RGD modified hydrogel and application thereof

Abstract

The invention provides RGD (arginine-glycine-aspartic acid) modified hydrogel and application thereof, relates to the technical field of biological materials, and has the technical key points that the RGD modified hydrogel is provided and is characterized in that the RGD modified hydrogel is prepared from a PEGDA (polyethylene glycol diacrylate) prepolymer, Irgacure 2959 and acryloylated RGD peptide. The application illustrates that aPL induces platelet aggregation, thrombosis and inflammatory response by activating alphaIIb beta3 integrin, and further damages the function of the trophoblast. The RGD functionalized hydrogel based on PEG and the alphaIIb beta3 antibody can significantly inhibit platelet aggregation (decreasing amplitude to 83%), delay fibrin clot formation (OD405 is reduced by 67%), and reduce release of inflammatory factors such as IL-6 / TNF-alpha (decreasing amplitude to 70-80%). More importantly, the intervention measures can protect the migration ability of the trophoblast, which is a key process of placenta vascular remodeling. The integrin targeting biological material strategy provides a new way for preventing and treating APS-related pregnancy complications through local antithrombotic and anti-inflammatory dual effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, in particular to RGD-modified hydrogel and applications thereof. Background Art

[0002] Antiphospholipid syndrome (APS) is an autoimmune thrombophilic disorder characterized by the presence of antiphospholipid antibodies (aPLs), which can lead to vascular thrombosis and pregnancy-related complications. In pregnant women, aPLs cross-react with anionic phospholipid-binding proteins on the surface of platelets and trophoblasts, triggering a hypercoagulable state, inflammation, and placental insufficiency. These pathophysiological changes underlie a range of obstetric complications, including recurrent miscarriage, preeclampsia, and fetal growth restriction.

[0003] Integrin αIIbβ3 is a core mediator of aPL-induced thrombosis. This platelet surface receptor mediates platelet-platelet aggregation by binding fibrinogen and ligands containing the RGD sequence. When activated by aPL or inflammatory signals, αIIbβ3 undergoes conformational changes that enhance adhesive interactions and promote thrombosis within placental vessels. This aggregation not only leads to local ischemia but also triggers the release of platelet-derived cytokines, further impairing trophoblast function and migration—processes crucial for spiral artery remodeling and placental development.

[0004] Although current treatments such as heparin and aspirin offer some protection, they carry the risk of systemic bleeding and do not directly target the molecular mechanisms of platelet activation. Therefore, novel strategies that can locally modulate platelet-integrin interactions may offer safer and more targeted treatment options. Summary of the Invention

[0005] The purpose of the present invention is to solve the problem in the prior art of lacking a strategy capable of locally regulating platelet-integrin interaction.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: An RGD-modified hydrogel is characterized in that it is prepared by using PEGDA prepolymer, Irgacure 2959 and acryloyl RGD peptide.

[0007] Preferably, its preparation method is as follows: S1: Take PEGDA prepolymer, Irgacure 2959 and acryloyl RGD peptide and mix them S2: The mixed solution in S1 was injected into the PDMS mold, and the RGD-modified hydrogel was obtained by UV cross-linking.

[0008] Preferably, the concentration of the PEGDA prepolymer in S1 is 20% (w / v), and the concentration of Irgacure 2959 is 0.05% (w / v).

[0009] Preferably, the final concentration of the acryloyl-RGD peptide is one of 10, 50 or 100 μmol / g.

[0010] Preferably, the conditions for UV cross-linking in S2 are: 365 nm, 5 mW / cm², 3 min.

[0011] Preferably, the RGD-modified hydrogel is washed in PBS for 24 hours before use.

[0012] The present application also provides an application of an RGD-modified hydrogel in the preparation and treatment of pregnancy complications, wherein the RGD-modified hydrogel is the hydrogel described above.

[0013] Preferably, the pregnancy complication is a pregnancy complication induced by antiphospholipid syndrome (APS).

[0014] Preferably, the RGD-modified hydrogel is used to reduce platelet activation and restore trophoblast in patients with antiphospholipid syndrome.

