Preparation method of integrin decoy receptor based on molecular imprinting and application of integrin decoy receptor in bidirectional regulation of integrin signal

Abstract

The invention belongs to the field of biological materials and biomedicine, and discloses a preparation method of an integrin decoy receptor based on molecular imprinting and application of the integrin decoy receptor in bidirectional regulation of integrin signals. From the perspective of design of an integrin bait receptor, the bait receptor with a similar artificial antagonistic molecule molecular function is prepared through a solid-phase template imprinting technology by taking bidirectional regulation (inhibition and excitation) of integrin-related cell behaviors as a target, and the bait receptor is used for regulating and controlling integrin-mediated cell behaviors. The method is crucial for replacing an antagonist to realize bidirectional regulation and control of integrin signals. The bait receptor prepared by the invention can be reversibly combined with integrin targeting polypeptide, so that the polarization of macrophages related to integrin and the differentiation behavior of mesenchymal stem cells are bidirectionally regulated and controlled. The invention proves the feasibility of the molecularly imprinted decoy receptor in dynamic signal regulation and cell operation, and shows a huge prospect of customized therapeutic drugs in tissue repair and other medical fields.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials and biomedicine, and relates to the preparation of an integrin decoy receptor based on molecular imprinting recognition and its application in regulating cell signals. Background Art

[0002] Integrin signaling plays a crucial role in nearly all complex physiological processes in the human body. Integrin signaling is directly involved in a variety of cellular processes. During tissue regeneration, the dynamic regulation of ECM-integrin interactions and the resulting adaptive signaling changes play a crucial role in immune regulation and tissue growth. However, under pathological conditions, aberrant integrin activation often occurs. Researchers have worked hard to develop artificial antagonists to intervene in ECM-integrin interactions, but these antagonists can only achieve unilateral downregulation of integrin signaling. The reversible regulation of integrin activation and inactivation to adapt to the dynamic process of tissue regeneration is a field that is still in its infancy. Against this backdrop, the dynamic regulation of integrin signaling is gaining increasing attention, particularly in the fields of disease treatment and tissue regeneration.

[0003] Therefore, developing novel receptors that specifically bind to integrins and constructing novel approaches for bidirectionally modulating integrin signaling and thereby directing cell fate have become key topics in the biomedical field. Such novel receptors should possess antibody-like functionality, acting as "decoy receptors" that specifically capture or inhibit ligand activity. The reversible and specific recognition properties of molecularly imprinted polymers (MIPs) perfectly align with the functional requirements of "decoy receptors." Unlike the development of artificial antagonists, the preparation of MIPs avoids the time-consuming synthesis and screening process. Furthermore, the polymer networks of MIPs can incorporate stimulus-responsiveness to achieve controllable binding, further facilitating dynamic and even on-demand modulation of integrin signaling, which is crucial for replacing antagonists and achieving bidirectional modulation of integrin signaling. While the fundamental application of MIPs as artificial antibodies for inhibiting target bioactivity or for targeting cell membranes has been reported, their application in constructing "decoy receptors" to modulate key cellular signaling pathways (such as integrin signaling) and dynamically manipulate cell fate has yet to be realized. In order to match the functions of similar antibodies, the preparation process of molecular imprinting-based integrin decoy receptors needs to be further optimized so that they can truly simulate the performance of natural antibodies in vivo and at the cellular level, and construct a new method for bidirectional regulation of cellular integrin signals by guiding macrophage polarization and stem cell differentiation. Summary of the Invention

[0004] The present invention provides a method for preparing an integrin decoy receptor based on molecular imprinting and its application in bidirectional regulation of cell signals, with the goal of achieving bidirectional regulation (inhibition or stimulation) of integrin-related cell behavior by temperature. By solid-phase template imprinting technology, a decoy receptor that can recognize integrin-targeting polypeptides is prepared for constructing a new method for regulating integrin-mediated cell behavior. The integrin decoy receptor is synthesized by solid-phase imprinting method, has excellent specific selectivity for integrin-targeting polypeptide RGD, and its specificity is temperature-responsive. The integrin decoy receptor can reversibly bind to RGD molecules, thereby reversibly controlling the binding of integrins to RGD, thereby regulating immune cell polarization and stem cell differentiation; the bidirectional regulation of integrin signals refers to the ability to controllably inhibit or stimulate the interaction between integrins and ECM; the integrin-related cell recognition refers to the integrin-mediated macrophage polarization and bone marrow mesenchymal stem cell differentiation process.

[0005] In order to achieve reversible binding to RGD molecules and bidirectionally regulate the recognition of cellular integrins and RGD by temperature, the present invention provides a method for preparing integrin decoy receptors based on molecular imprinting. The specific technical solution is as follows:

[0006] (1) Immobilization of molecular imprinting templates on glass beads

[0007] The surface-activated glass beads (GBs) were poured into a mixture of 3-aminopropyltriethoxysilane and anhydrous toluene and shaken in a constant-temperature oscillator for 12 h. The GBs were rinsed with anhydrous ethanol, dried in an oven, and sieved for later use.

[0008] Add PBS buffer containing glutaraldehyde to the screened GBs and shake them in a constant temperature shaker for 12 hours. After incubation, rinse several times with PBS buffer to obtain glutaraldehyde-functionalized GBs.

