Inflammation response type sustained release microsphere system of bionic ECM activating factor

Abstract

The invention relates to the technical field of biomedical materials, in particular to a biomimetic ECM activating factor inflammation response type sustained release microsphere system, which comprises: a) core-shell structure PLGA microspheres, the core of which is loaded with USAG-1 recombinant protein (the drug loading capacity is greater than or equal to 1.5 mu g / mg), and the shell of which is composed of an MMP-9 sensitive peptide crosslinking layer; b) co-modifying nano-hydroxyapatite (nHA) and RGD peptide on the surface of the microsphere, wherein the molar ratio of nHA to RGD is 1: 3; c) uniformly dispersing the BMP-2 in the PLGA matrix to realize zero-order slow-release kinetics; the precise time sequence controlled release and microenvironment responsiveness are as follows: 1) inflammatory stage targeted release: when the local MMP-9 concentration is greater than 15nM, the shell GPLGVRGD peptide crosslinking layer is rapidly degraded (release is greater than or equal to 65% USAG-1 within 24h), premature activation of BMP-2 is inhibited, and the ectopic ossification occurrence rate is reduced from 24.6% to 3.8% (p is less than 0.001); (2) continuously promoting regeneration in a repair period: slowly releasing BMP-2 (53.2 + / -2.1% accumulatively released in 56 days) by a PLGA core through zero-order dynamics, maintaining a stable osteogenesis signal, and increasing the volume ratio of new bones to 58.7% (22.3% of a control group);

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, in particular to an inflammation-responsive sustained-release microsphere system of a biomimetic ECM activating factor. Background Art

[0002] In the field of bone / tooth tissue regeneration and repair, precise controlled release of bioactive factors is the key to achieving efficient repair.

[0003] Microsphere systems using poly(lactic-co-glycolic acid) (PLGA) as a carrier are widely used for growth factor delivery, but they still face significant challenges: Inadequate microenvironmental responsiveness: The degradation mechanism of conventional PLGA microspheres relies solely on the material's own hydrolysis, failing to distinguish between physiological and pathological microenvironments. For example, during the early inflammatory phase of bone repair, local matrix metalloproteinase (MMP-9) concentrations significantly increase (typically >15nM), but existing PLGA microspheres are unable to respond to this signal, leading to premature or ectopic release of bone morphogenetic protein (BMP-2) and osteophyte formation rates exceeding 25% (see Biomaterials, 2018, 159: 1-12). Furthermore, poor cell adhesion properties: To enhance cell adhesion, existing technologies often use RGD peptides to modify the scaffold surface. However, due to the irrational spatial arrangement of peptide chains and the lack of biomimetic mineralization, single-RGD-modified scaffolds have a cell adhesion rate of less than 40% (Acta Biomater, 2020, 105: 100-110), significantly restricting seeded cell migration and osteogenic differentiation. Limitations of single-factor release: Some studies have constructed responsive systems using MMP-sensitive peptides, but these can only achieve the release of a single factor (such as BMP-2) and cannot coordinate the time-controlled release of antagonistic factors (such as USAG-1) (Adv Drug Deliv Rev, 2021, 173: 1-15). This single regulatory mechanism fails to mimic the synergistic effects of multiple factors in the natural extracellular matrix (ECM), resulting in blurred osteoblastic boundaries (error >300μm) and insufficient repair precision.

[0004] Therefore, we proposed an inflammation-responsive sustained-release microsphere system of biomimetic ECM activating factors to solve the above problems. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention discloses an inflammation-responsive sustained-release microsphere system of a biomimetic ECM activating factor, the technical solution of which comprises:

[0006] a) Core-shell PLGA microspheres: The core is loaded with USAG-1 recombinant protein (drug loading ≥1.5 μg / mg), and the shell is composed of a cross-linked layer of MMP-9 sensitive peptide. The MMP sensitive peptide in the shell is enzyme-responsive. The degradation rate of the core PLGA is regulated by molecular weight or copolymerization ratio to meet the zero-order release requirements of BMP-2 (traditional PLGA mostly has a first-order release).

[0007] b) The microsphere surface was co-modified with nanohydroxyapatite (nHA) and RGD peptide at a molar ratio of 1:3. nHA provided bone binding sites, while RGD promoted the adhesion of mesenchymal stem cells, increasing the cell adhesion rate to 82.5%.

[0008] c) BMP-2 is uniformly dispersed in the PLGA matrix, achieving zero-order sustained-release kinetics.

[0009] As a preferred technical solution of the present invention, the MMP-9 sensitive peptide sequence is GPLGVRGD. When the MMP-9 concentration in the microenvironment is greater than 15 nM, ≥60% of USAG-1 is released within 24 hours.

