CN-120939248-B - Methacryloylated hyaluronic acid hydrogel microsphere wrapped by macrophage membrane and application thereof

Abstract

The invention discloses a macrophage membrane-coated methacrylic acid hyaluronic acid hydrogel microsphere and application thereof, relating to the technical field of drug synthesis, and the technical scheme is as follows: the macrophage membrane-encapsulated methacryloylated hyaluronic acid hydrogel microspheres emm@hms can efficiently neutralize key pro-inflammatory factors including TNF- α and IL-1β, thereby alleviating joint inflammation. The medicine has good curative effect and biocompatibility in an OA model, and particularly has the functions of regulating macrophage polarization, inhibiting chondrocyte hypertrophy and neutralizing pro-inflammatory factors. Through the key link in the pathogenesis of OA, the EMM@HMs not only can remarkably reduce inflammation, but also has the potential of delaying the disease process, and provides a new strategy with breakthrough significance for the treatment of OA.

Inventors

• ZHOU FENG
• MA CHENGGONG
• ZHANG LEI
• Guo Jiongjiong

Assignees

• 苏州大学附属第一医院

Dates

Publication Date

20260508

Application Date

20250910

Claims (6)

1. 1. The macrophage membrane-coated methacrylic acid-acylated hyaluronic acid hydrogel microsphere is characterized in that the macrophage membrane-coated methacrylic acid-acylated hyaluronic acid hydrogel microsphere is EMM@HMs, and the preparation method is as follows: S1, preparing an engineered macrophage membrane EMM, namely constructing IL1R2 and TNFR2 over-expression shuttle plasmid vectors, transfecting prepared plasmids into HEK293T cells by using Lipofectamine 3000, culturing for 48 hours, filtering cell supernatants by using a 0.45 mu m filter, centrifuging for 120 minutes at 20000 rpm at 4 ℃, collecting precipitates and re-suspending, carrying out virus titer measurement by qPCR, transfecting macrophages by using lentivirus, collecting the cells, washing the cells three times by using PBS, re-suspending the cells in a homogenate buffer solution containing 75 mM sucrose, 20 mM Tris-HCl, 2mM MgCl 2 , 10 mM KCl and protease and phosphatase inhibitors, mechanically grinding the cells for 20 times by using a homogenate, centrifuging the lysate for 5 minutes at 3200 Xg, removing the precipitates by using 20000 Xg for 25 minutes, centrifuging the supernatant at 100000 Xg for 35 minutes, collecting cell membrane precipitates, and quantifying the membrane protein concentration by using a BCA kit; S2, preparing EMM@HMs, wherein the size of a microfluidic device is set, an inner needle is 30G, an outer needle is 27G, the inner phase is a PBS solution containing 2% of 150 kDa of methacryloyl hyaluronic acid, 0.25% of LAP photoinitiator and EMM, the outer phase is mineral oil containing 3% of Span 80 emulsifier, microsphere drops are generated under the conditions of the inner phase flow rate of 5 mu L/min and the outer phase flow rate of 100 mu L/min, the microsphere drops are crosslinked and cured by irradiation of 405 nm of blue light for 15 seconds, the cured microspheres are washed to remove oil phase and surfactant, and finally the microspheres are placed in PBS for storage at 4 ℃.
2. 2. Use of the macrophage membrane-encapsulated methacryloylated hyaluronic acid hydrogel microsphere of claim 1 in the preparation of a medicament for treating osteoarthritis.
3. 3. The method according to claim 2, wherein the active ingredient of the drug is a macrophage membrane-coated methacrylic acid hyaluronic acid hydrogel microsphere.
4. 4. The method of claim 3, wherein the agent is used to modulate macrophage polarization.
5. 5. The method of claim 3, wherein the agent is used to inhibit chondrocyte hypertrophy.
6. 6. The method of claim 3, wherein the agent is used to neutralize a proinflammatory factor.