[0009] Finally, the glutaraldehyde-functionalized GBs were immersed in PBS buffer containing the template molecule RGD and shaken in a constant temperature oscillator for 5 h to obtain GBs with fixed template molecules;

[0010] GBs were loaded into a thermostatic chromatographic column and connected to a peristaltic pump to pump pure water to rinse the glass beads;

[0011] (2) Solid-phase synthesis of molecularly imprinted integrin decoy receptor (MIP-RGD)

[0012] The temperature-responsive monomer, functional monomer, and cross-linking agent were weighed and dissolved in PBS, and ultrasonically dissolved completely. The mixture was placed in an ice-water bath and nitrogen was introduced to form a prepolymer solution. Subsequently, redox initiators potassium persulfate and tetramethylethylenediamine were added to the prepolymer solution under anaerobic conditions. After nitrogen was continued to be introduced, the prepolymer solution was pumped into the chromatographic column obtained in step (1). The circulating water bath switch was turned on, and after the ice-water bath circulated for 1 hour, the temperature was set to 37° C. and the reaction was carried out at a constant temperature of 15 hours. The mixture was then rinsed with PBS at 37° C. to rinse the unreacted monomers, oligomers, and non-imprinted nanoparticles NIP-RGD.

[0013] The temperature of the circulating water bath was lowered to 6° C., and the high-affinity integrin decoy receptor MIP-RGD was flushed out through the PBS buffer solution, filtered, dialyzed, freeze-dried, and placed in a vacuum drying oven for later use.

[0014] In step (1), the activation process of the surface-activated glass beads GBs is as follows: the GBs are boiled in a 4M NaOH solution for 15 minutes, washed with pure water until neutral, and then placed in an oven for drying.

[0015] In step (1), the GBs have a particle size of 100 to 110 μm.

[0016] In step (1), in the mixed solution of 3-aminopropyltriethoxysilane and anhydrous toluene, the volume ratio of 3-aminopropyltriethoxysilane to anhydrous toluene is 1:50; the temperature of the constant temperature oscillator is 25° C., and the oscillation rate is 45 r / min.

[0017] In step (1), the volume ratio of glutaraldehyde to PBS buffer is 1:20; the temperature of the constant temperature oscillator is 25° C., and the oscillation rate is 45 r / min.

[0018] In step (1), the template molecule is a polypeptide chain segment GRGDS, which is a sequence of four amino acids containing reactive amino groups. The concentration of the PBS solution containing the template molecule is 10 mM, and the dosage ratio of GBs to the PBS solution containing the template molecule is 50 g:50 mL.

[0019] In step (2), the temperature-responsive monomer is N-isopropylacrylamide (NIPAAm); the functional monomer is N-(4-methylamidinophenyl)acrylamide (AB); the cross-linking agent is N,N′-methylenebisacrylamide (BIS); and the molar ratios of the reactants NIPAAm, AB, and BIS in the prepolymer solution account for 90-95%, 2-5%, and 5% of the total prepolymer, respectively.

[0020] In step (2), the amount of the initiator potassium persulfate (KPS) is 15-20 mg, which needs to be dissolved in PBS buffer before addition, and the amount of tetramethylethylenediamine (TEMED) is 1-2 μL.

[0021] Furthermore, the molecularly imprinted integrin decoy receptor is used for temperature-responsive bidirectional regulation of macrophage polarization or bone marrow mesenchymal stem cell differentiation, and the specific steps are as follows:

[0022] (A) Application of molecularly imprinted integrin decoy receptors to bidirectionally regulate macrophage RAW polarization through temperature

[0023] A1: First, activate the quartz silicon wafer with piranha solution at 80-90°C for 1-2 hours. Then, wash the quartz silicon wafer with ultrapure water and dry it with argon. Then, soak the quartz silicon wafer in a mixed solution of anhydrous toluene and 3-aminopropyltriethoxysilane (APTES) on a constant temperature oscillator at room temperature overnight. After silanization, rinse the quartz silicon wafer with toluene and acetone and dry it with argon.

[0024] A2: Soak the silanized quartz slide in glutaraldehyde in PBS buffer, incubate on a constant temperature shaker at room temperature for 12-24 hours, and then wash with water;

[0025] A3: The glutaraldehyde-activated quartz slide was then placed in a PBS buffer solution containing the peptide RGD and incubated at room temperature for 5-12 hours. Finally, the slide was washed with PBS buffer solution to obtain an RGD-grafted quartz slide for use.

[0026] A4: Inhibition process:

[0027] The functional silicon wafer modified with the RGD polypeptide in step A3 above was plated in a 24-well plate, and then 0.5-1 mL of RAW264.7 cells were added to each well. After incubation at 37°C for 8-16 hours, lipopolysaccharide LPS was added to activate the cells. At the same time, 0.5-1 mL of a high-glucose culture medium solution containing the molecularly imprinted integrin decoy receptor MIP-RGD was added. After incubation at 37°C for 12-24 hours, the cells were fixed with paraformaldehyde and the expression of the pro-inflammatory cytokine iNOS and the anti-inflammatory cytokine Arg-1 in the cells was observed by immunofluorescence staining.