[0010] As a preferred technical solution of the present invention, the C-terminus of the USAG-1 recombinant protein is fused with a heparin binding domain (sequence KKKRK), so that the retention time at the defect site is ≥72 hours.

[0011] As a preferred technical solution of the present invention, the system is used in combination with a gradient scaffold for bone / tooth tissue regeneration and repair.

[0012] As a preferred technical solution of the present invention, a method for preparing an inflammation-responsive sustained-release microsphere system of a biomimetic ECM activating factor is characterized by comprising the following steps:

[0013] S1. Preparation of PLGA microspheres using W / O / W emulsion method:

[0014] 1) Internal aqueous phase: USAG-1 / PBS (2 mg / mL) mixed with BMP-2 (1 mg / mL);

[0015] 2) Oil phase: PLGA (50 mg / mL) dissolved in dichloromethane (DCM) and 0.1% Span80 added;

[0016] 3) External aqueous phase: 2% PVA solution containing 5 mM EDC / NHS crosslinker.

[0017] S2, MMP peptide modification:

[0018] GPLGVRGD peptide (5 mg / mL) was mixed with nHA / RGD (1:3) and grafted onto the microsphere surface through amide bonds.

[0019] S3. Post-processing: freeze-dried and then sterilized by gamma irradiation (25 kGy).

[0020] As a preferred technical solution of the present invention, targeted release is performed during the inflammatory period (0-7 days). When MMP-9 is greater than 15nM, the shell degrades and bursts out to release USAG-1. The release time is 24h and the release amount is ≥65%.

[0021] As a preferred technical solution of the present invention, zero-order sustained release is achieved during the repair period (8-56 days), and the PLGA body degrades at a rate of 0.8 μg / day, thereby achieving zero-order sustained release of BMP-2.

[0022] Beneficial effects of the present invention:

[0023] 1. Precise time-controlled release and microenvironmental responsiveness:

[0024] 1) Targeted release during the inflammatory phase: When the local MMP-9 concentration is >15 nM, the outer GPLGVRGD peptide cross-linked layer rapidly degrades (≥65% USAG-1 is released within 24 hours), inhibiting the premature activation of BMP-2 and reducing the incidence of heterotopic ossification from 24.6% to 3.8% (p < 0.001);

[0025] 2) Continuous promotion of regeneration during the repair period: The PLGA core sustained release of BMP-2 via zero-order kinetics (83.2±2.1% cumulative release over 56 days), maintaining a stable osteogenic signal and increasing the proportion of new bone volume to 58.7% (22.3% in the control group);

[0026] 3) Dual-factor antagonistic regulation: USAG-1 and BMP-2 formed a dynamic concentration gradient (1:8→1:25), spatially limiting the osteogenesis area with a boundary accuracy of 132±28μm (315±45μm in the control group).

[0027] 2. Bionic surface modification enhances cell response:

[0028] 1) nHA / RGD synergistic effect: Nanohydroxyapatite (nHA) provides a biomimetic mineralized interface, while RGD peptide optimizes integrin binding sites, increasing MC3T3 cell adhesion to 82.5% (unmodified group: 35.4%) and the spreading area by 2.3 times (ImageJ analysis). Synergistic nHA / RGD modification optimizes mechanical support and biological signal transmission, resulting in a 107% increase in cell adhesion.

[0029] 2) Optimization of mechanical properties: AFM detection showed that the cell adhesion force after composite modification reached 8.7nN, which was significantly higher than that of the single modification group (4.2nN), promoting cytoskeleton reorganization and osteogenic differentiation.

[0030] 3. Clinical transformation advantages:

[0031] 1) Doubled bone regeneration efficiency: Animal experiments showed that the combined use of gradient scaffolds increased bone regeneration speed by 2.6 times compared to FDA-approved products, and the repair cycle was shortened by 50%;

[0032] 2) Safety assurance: Through the synergistic effect of early burst release of USAG-1 and sustained release of BMP-2, the risk of heterotopic ossification is reduced by 84.5% (p < 0.001), and gamma irradiation sterilization (25kGy) ensures the sterility of the microspheres.

[0033] 4. Process Scalability: Core-shell microspheres were prepared using a W / O / W emulsion method, with BMP-2 dispersed in the inner aqueous phase and USAG-1 loaded onto the PLGA shell. The microspheres had a uniform particle size (PDI = 0.15) and a precisely controllable drug loading (USAG-1: 1.82 ± 0.15 μg / mg, BMP-2: 0.95 ± 0.08 μg / mg), demonstrating potential for large-scale production. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0035] The present invention discloses an inflammation-responsive sustained-release microsphere system of a biomimetic ECM activating factor, and the technical solution adopted is as follows:

[0036] a) Core-shell PLGA microspheres, with the core loaded with USAG-1 recombinant protein (drug loading ≥ 1.5 μg / mg) and the shell composed of a cross-linked layer of MMP-9 sensitive peptide;

[0037] b) The microsphere surface was co-modified with nanohydroxyapatite (nHA) and RGD peptide at a molar ratio of 1:3. nHA provided bone binding sites, while RGD promoted the adhesion of mesenchymal stem cells, increasing the cell adhesion rate to 82.5%.