[0015] Compared with the prior art, this application has the following beneficial effects: This application demonstrates that aPL induces platelet aggregation, thrombosis, and inflammation by activating the αIIbβ3 integrin, thereby impairing trophoblast function. PEG-based RGD-functionalized hydrogels and αIIbβ3 antibodies significantly inhibited platelet aggregation (by 83%), delayed fibrin clot formation (OD405 decreased by 67%), and reduced the release of inflammatory factors such as IL-6 and TNF-α (by 70-80%). Importantly, these interventions preserved trophoblast migration, a key process in placental vascular remodeling. This integrin-targeted biomaterial strategy, through its dual local antithrombotic and anti-inflammatory effects, offers a novel approach for preventing and treating APS-related pregnancy complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of aPL-induced platelet activation and integrin αIIbβ3-mediated placental thrombosis in one embodiment of the present application; Figure 2Comparison of the binding affinity of integrin αIIbβ3 to PEGDA-RGD hydrogels with different RGD densities in one embodiment of the present application, used to demonstrate the dose-dependent effect of RGD-functionalized hydrogels on integrin αIIbβ3 binding; Figure 3 This is a comparison diagram of the inhibitory effects of PEG hydrogel and αIIbβ3 antibody on aPL-induced platelet aggregation in one embodiment of the present application; Figure 4 This is a comparison diagram of the fibrin clot formation kinetics in different treatment groups in one embodiment of the present application; Figure 5 This is a comparison diagram of the effects of different treatment groups on the release of inflammatory factors from trophoblast cells in one embodiment of the present application; Figure 6 This is a comparison chart of the effects of different treatment groups on the migration ability of trophoblast cells in one embodiment of the present application. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to specific embodiments.

[0018] This application provides an RGD-modified hydrogel, the preparation method of which is as follows: S1: Hydrogel preparation: 20% (w / v) PEGDA prepolymer was mixed with 0.05% (w / v) Irgacure 2959 and acryloyl-RGD peptide (final concentration 0, 10, 50, or 100 μmol / g).

[0019] The mass ratio of the PEGDA prepolymer, Irgacure 2959, and acryloyl-RGD peptide is (20-30):0.05:(0.1-0.5)". For example, in a preferred embodiment, the mass ratio can be 20:0.05:0.1, and the final concentration of the acryloyl-RGD peptide is 10 μmol / g; when the mass ratio is 20:0.05:0.5, the final concentration of the acryloyl-RGD peptide is 100 μmol / g.

[0020] The solution was injected into a PDMS mold in a 96-well plate and cross-linked by UV light (365 nm, 5 mW / cm², 3 min).

[0021] Before use, hydrogels were washed in PBS for 24 h.

[0022] In addition, the present application also provides the use of RGD-modified hydrogels in the preparation and treatment of pregnancy complications, wherein the pregnancy complications are pregnancy complications induced by antiphospholipid syndrome (APS), and the RGD-modified hydrogels are used to reduce platelet activation and restore the trophoblast in patients with antiphospholipid syndrome.

[0023] See also Figure 1 The pathophysiological process begins when circulating antiphospholipid antibodies (aPL) interact with the platelet surface, triggering platelet activation. Activated platelets upregulate the expression of integrin αIIbβ3, a receptor that mediates platelet-platelet binding via a fibrinogen bridge. This enhanced adhesion leads to platelet aggregation and ultimately to the formation of intraplacental thrombi. This schematic highlights the role of integrin αIIbβ3 as a central mediator of aPL-driven placental thrombotic pathology.

[0024] Figure 1 The molecular mechanism by which antiphospholipid antibodies (aPL) induce thrombotic events in pregnancy is revealed. In patients with antiphospholipid syndrome (APS), circulating antibodies target phospholipid-binding proteins on the platelet surface, leading to pathological activation. This process promotes increased surface expression of integrin αIIbβ3, an adhesion receptor that drives irreversible platelet aggregation by mediating platelet cross-linking and fibrinogen bridging. This cascade of reactions within the placental vessels can trigger local thrombosis, impair maternal-fetal blood exchange, and ultimately lead to miscarriage, preeclampsia, and fetal growth restriction. This pathway illustrates that integrin αIIbβ3 can serve as a rational therapeutic target: biomaterials such as RGD-functionalized hydrogels or antibody-loaded drug platforms can intervene in thrombosis by specifically binding to or blocking αIIbβ3 without inducing systemic anticoagulation.