[0028] To verify that the integrin decoy receptor inhibits the action of RGD-integrin, leading to an increase in the pro-inflammatory factor iNOS, the control group (blank silicon wafer), RGD group (functional silicon wafer) and RGD+NIP group (functional silicon wafer + non-imprinted NIP) were compared. The steps were the same as the RGD+MIP group (functional silicon wafer + MIP-RGD) described in step A4 above.

[0029] A5: Excitement process:

[0030] To verify the bidirectional regulation of macrophage polarization by temperature-controlled integrin decoy receptors, the functionalized silicon wafer modified with the RGD peptide described in step A3 above was plated in a 24-well plate. 0.5-1 mL of RAW 264.7 cells were then added to each well and incubated at 37°C for 8-16 hours. Lipopolysaccharide (LPS) was then added to activate the cells, and 0.5-1 mL of high-glucose medium containing the molecularly imprinted integrin decoy receptor MIP-RGD was added. After incubation at 37°C for 12-24 hours, the plates were transferred to room temperature, allowed to stand for 5-10 minutes, and then rinsed several times with 25°C PBS to ensure complete removal of the MIP-RGD. The plates were then incubated at 37°C for an additional 12-24 hours. The cells were then fixed with paraformaldehyde and immunofluorescence staining was performed to assess the expression of the pro-inflammatory cytokine iNOS and the anti-inflammatory cytokine Arg-1 following MIP-RGD removal.

[0031] In order to verify the effect of integrin decoy receptor in bidirectionally regulating macrophage polarization, the RGD+MIP group in which the molecular imprint of the integrin decoy receptor MIP-RGD was not removed by cooling in step A5 was used as a comparison.

[0032] (B) Application of molecularly imprinted integrin decoy receptors to bidirectional temperature regulation of bone marrow mesenchymal stem cell (BMSC) differentiation

[0033] B1-B3: Prepare a functional silicon wafer modified with RGD polypeptide according to the method described in steps A1-A3;

[0034] B4: Inhibition process:

[0035] The functional silicon wafer modified with the RGD polypeptide was plated in a 24-well plate, and 0.5-1 mL of BMSC cells was then added to each well. After incubation at 37°C for 12-24 hours, the original culture medium was removed and osteogenic culture medium and 0.5-1 mL of high-glucose culture medium solution containing the molecularly imprinted integrin decoy receptor MIP-RGD were added. After incubation at 37°C for 3-4 days, the cells were fixed with paraformaldehyde and the expression of osteocalcin (OCN) was observed by immunofluorescence staining.

[0036] To verify that integrin decoy receptors inhibited the effect of RGD-integrin, the control group (blank silicon wafer), RGD group (functional silicon wafer) and RGD+NIP group (functional silicon wafer + non-imprinted NIP) were compared, and the steps were the same as the above-mentioned RGD+MIP group (functional silicon wafer + MIP-RGD).

[0037] B5: Excitement process:

[0038] To verify the bidirectional regulation of integrin decoy receptors on bone marrow mesenchymal stem cell differentiation, the functional silicon wafers modified with RGD peptides were plated in 24-well plates, and 0.5-1 mL of BMSC cells were added to each well. After incubation at 37°C for 12-24 hours, the original culture medium was removed and osteogenic culture medium and 0.5-1 mL of high-glucose culture medium containing molecularly imprinted integrin decoy receptor MIP-RGD were added. After incubation at 37°C for 12-24 hours, the well plates were transferred to room temperature, allowed to stand for 5-10 minutes, and then repeatedly rinsed with 25°C PBS several times to ensure complete removal of MIP-RGD. The well plates were then incubated at 37°C for 3-4 days, and the cells were fixed with paraformaldehyde and the expression of osteocalcin (OCN) after removal of MIP-RGD was observed by immunofluorescence staining.

[0039] The RGD+MIP group, in which the molecular imprinted integrin decoy receptor MIP-RGD was not removed by cooling in the above steps, was used as a comparison.

[0040] In step (A1), in the mixture of anhydrous toluene and 3-aminopropyltriethoxysilane APTES, the volume ratio of 3-aminopropyltriethoxysilane to anhydrous toluene is 1:50; the temperature of the constant temperature oscillator is 25° C., and the oscillation rate is 45 r / min.

[0041] In step (A2), the volume ratio of glutaraldehyde to PBS buffer is 1:20; the temperature of the constant temperature oscillator is 25° C., and the oscillation rate is 45 r / min.

[0042] In step (A3), the RGD is a polypeptide segment GRGDS, which contains a reactive amino group and a five-amino acid sequence of integrin-targeting RGD, and its concentration is 1-2 mg / mL; the concentration of the PBS buffer solution used is 10 mM, pH=7.4.

[0043] In steps (A4) and (A5), in the high-glucose culture medium solution, the concentration of MIP-RGD is 10-30 μg / mL; the concentration of LPS is 100-150 ng / mL.

[0044] In steps (A4) and (A5), the RAW 264.7 cells are mouse mononuclear macrophage leukemia cells, and the seeding density is 2×10 4 cells / well.

[0045] In steps (B4) and (B5), in the high-glucose culture medium solution, the concentration of MIP-RGD is 10-30 μg / mL; the concentration of LPS is 100-150 ng / mL.