[0038] c) BMP-2 is uniformly dispersed in the PLGA matrix, achieving zero-order sustained-release kinetics.

[0039] As a preferred technical solution of the present invention, the MMP-9 sensitive peptide sequence is GPLGVRGD. When the MMP-9 concentration in the microenvironment is greater than 15 nM, ≥60% of USAG-1 is released within 24 hours.

[0040] As a preferred technical solution of the present invention, the C-terminus of the USAG-1 recombinant protein is fused with a heparin binding domain (sequence KKKRK), so that the retention time at the defect site is ≥72 hours.

[0041] As a preferred technical solution of the present invention, the system is used in combination with a gradient scaffold for bone / tooth tissue regeneration and repair.

[0042] As a preferred technical solution of the present invention, a method for preparing an inflammation-responsive sustained-release microsphere system of a biomimetic ECM activating factor is characterized by comprising the following steps:

[0043] S1. Preparation of PLGA microspheres using W / O / W emulsion method:

[0044] 1) Internal aqueous phase: USAG-1 / PBS (2 mg / mL) mixed with BMP-2 (1 mg / mL);

[0045] 2) Oil phase: PLGA (50 mg / mL) dissolved in dichloromethane (DCM) and 0.1% Span80 added;

[0046] 3) External aqueous phase: 2% PVA solution containing 5 mM EDC / NHS crosslinker.

[0047] S2, MMP peptide modification:

[0048] GPLGVRGD peptide (5 mg / mL) was mixed with nHA / RGD (1:3) and grafted onto the microsphere surface through amide bonds.

[0049] S3. Post-processing: freeze-dried and then sterilized by gamma irradiation (25 kGy).

[0050] As a preferred technical solution of the present invention, targeted release is performed during the inflammatory period (0-7 days). When MMP-9 is greater than 15nM, the shell degrades and bursts out to release USAG-1. The release time is 24h and the release amount is ≥65%.

[0051] As a preferred technical solution of the present invention, zero-order sustained release is achieved during the repair period (8-56 days), and the PLGA body degrades at a rate of 0.8 μg / day, thereby achieving zero-order sustained release of BMP-2.

[0052] Synergistic mechanism

[0053] The nHA / RGD complex increased the adhesion rate of MC3T3 cells to 82.5% (35.4% in the control group);

[0054] USAG-1 and BMP-2 form a concentration gradient antagonism (1:8→1:25), spatially limiting the osteogenic area (boundary accuracy <150μm).

[0055] In vitro performance testing

[0056] Release Kinetics:

[0057] In the presence of MMP-9: 24h burst release of USAG-1 was 67.3±3.8%, and 56-day cumulative release of BMP-2 was 83.2±2.1%;

[0058] The release curve was consistent with the Korsmeyer-Peppas model (n=0.43, Fickian diffusion mechanism).

[0059] Cell experiments:

[0060] Osteogenesis inhibition experiment: USAG-1 (8 ng / mL) reduced BMP-2-induced ALP activity by 81% (p < 0.001);

[0061] Adhesion assay: The cell spreading area in the nHA / RGD group increased 2.3-fold (ImageJ analysis).

[0062] Animal experiments

[0063] The beagle mandibular defect model shows:

[0064] In the experimental group (combined patent 1 scaffold), the new bone volume accounted for 58.7% at 6 weeks (22.3% in the control group);

[0065] The incidence of heterotopic ossification was 3.8% (24.6% in the control group), and Micro-CT showed clear osteoblastic boundaries (error 132±28μm).

[0066] Characterization of the physicochemical properties of microspheres

[0067] Table 1: Microsphere particle size and drug loading determination

[0068] parameter Measured value method Average particle size (μm) 12.5 ± 2.3 Laser particle size analyzer (Malvern) Drug loading (USAG-1) 1.82 ± 0.15 μg / mg BCA protein quantification Drug loading (BMP-2) 0.95 ± 0.08 μg / mg ELISA test Surface zeta potential (mV) -23.4 ± 1.7 Zeta Potential Instrument

[0069] Description: The microspheres have uniform particle size distribution (PDI=0.15), negative surface potential is conducive to stable dispersion, and the drug loading capacity meets the design requirements.

[0070] MMP-9 concentration-dependent release experiment

[0071] Table 2: USAG-1 release rate at different MMP-9 concentrations (24h)

[0072] MMP-9 concentration (nM) USAG-1 release rate (%) Significance (p-value) 0 (control group) 5.2 ± 1.1 - 10 18.4 ± 2.3 0.12 15 67.3 ± 3.8 <0.001 20 72.6 ± 4.1 <0.001

[0073] Note: When the MMP-9 concentration is ≥15nM, the USAG-1 release rate is significantly increased (p<0.001), demonstrating the inflammatory response properties of the shell.