[0025] The above contents are described below with reference to specific embodiments: Materials and sources: Polyethylene glycol diacrylate (PEGDA, molecular weight ~20000 Da), photoinitiator Irgacure 2959, and human fibrinogen were purchased from Sigma-Aldrich.

[0026] Acryloyl-RGD peptide (Ac-GRGDSC) was synthesized by cysteine ​​thiol modification with a purity of >95% and terminal acryloylation.

[0027] Recombinant human integrin αIIbβ3 (His tag) and IL-6 and TNF-α ELISA kits were purchased from R&D Systems.

[0028] Human trophoblast cells HTR-8 / SVneo were purchased from ATCC.

[0029] Platelets were freshly isolated from healthy donors (approved by the ethics committee).

[0030] All reagents are endotoxin-free. PBS, DMEM / F12 medium, fetal bovine serum (FBS), and other cell culture materials were purchased from Gibco.

[0031] Example 1: Preparation of RGD-functionalized PEGDA hydrogel and integrin binding assay S1: Hydrogel preparation and storage: A 20% (w / v) solution of PEGDA prepolymer and a 0.05% (w / v) solution of Irgacure 2959 were prepared in sterile water and then mixed thoroughly with the acryloyl-RGD peptide before proceeding. At room temperature, the 20% (w / v) PEGDA prepolymer, 0.05% (w / v) Irgacure 2959 solution, and acryloyl-RGD peptide (final concentrations of 0, 10, 50, or 100 μmol / g) were placed in a clean container and stirred at 500-800 rpm for 3-5 minutes to thoroughly mix the components and avoid air bubbles. Maintain a clean environment during mixing to prevent impurities from contaminating the hydrogel. Finally, the mixed solution was poured into a 96-well PDMS mold and UV-crosslinked (365 nm, 5 mW / cm², 3 minutes). Before use, hydrogels were washed in PBS for 24 h.

[0032] The prepared RGD-modified hydrogel should be stored at 4°C, sealed, and protected from light before use. After washing in PBS, it should be used as soon as possible or stored short-term according to the above conditions to ensure its stability and bioactivity. Long-term storage may cause changes in the hydrogel's physicochemical properties, affecting its effectiveness in treating pregnancy complications.

[0033] S2: Integrin binding: Recombinant integrin αIIbβ3 (1 μg / mL) was incubated with the hydrogel at 37°C for 1 hour. After washing, anti-His HRP-conjugated antibody (1:1000, 1 hour) was added, followed by TMB chromogenic substrate. OD450 was measured to quantify binding.

[0034] The results are as follows Figure 2As shown, the binding capacity of integrin αIIbβ3 showed a significant positive correlation with the density of RGD ligands within the PEGDA hydrogel matrix. At an RGD concentration of 0 μmol / g, baseline binding was extremely low (OD450 ≈ 0.1), indicating negligible nonspecific interactions. When the RGD concentration increased to 10 μmol / g, the OD450 increased to approximately 0.35; further increases to 50 μmol / g and 100 μmol / g reached OD450s of ~0.75 and ~0.95, respectively. These results demonstrate that increasing the degree of RGD functionalization in the hydrogels significantly enhances the binding affinity of αIIbβ3 in a dose-dependent manner.

[0035] These experimental results confirm that ligand density is a key parameter in regulating integrin-material interactions. The αIIbβ3 integrin plays a central role in platelet aggregation by recognizing the RGD motif of adhesion proteins such as fibrinogen. By increasing the RGD density of PEGDA hydrogels (0-100 μmol / g), we observed a dose-dependent increase in receptor binding affinity. This finding has important biomedical engineering implications: at moderate RGD densities (10-50 μmol / g), the hydrogels act as competitive decoys, partially sequestering αIIbβ3 and attenuating platelet activation; whereas a high RGD density (100 μmol / g) achieves near-saturated integrin binding, making it suitable for the local prevention of aPL-induced placental thrombosis. This study provides a molecular basis for the rational design of antithrombotic biomaterials.

[0036] Example 2: Platelet aggregation experiment under aPL stimulation 1. Platelet Preparation: After centrifugation of sodium citrate anticoagulated whole blood, platelets (2 × 10 8 / mL).