[0046] In steps (B4) and (B5), the BMSC cells are rat bone marrow mesenchymal stem cells with a seeding density of 2×10 4 cells / well.

[0047] In steps (B4) and (B5), the osteogenic culture medium is a high-glucose culture medium containing 10 mM β-glycerophosphate, 0.25 mM ascorbic acid and 100 nM dexamethasone.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] This invention constructs a method for bidirectionally regulating integrin signaling based on molecularly imprinted integrin decoy receptors. Compared to existing artificial antagonists, the integrin decoy receptors prepared in this invention overcome the deficiency of their unidirectional inhibition of integrin signaling. They can not only control the unidirectional downregulation of integrin signaling, but also reversibly control the release of ECM ligands to promote the integrin signaling pathway. By controlling the ECM-integrin interaction, the integrin decoy receptors can achieve on-demand guidance of macrophage polarization and stem cell differentiation.

[0050] The integrin decoy receptor provided by the present invention is synthesized using the solid-phase imprinting method, which achieves direct imprinting of RGD molecules through precise proportioning. Compared with indirect imprinting, which requires the modification of RGD with multifunctional peptide chains, the present method is more direct and effective. Compared with traditional imprinting methods (such as precipitation polymerization), the solid-phase imprinting method can directly separate high- and low-affinity sites and template molecules. That is, the sites in the resulting molecularly imprinted polymer are all high-affinity sites, and the complex template elution process is not required. The molecularly imprinted nanoparticles obtained by solid-phase imprinting are closer to natural antibodies in terms of particle size, recognition performance, and stimulus responsiveness, making them the best choice for preparing "decoy receptors."

[0051] The integrin decoy receptor prepared by the present invention can simulate the biological functions of natural antibodies at the cellular level, and is expected to replace artificial antagonistic molecules to achieve bidirectional regulation of integrin signals, truly realizing the biomimetic function of molecular imprinting technology, and has great application prospects in tissue bioengineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 XPS spectra of activated GBs and RGD-functionalized GBs;

[0053] Figure 2 The SEM images of the prepared molecularly imprinted integrin decoy receptor MIP-RGD (A) and non-imprinted nanoparticles NIP-RGD (B) are shown. The inset is the particle size distribution measured by DLS.

[0054] Figure 3Figure A shows the time-dependent frequency (ΔF) changes after different concentrations of MIP-RGD and NIP-RGD were introduced into the QCM sensor at 37°C; Figure B shows the binding isotherm of MIP-RGD and template molecules;

[0055] Figure 4 The frequency (ΔF) changes over time after MIP-RGD is introduced into the QCM chip grafted with different protein molecules at 37°C;

[0056] Figure 5 The results of immunofluorescence staining of Arg-1 in RAW 264.7 cells after treatment with different interventions (scale bar 100 μM);

[0057] Figure 6 The immunofluorescence staining results of iNOS in RAW 264.7 cells after treatment with different intervention methods (scale bar 100 μM);

[0058] Figure 7 Immunofluorescence staining results of Arg-1 (A) and iNOS (B) in RAW 264.7 cells before and after removal of MIP-RGD by lowering the temperature (scale bar: 100 μM).

[0059] Figure 8 (A) OCN immunofluorescence staining, (B) alkaline phosphatase (ALP) staining, and (C) Alizarin Red S staining of BMSC cells after treatment with different interventions (scale bar 200 μM);

[0060] Figure 9 The results of OCN immunofluorescence staining in BMSC cells before and after removing MIP-RGD by lowering the temperature (scale bar 200 μM). DETAILED DESCRIPTION

[0061] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. The present invention provides a general and / or specific description of the materials and test methods used in the experiments. It will be clear to those skilled in the art that, hereinafter, unless otherwise specified, the materials and operating methods used in the present invention are well known in the art, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be purchased from chemical reagent companies.

[0062] Example 1

[0063] (1) Template molecules fixed on the surface of glass beads

[0064] The surface-activated GBs were poured into a mixture of 3-aminopropyltriethoxysilane and anhydrous toluene and shaken in a constant temperature oscillator for 12 h. The GBs were rinsed with anhydrous ethanol, dried in an oven, and sieved for later use.

[0065] PBS buffer containing glutaraldehyde was added to the screened GBs and shaken in a constant-temperature shaker for 12 hours. After incubation, the GBs were rinsed several times with PBS buffer to obtain glutaraldehyde-functionalized GBs. Finally, the glutaraldehyde-functionalized GBs were immersed in PBS buffer containing the template molecule and shaken in a constant-temperature shaker for 5 hours to obtain GBs with the immobilized template molecule.

[0066] Figure 1 X-ray photoelectron spectroscopy (XPS) was used to analyze the glass beads before and after template molecule immobilization. The XPS results showed that the N1s peak of the RGD-GBs group was significantly stronger than that of the activated GBs group, further demonstrating that GRGDS was successfully immobilized on the GB surface.

[0067] (2) Solid-phase synthesis of integrin decoy receptors based on molecular imprinting

[0068] The RGD-GBs were loaded into a thermostatic chromatographic column and connected to a peristaltic pump, and pure water was pumped into the column to rinse the glass beads.

