano delivery systems are degraded in endolysosomes after being endocytosed by cells, so that the reprogramming proteins with biological activity cannot be effectively delivered to the action target point of the cell nucleus. Disclosure of Invention The present invention has been made in view of the above-described problems occurring in the prior art. Therefore, the invention provides a protein delivery method for targeted reversal of nucleus pulposus cell aging, which solves the key problems that in the prior art, the reprogramming protein is low in nuclear delivery efficiency and cannot effectively function due to poor in-vivo stability and difficult to efficiently cross multiple biological barriers. In order to solve the technical problems, the invention provides the following technical scheme: in a first aspect, the present invention provides a protein delivery method for targeted reversal of nuclear cell senescence comprising preparing a recombinant OSK reprogramming protein cocktail of OCT4 protein, SOX2 protein, and KLF4 protein; The recombinant OSK reprogramming protein mixed solution is used for loading on carboxymethyl chitosan nano-particles functionalized by nuclear localization signal peptide and endosome escape peptide L17E through electrostatic adsorption and hydrophobic action, so as to form a nuclear targeting protein drug-carrying core; coating the core targeting protein drug-carrying core and membrane vesicles extracted from M1 polarized macrophages by ultrasonic fusion and sequential extrusion technology to prepare a bionic delivery unit; Applying the bionic delivery unit into a pathological change intervertebral disc of an intervertebral disc degeneration model animal in an intra-intervertebral-disc injection mode, and carrying out targeted delivery on nucleus pulposus cells; the bionic delivery unit neutralizes inflammatory factors and weakens inflammatory microenvironment in the pathological change intervertebral disc through M1 polarized macrophage membrane vesicles, and realizes endosome escape through endosome escape peptide L17E after being internalized by nucleus pulposus cells; the nuclear localization signal peptide enters the nucleus, the recombinant OSK reprogramming protein mixed solution is interpreted and released in the environment of high concentration glutathione in the nucleus, and the partial reprogramming program is started to reverse the aging of the nucleus cell. As a preferred embodiment of the protein delivery method for targeted reversal of aging of nucleus pulposus cells of the present invention, a recombinant OSK reprogramming protein mixture of OCT4 protein, SOX2 protein and KLF4 protein is prepared comprising the steps of: respectively transforming plasmids encoding OCT4 protein, SOX2 protein and KLF4 protein genes into expression hosts; Culturing and inducing expression of the transformed escherichia coli competent cells of the gene plasmids of the OCT4 protein, the SOX2 protein and the KLF4 protein to obtain bacteria expressing the OCT4 protein, bacteria expressing the SOX2 protein and bacteria expressing the KLF4 protein; Respectively lysing the thalli expressing OCT4 protein, t