[0037] 2. Experimental groups: aPL only (APS patient purified IgG, 100 μg / mL), aPL + PEG hydrogel (pre-incubated), aPL + αIIbβ3 antibody (10 μg / mL) 3. Aggregation Assay: Platelet aggregation was measured using a turbidimetric method (Chrono-log aggregometer). 10 μM ADP was used as a positive control, and the maximum aggregation rate (%) was calculated.

[0038] like Figure 3As shown, aPL stimulation significantly enhanced platelet aggregation to approximately 90%, compared to a very low baseline aggregation rate in the control group (~5%). Addition of PEG hydrogel reduced the aggregation rate to ~60%, demonstrating a partial but significant inhibitory effect. More strikingly, treatment with a specific integrin αIIbβ3 antibody further reduced platelet aggregation to approximately 15%, demonstrating a more effective blockade of the platelet-platelet adhesion pathway. These results demonstrate that both biomaterials and molecular interventions can effectively mitigate the prothrombotic effects of aPL.

[0039] pass Figure 3 Experimental results validated the effectiveness of biomaterials in intervening in aPL pathology. The near-complete aggregation (~90%) observed in the "aPL-only" group demonstrates the potent aggregation-promoting activity of antiphospholipid antibodies through activation of αIIbβ3. Despite being an inert material, PEG hydrogel reduced the aggregation rate to ~60%, likely through steric interference with platelet interactions. The αIIbβ3 antibody group exhibited only ~15% aggregation, demonstrating the effectiveness of specific blockade of receptor-ligand interactions. These findings offer new insights into local antithrombotic therapy for APS during pregnancy—one that can mitigate systemic bleeding risks while precisely intervening in the pathological process.

[0040] In addition, if Figure 4 As shown, the OD405 value of the aPL-treated group alone increased rapidly and significantly, reaching a peak of approximately 1.8 at 25 minutes, indicating accelerated fibrin clot formation. In contrast, the OD405 value of the control group without aPL stimulation increased slowly, ultimately stabilizing at approximately 1.1. The coagulation kinetics of the aPL+PEG hydrogel-treated group were intermediate between the two groups, with a slower OD405 increase, reaching ~1.3 at 25 minutes. Notably, the OD405 value of the aPL+αIIbβ3 antibody-treated group decreased significantly throughout the time course, reaching a final value of only ~0.6, reflecting a significant inhibition of fibrin polymerization. Figure 4 The study revealed a mechanism by which aPL accelerates thrombin generation and fibrin polymerization through platelet activation (peak OD405 in the aPL group reached 1.8). PEG hydrogel (OD405 ~1.3) likely slows this process by altering the local coagulation microenvironment, while αIIbβ3 antibodies (OD405 ~0.6) directly inhibit platelet aggregation and reduce the formation of a procoagulant surface. This suggests two strategies for managing aPL-associated placental thrombosis: material-mediated physical intervention and molecularly targeted therapy.

[0041] Example 3: Cytokine release assay of trophoblast cells 1. Cell culture: HTR-8 / SVneo cells (1 × 10 5 / well) were seeded in 24-well plates. After 24 hours, the platelet-conditioned medium of different treatment groups was replaced (see Figure 3 ).

[0042] 2. ELISA test: After 24 h of incubation, the levels of IL-6 and TNF-α in the supernatant were detected according to the manufacturer's instructions.

[0043] like Figure 5 As shown, baseline cytokine levels in the control group were low (IL-6 ~20 pg / mL, TNF-α ~10 pg / mL). Following aPL stimulation, IL-6 and TNF-α levels increased dramatically to ~150 pg / mL and ~120 pg / mL, respectively, suggesting that platelet activation may mediate a robust inflammatory response. Cytokine release was partially attenuated when platelet-conditioned medium from the aPL+PEG hydrogel group was used (IL-6 ~100 pg / mL, TNF-α ~85 pg / mL). Cytokine levels in the aPL+αIIbβ3 antibody-treated group decreased even more significantly (IL-6 ~35 pg / mL, TNF-α ~25 pg / mL), approaching control levels.

[0044] Figure 5 They demonstrated that aPL-activated platelets exacerbate trophoblast inflammation through a paracrine mechanism (IL-6 levels increased by 7.5-fold and TNF-α levels increased by 12-fold). PEG hydrogels partially alleviated the cytokine storm (reduced by 30-40%), while αIIbβ3 antibodies almost completely reversed the inflammatory response, confirming that integrin-mediated platelet degranulation is the core mechanism. This finding sheds light on a novel pathway of coagulation-immunity crosstalk in APS.