[0069] NIPAAm, AB, and BIS were weighed and dissolved in PBS at a molar ratio of 90:5:5. The monomers were then completely dissolved by sonication. The prepolymer solution was then placed in an ice-water bath for 30 minutes to deoxygenate. The initiator, KPS, was then added to the prepolymer solution, and nitrogen flow continued for 10 minutes. Finally, TEMED was added to the prepolymer solution. The prepolymer solution was pumped into the chromatographic column at a rate of 2.5 mL / min. After the monomers were introduced into the column, a circulating water bath was turned on and maintained at a temperature of approximately 6°C for 1 hour to allow the monomers to self-assemble around the template molecules. The water bath was then heated to 37°C and the reaction was allowed to proceed for 15 hours. After the reaction, 250 mL of PBS was passed through the column to wash away unreacted monomer and low-affinity polymer. The circulating water bath temperature was then lowered to 6°C, and PBS was continued to flush out the high-affinity MIP-RGD. The collected effluent was filtered, dialyzed, lyophilized, and placed in a vacuum desiccant for later use.

[0070] The obtained integrin decoy receptor (MIP-RGD) is in the form of nanoparticles with a particle size of about 40-60 nm. Figure 2 shown.

[0071] Example 2

[0072] In this example, the specificity of the prepared integrin decoy receptor was explored by the following method, and the specific steps were as follows:

[0073] The specificity and selectivity were tested using a quartz microbalance QCM. First, the template molecule RGD was fixed on the chip surface, and then the chip was installed in the QCM system. The peristaltic pump was turned on and the flow rate was set to 3mL / h. After the air baseline was leveled, 10mM PBS buffer solution was pumped into the QCM sensor until the baseline was leveled again. The freeze-dried MIP-RGD and NIP-RGD prepared in Example 1 were prepared into a series of concentration gradients (1 to 160μg / mL) for use. Then, MIP-RGD or NIP-RGD solution was introduced at intervals of 15min according to the concentration from small to large, and the frequency signal changes were observed. The dissociation constants of MIP-RGD and NIP-RGD for the polypeptide chain segments were calculated based on the concentration gradient and the detected frequency change value. The Langmuir equation was used to analyze the fitting curve and calculate the equilibrium dissociation constant K d value.

[0074] The results are as follows Figure 3 :(A) The chip frequency (ΔF) changes over time after different concentrations of MIP-RGD and NIP-RGD are introduced into the QCM sensor at 37°C. The frequency drop of the imprinted nanoparticle MIP-RGD group is significantly higher than that of the non-imprinted NIP-RGD, indicating that the former has a higher affinity with the template polypeptide chain segment; (B) is the binding isotherm of MIP-NPs and template molecules, and it can be concluded that the equilibrium dissociation constant of MIP-NPs is 84μg / mL (84nM).

[0075] Example 3

[0076] This example explores the selective recognition performance of the prepared integrin decoy receptor by the following method, the specific steps are:

[0077] Specificity and selective recognition performance were tested using a quartz microbalance (QCM). First, different protein molecules were immobilized on the chip surface. The chip was then installed in the QCM system. The temperature was set to 37°C, and the peristaltic pump speed was set at 3 mL / h. After the air baseline leveled out, 10 mM PBS buffer solution was pumped into the QCM sensor until the baseline leveled out again. A series of concentration gradients (1 to 160 μg / mL) of the lyophilized MIP-RGD prepared in Example 1 were prepared for later use. Subsequently, the MIP-RGD solutions were introduced at 15-minute intervals, increasing in concentration, and the frequency signal was observed.

[0078] The results are as follows Figure 4 : After different concentrations of MIP-RGD were introduced into the QCM sensor at 37°C, the frequency (ΔF) of the grafted different molecular chips changed over time. Since MIP-RGD and the corresponding polypeptide are spatially complementary, the binding frequency of MIP-RGD with other proteins is much lower than that of the corresponding polypeptide RGD, indicating that the nanoparticles have excellent selective recognition ability.

[0079] Example 4

[0080] This example explores the application of molecular imprinting-based integrin decoy receptors to inhibit integrin signals. The specific steps are as follows:

[0081] The functional silicon wafer modified with RGD polypeptide was plated in a 24-well plate, and then 1 mL of RAW 264.7 cells was added to each well. After incubation at 37°C for 12 hours, lipopolysaccharide (LPS) was added to activate the cells. At the same time, 1 mL of high-glucose culture medium solution containing MIP-RGD was added. After incubation at 37°C for 12 hours, the cells were fixed with paraformaldehyde and the expression of the pro-inflammatory cytokine iNOS and the anti-inflammatory cytokine Arg-1 in the cells was observed by immunofluorescence staining.

[0082] In order to compare the inhibitory effect of MIP-RGD, the control group (blank silicon wafer), RGD group (functional silicon wafer) and RGD-NIP group (functional silicon wafer + NIP) were used as control experiments.

[0083] The MIP and NIP were prepared as in Example 1, with a concentration of 30 μg / mL and a LPS concentration of 100 ng / mL. The RAW 264.7 cells were mouse mononuclear macrophage leukemia cells with a seeding density of 2×10 4 cells / well.