[0074] Cell adhesion and proliferation assay

[0075] Table 3: Effects of different surface modifications on MC3T3 cell adhesion rate (24h)

[0076] Group Cell adhesion rate (%) Spreading area (μm² / cell) Unmodified PLGA 35.4 ± 4.2 520 ± 45 RGD single modification 58.1 ± 5.6 890 ± 68 nHA / RGD co-modification 82.5 ± 3.9 1190 ± 92

[0077] Note: The adhesion rate and spreading area of ​​the nHA / RGD co-modified group were significantly better than those of the control group (p<0.001), indicating that the composite modification synergistically enhanced the cell response.

[0078] Comparison of bone regeneration effects in vivo

[0079] Table 4: 6-week repair effect of beagle mandibular defect model

[0080] parameter Experimental group (combined stent) Control group (traditional stent) p-value New bone volume percentage (%) 58.7 ± 4.3 22.3 ± 3.1 <0.001 Incidence of heterotopic ossification (%) 3.8 ± 0.9 24.6 ± 2.5 <0.001 Osteogenesis boundary error (μm) 132 ± 28 315 ± 45 <0.001

[0081] Note: The bone regeneration efficiency of the experimental group increased by 2.6 times, and the accuracy of the bone formation boundary was significantly improved (error <150μm).

[0082] Experimental data description

[0083] Statistical methods: All data were analyzed by one-way analysis of variance (ANOVA) using SPSS 26.0, and the significance level was set at p < 0.05.

[0084] Experimental reproducibility: Each experiment was repeated 3 times, and the data were expressed as mean ± standard deviation.

[0085] Components not described in detail herein are prior art.

[0086] Although the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by ordinary technicians in this field, various changes can be made without departing from the purpose of the present invention. Modifications or deformations that do not involve creative work are still within the scope of protection of the present invention.

Claims

1. An inflammatory response sustained-release microsphere system of a biomimetic ECM activator, characterized in that: include: a) Core-shell PLGA microspheres, with the core loaded with USAG-1 recombinant protein (drug loading ≥ 1.5 μg / mg) and the shell composed of a cross-linked layer of MMP-9 sensitive peptide; b) The microsphere surface was co-modified with nanohydroxyapatite (nHA) and RGD peptide, with a nHA:RGD molar ratio of 1:3; c) BMP-2 is uniformly dispersed in the PLGA matrix, achieving zero-order sustained-release kinetics.

2. The inflammatory response sustained-release microsphere system of biomimetic ECM activator according to claim 1, characterized in that: The MMP-9 sensitive peptide sequence is GPLGVRGD. When the MMP-9 concentration in the microenvironment is greater than 15 nM, ≥60% of USAG-1 is released within 24 hours.

3. The inflammatory response sustained-release microsphere system of biomimetic ECM activator according to claim 1, characterized in that: The C-terminus of the USAG-1 recombinant protein is fused with a heparin binding domain (sequence KKKRK), so that the protein stays at the defect site for ≥72 hours.

4. The inflammatory response sustained-release microsphere system of biomimetic ECM activator according to claims 1-3, characterized in that: The system is used in combination with a gradient scaffold for bone / tooth tissue regeneration and repair.

5. The method for preparing the inflammation-responsive sustained-release microsphere system of the biomimetic ECM activator according to claim 4, characterized in that: The steps include: S1. Preparation of PLGA microspheres using W / O / W emulsion method: 1) Internal aqueous phase: USAG-1 / PBS (2 mg / mL) mixed with BMP-2 (1 mg / mL); 2) Oil phase: PLGA (50 mg / mL) dissolved in dichloromethane (DCM) and 0.1% Span80 added; 3) External aqueous phase: 2% PVA solution containing 5mM EDC / NHS crosslinker. S2, MMP peptide modification: GPLGVRGD peptide (5 mg / mL) was mixed with nHA / RGD (1:3) and grafted onto the microsphere surface through amide bonds. S3. Post-processing: freeze-dried and then sterilized by gamma irradiation (25 kGy).

6. The method for preparing the inflammation-responsive sustained-release microsphere system of biomimetic ECM activator according to claim 5, characterized in that: When MMP-9>15nM, the shell degrades and bursts out to release USAG-1, the release time is 24h, and the release amount is ≥65%.

7. The method for preparing the inflammation-responsive sustained-release microsphere system of biomimetic ECM activator according to claim 5, characterized in that: When the PLGA body degrades at a rate of 0.8 μg / day, zero-order sustained release of BMP-2 can be achieved.