[0045] Example 4: Transwell migration assay 1. Experimental setup: HTR-8 / SVneo cells (5 × 10 4 Platelet-conditioned medium (600 μL) was added to the upper chamber of a 24-well Transwell insert (8 μm pore size, Corning).

[0046] 2. Migration analysis: After 24 hours, the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The number of migrated cells was counted in five randomly selected fields under an inverted microscope.

[0047] like Figure 6As shown, the number of trophoblast cells migrating in the control group reached ~220 per field of view, indicating a high basal migration capacity. However, platelet-conditioned medium stimulated with aPL significantly reduced the number of migrating cells to ~80 per field of view, indicating a strong inhibitory effect on trophoblast cell motility. The number of migrating cells in the aPL + PEG hydrogel group was moderately restored to ~140 per field of view, while the aPL + αIIbβ3 antibody group almost completely restored the number to the control level (~200 per field of view). These results indicate that both interventions can alleviate aPL-induced inhibition of migration, with the antibody having a more pronounced effect.

[0048] Figure 6 The study demonstrated that aPL, through platelet-derived signaling, reduced trophoblast migration by 64% (from 220 cells / field to 80 cells / field). PEG hydrogel partially restored migration (140 cells / field), while the αIIbβ3 antibody nearly completely rescued this effect (200 cells / field). This finding has important clinical implications: maintaining trophoblast migration is crucial for placental vascular remodeling, and integrin-targeted intervention can effectively disrupt the vicious cycle of aPL, platelet activation, and trophoblast dysfunction.

[0049] In summary, this application demonstrates that aPL induces platelet aggregation, thrombosis, and inflammation by activating the αIIbβ3 integrin, thereby impairing trophoblast function. PEG-based RGD-functionalized hydrogels and αIIbβ3 antibodies significantly inhibited platelet aggregation (by 83%), delayed fibrin clot formation (OD405 decreased by 67%), and reduced the release of inflammatory factors such as IL-6 and TNF-α (by 70-80%). Importantly, these interventions preserved trophoblast migration, a key process in placental vascular remodeling. Through dual local antithrombotic and anti-inflammatory effects, this integrin-targeted biomaterial strategy offers a novel approach for preventing and treating APS-related pregnancy complications.

Claims

1. An RGD-modified hydrogel, characterized in that: It is prepared from PEGDA prepolymer, Irgacure 2959 and acryloyl-RGD peptide.

2. The RGD-modified hydrogel according to claim 1, wherein: The preparation method is as follows: S1: Take PEGDA prepolymer, Irgacure 2959 and acryloyl RGD peptide and mix; S2: The mixed solution in S1 was injected into the PDMS mold, and the RGD-modified hydrogel was obtained by UV cross-linking.

3. The RGD-modified hydrogel according to claim 2, wherein: The concentration of the PEGDA prepolymer in the S1 was 20% (w / v), and the concentration of the Irgacure 2959 was 0.05% (w / v).

4. The RGD-modified hydrogel according to claim 3, wherein: The final concentration of the acryloyl-RGD peptide is one of 10, 50 or 100 μmol / g.

5. The RGD-modified hydrogel according to claim 5, characterized in that: The conditions for UV cross-linking in S2 are: 365 nm, 5 mW / cm², 3 min.

6. The RGD-modified hydrogel according to claim 5, characterized in that: The RGD-modified hydrogels were washed in PBS for 24 hours before use.

7. Use of an RGD-modified hydrogel in the preparation and treatment of pregnancy complications, characterized in that: The RGD-modified hydrogel is the hydrogel according to any one of claims 1 to 6.

8. Use of an RGD-modified hydrogel according to claim 7 in preparing a method for treating pregnancy complications, characterized in that: The pregnancy complication is a pregnancy complication induced by antiphospholipid syndrome (APS).

9. Use of an RGD-modified hydrogel according to claim 8 in preparing a method for treating pregnancy complications, characterized in that: The RGD-modified hydrogel is used to reduce platelet activation and restore the trophoblast in patients with antiphospholipid syndrome.

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