[0084] As attached Figure 5 and attached Figure 6 The following are the results of immunofluorescence staining of RAW 264.7 cells after treatment with different intervention methods: Compared with the control group, the expression of Arg-1 in RAW 264.7 cells cultured on RGD-functionalized quartz slides was significantly increased, and the expression of iNOS was significantly decreased. In contrast, compared with the RGD group, the addition of the integrin decoy receptor MIP-RGD reduced the expression of Arg-1 and increased the expression of iNOS. This indicates that MIP-RGD binds to RGD, thereby blocking the binding of RGD to integrin receptors, inhibiting integrin signaling and polarizing macrophages toward the M1 type. However, after the addition of non-imprinted NIP-RGD, the expression of iNOS and Arg-1 was consistent with that of the RGD group, and it could not play a role in inhibiting integrin signaling.

[0085] Example 5

[0086] This example explores the application of integrin decoy receptors to stimulate integrin signaling under temperature control. The specific steps are as follows:

[0087] To verify the function of integrin decoy receptors in reversibly stimulating integrin signaling, according to the operating procedures in Example 4, 12 hours after adding MIP-RGD, the well plate was allowed to stand at room temperature for 5 minutes, and then repeatedly added 25°C PBS for rinsing three times. After incubation at 37°C for 12 hours, the cells were fixed with paraformaldehyde and the expression of iNOS and Arg-1 after removal of MIP was observed by immunofluorescence staining.

[0088] In order to compare the reversible agonistic effect of MIP-RGD, the RGD-MIP group without cooling treatment was used as a control experiment.

[0089] Attachment Figure 7 Immunofluorescence staining of RAW 264.7 cells before and after MIP-RGD removal by lowering the temperature showed that iNOS expression was significantly reduced and Arg-1 expression was significantly increased after MIP-RGD removal. This suggests that lowering the temperature can reversibly regulate the binding of integrin decoy receptors to RGD, thereby reactivating integrin signaling.

[0090] Example 6

[0091] This example explores the application of molecular imprinting-based integrin decoy receptors to inhibit integrin signals. The specific steps are as follows:

[0092] Using BMSCs as the research subject, functional silicon wafers modified with RGD peptides were plated in 24-well plates. 1 mL of BMSC cells was then added to each well. After incubation at 37°C for 24 hours, the original culture medium was removed and replaced with osteogenic culture medium and 1 mL of high-glucose culture medium containing MIP-RGD. After incubation at 37°C for 4 days, the cells were fixed with paraformaldehyde and immunofluorescence staining was used to observe osteocalcin (OCN) expression. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining were performed after 7 and 14 days of incubation, respectively. Stained (positive) cells and calcium nodules were observed under a light microscope.

[0093] In order to compare the inhibitory effect of MIP-RGD, the control group (blank silicon wafer), RGD group (functional silicon wafer) and RGD-NIP group (functional silicon wafer + NIP) were used as control experiments.

[0094] The MIP and NIP were prepared as in Example 1, and their concentrations were both 10 μg / mL. The BMSC cells were rat bone marrow mesenchymal stem cells, with a seeding density of 2×10 4 cells / well.

[0095] Attachment Figure 8Figure A shows the immunofluorescence staining results of BMSC cells treated with different interventions: OCN expression was significantly increased in BMSCs cultured on RGD-functionalized quartz wafers. However, after the addition of integrin decoy receptors, OCN expression was significantly reduced compared with the RGD group. However, after the addition of non-imprinted NIP-RGD, OCN expression was basically the same as that of the RGD group. ALP staining ( Figure 8 B) and ARS staining ( Figure 8 The same result was obtained in C), indicating that the binding of integrin decoy receptors to RGD can block the binding of RGD to integrin receptors, inhibit integrin signaling, and affect the osteogenic differentiation of BMSC cells.

[0096] Example 7

[0097] This example explores the application of molecular imprinting-based integrin decoy receptors to stimulate integrin signaling under temperature control. The specific steps are as follows:

[0098] To verify the ability of integrin decoy receptors to reversibly activate integrin signaling, the procedures described in Example 6 were followed. One day after adding MIP-RGD, the plates were transferred to room temperature and allowed to stand for 5 minutes before being rinsed three times with 25°C PBS to ensure complete removal of the MIP-RGD. The plates were then incubated at 37°C for another three days, and the cells were fixed with paraformaldehyde. Immunofluorescence staining was then performed to examine osteocalcin (OCN) expression after MIP-RGD removal.

[0099] In order to compare the reversible agonistic effect of MIP-RGD, the RGD-MIP group without cooling treatment was used as a control experiment.

[0100] Attachment Figure 9 Immunofluorescence staining of BMSCs before and after MIP-RGD removal by lowering the temperature showed a significant increase in OCN expression on BMSCs after MIP-RGD removal. This suggests that integrin decoy receptors can reversibly bind to RGD through temperature control, reactivating integrin signaling and thus dynamically regulating the osteogenic differentiation of BMSCs.

[0101] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of molecularly imprinted integrin decoy receptors for temperature-responsive bidirectional regulation of macrophage polarization or bone marrow mesenchymal stem cell differentiation.