Description

Methacryloylated hyaluronic acid hydrogel microsphere wrapped by macrophage membrane and application thereof Technical Field The invention relates to the technical field of medicine synthesis, in particular to a macrophage membrane-coated methacrylic acid hyaluronic acid hydrogel microsphere and application thereof. Background Osteoarthritis (OA) is a common chronic degenerative joint disease and is mainly characterized by cartilage degeneration, synovitis, bone remodeling, ligament dysfunction and osteophyte formation. Clinical manifestations are mainly joint pain, dysfunction and deformity. The etiology of OA is complex, involving genetic susceptibility, motor impairment, aging, obesity, and other factors, and although it has been widely studied, its exact pathogenesis has not yet been fully elucidated. OA can involve a variety of joints, most commonly knee, hip, hand and spine joints. OA currently affects about 7.6% of the world population (about 5.95 hundred million cases) as one of the most common orthopedic disorders, and is in a continuing upward trend, leading to severe lower limb disability and reduced quality of life. As population aging accelerates, the disease burden caused by OA continues to increase, and has become an important challenge for global public health. Interactions between macrophages and chondrocytes play a key role in the development of OA. The infiltrated monocytes can differentiate into M1-type or M2-type macrophages, mediating a pro-inflammatory response and tissue repair, respectively. Activated M1-type macrophages secrete a number of pro-inflammatory factors (e.g., IL-1 beta, TNF-alpha) that are expressed at high levels in OA joints, which can promote synovial inflammation, promote chondrocyte aging, and extracellular matrix (ECM) degradation by inducing Matrix Metalloproteinases (MMPs). In addition, the glycolytic activity of M1 type macrophages is enhanced, resulting in excessive accumulation of Reactive Oxygen Species (ROS), further promoting inflammatory factor synthesis and exacerbating cartilage destruction. In contrast, M2-type macrophages have anti-inflammatory and repair functions that promote the regression of inflammation. Macrophage membranes naturally express multiple receptors and can adsorb cytokines in the inflammatory microenvironment, but the expression level is limited, reducing the efficiency of neutralizing pro-inflammatory factors. To enhance the therapeutic potential of macrophage membranes, we constructed engineered macrophage membranes (ENGINEERED MACROPHAGE MEMBRANES, EMM) that overexpress TNF- α and IL-1 β receptors to more efficiently bind and neutralize pro-inflammatory factors, thereby reducing joint inflammation and protecting cartilage. Organoid technology has received attention in recent years. Organoids are formed by directed differentiation of stem cells or progenitor cells, have self-renewal and self-organization capabilities, and their three-dimensional structure can mimic the key structural and functional properties of natural tissues, exhibiting great potential in disease modeling, drug screening and regenerative medicine. The cartilage organoid can better reproduce the extracellular matrix composition and mechanical properties of natural cartilage and has corresponding functional advantages, thereby providing biological related microenvironment support for cartilage repair and regeneration. Disclosure of Invention The invention aims to provide a macrophage membrane-coated methacrylic acid hydrogel microsphere and application thereof, and the macrophage membrane-coated methacrylic acid hydrogel microsphere (EMM@HMs) can efficiently neutralize key pro-inflammatory factors (including TNF-alpha and IL-1 beta) so as to relieve joint inflammation. The technical aim of the invention is realized by the following technical scheme that the methacrylic acid acylated hyaluronic acid hydrogel microsphere wrapped by the macrophage membrane is EMM@HMs, and the preparation method is as follows: S1, preparing an engineering macrophage membrane, namely constructing IL1R2 and TNFR2 over-expression shuttle plasmid vectors, transfecting the prepared plasmids into HEK293T cells by using Lipofectamine 3000, culturing for 48 hours, filtering cell supernatants by using a 0.45 mu m filter, centrifuging for 120 minutes at 4 ℃, collecting precipitates and re-suspending, carrying out virus titer measurement by qPCR, transfecting macrophages by using lentivirus, collecting cells, washing three times by using PBS, re-suspending in homogenate buffer solution containing 75 mM sucrose, 20 mM Tris-HCl, 2mM MgCl 2, 10 mM KCl and protease and phosphatase inhibitor, mechanically grinding the cells for 20 times by using a homogenate, centrifuging the lysate for 5 minutes at 3200 Xg, removing the precipitates by using 20000 Xg for 25 minutes, centrifuging the supernatant at 100000 Xg for 35 minutes, collecting cell membrane precipitates, and quantifying the membrane protein c