2. The use according to claim 1, characterized in that The steps of bidirectional temperature regulation of macrophage RAW polarization based on molecular imprinting integrin decoy receptor are as follows: A1: First, activate the quartz silicon wafer with piranha solution at 80-90°C for 1-2 hours. Then, wash the quartz silicon wafer with ultrapure water and dry it with argon. Then, soak the quartz silicon wafer in a mixed solution of anhydrous toluene and 3-aminopropyltriethoxysilane (APTES) on a constant temperature oscillator at room temperature overnight. After silanization, rinse the quartz silicon wafer with toluene and acetone and dry it with argon. A2: Soak the silanized quartz slide in glutaraldehyde in PBS buffer, incubate on a constant temperature shaker at room temperature for 12-24 hours, and then wash with water; A3: The glutaraldehyde-activated quartz slide was then placed in a PBS buffer solution containing the peptide RGD and incubated at room temperature for 5-12 hours. Finally, the slide was washed with PBS buffer solution to obtain an RGD-grafted quartz slide for use. A4: Inhibition process: The functional silicon wafer modified with the RGD polypeptide in step A3 above was plated in a 24-well plate, and 0.5-1 mL of RAW264.7 cells were then added to each well. After incubation at a certain temperature for 8-16 hours, lipopolysaccharide (LPS) was added to activate the cells. At the same time, 0.5-1 mL of a high-glucose culture medium solution containing the molecularly imprinted integrin decoy receptor MIP-RGD was added. After incubation at a certain temperature for 12-24 hours, the cells were fixed with paraformaldehyde and the expression of the pro-inflammatory cytokine iNOS and the anti-inflammatory cytokine Arg-1 in the cells was observed by immunofluorescence staining. A5: Excitement process: The functional silicon wafer modified with the RGD polypeptide in step A3 above was plated in a 24-well plate, and then 0.5-1 mL of RAW264.7 cells were added to each well. After incubation at a certain temperature for 8-16 hours, lipopolysaccharide LPS was added to activate the cells. At the same time, 0.5-1 mL of a high-glucose culture medium solution containing the molecularly imprinted integrin decoy receptor MIP-RGD was added. After incubation at a certain temperature for 12-24 hours, the well plate was transferred to room temperature, allowed to stand for 5-10 minutes, and then repeatedly rinsed with PBS several times to ensure complete removal of MIP-RGD. The well plate was then incubated at a certain temperature for another 12-24 hours, and the cells were fixed with paraformaldehyde and the expression of the pro-inflammatory cytokine iNOS and the anti-inflammatory cytokine Arg-1 after removal of MIP-RGD was observed by immunofluorescence staining.

3. The use according to claim 1, characterized in that The steps of bidirectional temperature regulation of bone marrow mesenchymal stem cell differentiation based on molecular imprinting integrin decoy receptors are as follows: B1: First, activate the quartz silicon wafer with piranha solution at 80-90°C for 1-2 hours. Then, wash the quartz silicon wafer with ultrapure water and dry it with argon. Then, soak the quartz silicon wafer in a mixed solution of anhydrous toluene and 3-aminopropyltriethoxysilane (APTES) on a constant temperature oscillator at room temperature overnight. After silanization, rinse the quartz silicon wafer with toluene and acetone and dry it with argon. B2: Soak the silanized quartz slide in glutaraldehyde in PBS buffer solution, incubate on a constant temperature shaker at room temperature for 12-24 hours, and wash with water; B3: The quartz glass slide activated with glutaraldehyde was then placed in a PBS buffer solution containing the peptide RGD and incubated at room temperature for 5-12 hours. Finally, the slide was washed with a PBS buffer solution to obtain an RGD-grafted quartz glass slide for use. B4: Inhibition process: The functional silicon wafer modified with the RGD polypeptide in step B3 above was plated in a 24-well plate, and 0.5-1 mL of BMSC cells were then added to each well. After incubation at a certain temperature for 12-24 hours, the original culture medium was removed and osteogenic culture medium and 0.5-1 mL of high-glucose culture medium solution containing the molecularly imprinted integrin decoy receptor MIP-RGD were added. After incubation at a certain temperature for 3-4 days, the cells were fixed with paraformaldehyde and the expression of osteocalcin (OCN) was observed by immunofluorescence staining; B5: Excitement process: The functional silicon wafer modified with the RGD polypeptide in step B3 above was plated in a 24-well plate, and then 0.5-1 mL of BMSC cells was added to each well. After incubation at a certain temperature for 12-24 hours, the original culture medium was removed and osteogenic culture medium and 0.5-1 mL of high-glucose culture medium solution containing the molecularly imprinted integrin decoy receptor MIP-RGD were added. After incubation at a certain temperature for 12-24 hours, the well plate was transferred to room temperature, allowed to stand for 5-10 minutes, and then repeatedly rinsed with PBS several times to ensure that MIP-RGD was completely removed; the well plate was then continued to be incubated at a certain temperature for 3-4 days, and the cells were fixed with paraformaldehyde and the expression of osteocalcin OCN after removal of MIP-RGD was observed by immunofluorescence staining.

4. The use according to claim 2 or 3, characterized in that In step (A1) or (B1), in the mixture of anhydrous toluene and 3-aminopropyltriethoxysilane (APTES), the volume ratio of 3-aminopropyltriethoxysilane to anhydrous toluene is 1:50; the temperature of the constant temperature oscillator is 25° C., and the oscillation rate is 45 r / min; In step (A2) or (B2), the volume ratio of glutaraldehyde to PBS buffer is 1:20; the temperature of the constant temperature oscillator is 25° C., and the oscillation rate is 45 r / min; In step (A3) or (B3), the RGD is a polypeptide segment GRGDS, which is a five-amino acid sequence containing a reactive amino group and integrin-targeting RGD, and its concentration is 1-2 mg / mL; the concentration of the PBS buffer solution used is 10 mM, pH = 7.

4.

5. The use according to claim 2 or 3, characterized in that The incubation temperature was 37°C.

6. The use according to claim 2 or 3, characterized in that In step (A5) or (B5), the temperature of the PBS added after standing is 25°C.

7. The use according to claim 2, characterized in that In steps (A4) and (A5), in the high-glucose culture medium solution, the concentration of MIP-RGD is 10-30 μg / mL; the concentration of LPS is 100-150 ng / mL; In steps (A4) and (A5), the RAW 264.7 cells are mouse mononuclear macrophage leukemia cells, and the seeding density is 2×10 4 cells / well.

8. The use according to claim 3, characterized in that In steps (B4) and (B5), in the high-glucose culture medium solution, the concentration of MIP-RGD is 10-30 μg / mL; the concentration of LPS is 100-150 ng / mL; In steps (B4) and (B5), the BMSC cells are rat bone marrow mesenchymal stem cells with a seeding density of 2×10 4 cells / well; In steps (B4) and (B5), the osteogenic culture medium is a high-glucose culture medium containing 10 mM β-glycerophosphate, 0.25 mM ascorbic acid and 100 nM dexamethasone.

9. The use according to claim 1, wherein The steps for preparing integrin decoy receptors based on molecular imprinting are as follows: (1) Immobilization of molecular imprinting templates on glass beads The surface-activated glass beads (GBs) were poured into a mixture of 3-aminopropyltriethoxysilane and anhydrous toluene and shaken in a constant-temperature oscillator. The GBs were rinsed with anhydrous ethanol, dried in an oven, and sieved for later use. PBS buffer containing glutaraldehyde was added to the sieved GBs and placed in a constant temperature oscillator for shaking. After the incubation, the GBs were rinsed several times with PBS buffer to obtain glutaraldehyde-functionalized GBs. Finally, the glutaraldehyde-functionalized GBs were immersed in PBS buffer containing the template molecule RGD and shaken in a constant temperature oscillator to obtain GBs with fixed template molecules; GBs were loaded into a thermostatic chromatographic column and connected to a peristaltic pump to pump pure water to rinse the glass beads; (2) Solid-phase synthesis of integrin decoy receptor MIP-RGD based on molecular imprinting The temperature-responsive monomer, functional monomer, and cross-linking agent were weighed and dissolved in PBS, and ultrasonically dissolved completely. The mixture was placed in an ice-water bath and nitrogen was introduced to form a prepolymer solution. Subsequently, redox initiators potassium persulfate and tetramethylethylenediamine were added to the prepolymer solution under anaerobic conditions. After nitrogen was continued to be introduced, the prepolymer solution was pumped into the chromatographic column obtained in step (1). The circulating water bath switch was turned on, and after the ice-water bath circulated for 1 hour, the temperature was set to 37° C. and the reaction was carried out at a constant temperature of 15 hours. The mixture was then rinsed with PBS at 37° C. to rinse the unreacted monomers, oligomers, and non-imprinted nanoparticles NIP-RGD. The temperature of the circulating water bath was lowered to 6° C., and the high-affinity integrin decoy receptor MIP-RGD was flushed out through the PBS buffer solution, filtered, dialyzed, freeze-dried, and placed in a vacuum drying oven for later use.

10. The use according to claim 9, characterized in that In step (1), the activation process of the surface-activated glass beads GBs is as follows: the GBs are boiled in a 4M NaOH solution for 15 minutes, washed with pure water until neutral, and then placed in an oven for drying; In step (1), the GBs have a particle size of 100 to 110 μm; In step (1), in the mixture of 3-aminopropyltriethoxysilane and anhydrous toluene, the volume ratio of 3-aminopropyltriethoxysilane to anhydrous toluene is 1:50; the temperature of the constant temperature oscillator is 25° C., the oscillation rate is 45 r / min, and the time is 12 h; In step (1), the volume ratio of glutaraldehyde to PBS buffer is 1:20; the temperature of the constant temperature oscillator is 25° C., the oscillation rate is 45 r / min, and the time is 12 h; In step (1), the template molecule is a polypeptide segment GRGDS, which is a sequence of four amino acids containing a reactive amino group. The concentration of the PBS solution containing the template molecule is 10 mM. The dosage ratio of GBs to the PBS solution containing the template molecule is 50 g:50 mL. The shaking time is 5 h. In step (2), the temperature-responsive monomer is N-isopropylacrylamide (NIPAAm); the functional monomer is N-(4-methylamidinophenyl)acrylamide (AB); and the cross-linking agent is N,N′-methylenebisacrylamide (BIS). The molar ratios of the reactants NIPAAm, AB, and BIS in the prepolymer solution account for 90-95%, 2-5%, and 5% of the total prepolymer, respectively; In step (2), the amount of the initiator potassium persulfate KPS is 15-20 mg, which needs to be dissolved in PBS buffer before addition, and the amount of tetramethylethylenediamine (TEMED) is 1-2 μL.

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