Targeting system based on biological orthogonal click chemical reaction as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a targeting system based on bio-orthogonal chemistry, the targeting system comprises gel GelMA-DBCO modified with a targeting receptor DBCO and nanoparticles NV-N3 modified with a targeting ligand azide group, the preparation method comprises the following steps: respectively dissolving GelMA and DBCO-NHS, mixing, adding triethylamine, and stirring at room temperature for reaction to obtain GelMA-DBCO; the preparation method comprises the following steps: adding GelMA-DBCO into a solution containing a photoinitiator, and carrying out ultraviolet crosslinking to obtain GelMA-DBCO gel; the preparation method comprises the following steps: adding Ac4ManNAz into a culture medium of MSC to obtain mesenchymal stem cells MSC with N3 modified on the surface, digesting and collecting cells, and extruding the MSC by using a liposome extruder to pass through a filter membrane to obtain nano-particles with azido modified on the surface; and purifying by centrifuging, and resuspending to obtain the NV-N3. The invention also provides application of the targeting system in preparation of products for treating spinal cord injury. The targeting system provided by the invention has strong specificity and stability, good biocompatibility and biodegradability, and effectively improves the targeting ability of the nanoparticles in a spinal cord injury area.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of preparation materials and regenerative medicine, and in particular relates to a targeting system based on bioorthogonal click chemistry reaction and a preparation method and application thereof. Background Art

[0002] Spinal cord injury is a serious disease of the central nervous system, mostly caused by external impact or other risk factors. After a patient suffers from spinal cord injury, the nerve function of the spinal cord is temporarily or permanently abnormal, and motor and sensory functions are lost, which can lead to death in severe cases. Based on the pathological characteristics of spinal cord injury, in the early stage of injury, the reactive oxygen and inflammatory factors in the microenvironment of the damaged area increase significantly, further causing more serious secondary injuries, including necrosis of neural tissue, formation of glial scars, and the occurrence of neural cavities. Therefore, the local microenvironment can be alleviated by delivering drugs to the injured area to achieve a therapeutic effect.

[0003] At present, drug treatment approaches for spinal cord injury can be divided into two categories. One is to achieve drug delivery through a local implant system, that is, by encapsulating the drug in a polymer biomaterial, such as hyaluronic acid, gelatin, etc., cross-linking to form a semi-solid with a three-dimensional network structure, and then implanting it into the spinal cord injury area. Local implantation solves the problem of drug distribution in the body, and this strategy can achieve better drug utilization. However, since this method of administration requires opening the lamina around the spinal cord for administration, it causes great damage to the organism, so it is generally only given once. And the cavity at the site of injury is between a few microliters and tens of microliters. Therefore, the number and space of local implantation administration are limited, and the amount of administration is greatly limited. The other is to achieve drug delivery through a non-local implant system, including intravenous injection, intrathecal injection, and nasal administration. Although these methods of administration are less invasive and can achieve continuous drug delivery at different time points, the drug lacks targeting to the injury site after entering the body through the above methods, and the enrichment in the injury area is low, and the efficacy of drug delivery is still limited. Therefore, how to combine the advantages of the two may become a solution to the drug delivery problem.

[0004] Bioorthogonal chemistry refers to chemical reactions that can be carried out in living cells or tissues without interfering with the biochemical reactions of the organism itself. Among them, the reaction conditions of azide and dibenzocyclooctene (DBCO) are mild, rapid and highly specific, and can be used as a target inside the organism. Summary of the invention

[0005] The purpose of the present invention is to provide a targeting system based on bioorthogonal click chemistry reaction and its preparation method and application. The targeting system has strong specificity and stability, good biocompatibility and biodegradability, and effectively improves the targeting of nanoparticles in the spinal cord injury area.

[0006] The present invention adopts the following technical solution:

[0007] A method for preparing a targeting system based on a bioorthogonal click chemistry reaction, the targeting system comprising a gel (GelMA-DBCO) modified with a targeting receptor dibenzocyclooctene (DBCO) and a nanoparticle (NV-N3) modified with a targeting ligand azido (N3), the preparation method comprising:

[0008] (1) Preparation of GelMA-DBCO

[0009] (1-1) Gelatin methacrylate (GelMA) and dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS) are dissolved separately and then mixed, triethylamine is added, and the mixture is stirred at room temperature to obtain a gel GelMA-DBCO modified with a targeting receptor;

[0010] (1-2) adding GelMA-DBCO to a solution containing a photoinitiator to obtain a GelMA-DBCO solution, and subjecting the solution to UV crosslinking to obtain a GelMA-DBCO gel;

[0011] (2) Preparation method of NV-N3

[0012] (2-1) adding azide-modified mannose (Ac4ManNAz) to the culture medium of MSC to obtain mesenchymal stem cells (MSC) surface-modified with N3, digesting and collecting the cells, and extruding the MSC through a filter membrane using a liposome extruder to obtain nanoparticles surface-modified with azide groups;

[0013] (2-2) The nanoparticles modified with azide groups on the surface are purified by centrifugation and resuspended to obtain NV-N3.

[0014] In step (1-1), the mass ratio of GelMA to DBCO-NHS is 16-24:1, and the reaction is carried out at 10-30°C for 16-24h. The reaction process is as follows:

[0015]

[0016] In step (1-2), the mass concentration of GelMA-DBCO in the GelMA-DBCO solution is 5 to 15%.

[0017] Preferably, in step (1-2), the mass concentration of the photoinitiator LAP solution is 0.25%, and it is dissolved by stirring at 20-60°C.

[0018] The preparation method further comprises: dialysis purification of the reaction product obtained in step (1-1), freeze drying to obtain a white solid, which is a gel GelMA-DBCO modified with a targeting receptor, and then adding it to a solution containing a photoinitiator. The specific process is: the product is placed in a dialysis bag, dialyzed in pure water, the dialysis time is 72 hours, and the pure water is replaced every 12 hours.

[0019] In step (2-1), the final concentration of Ac4ManNAz in the MSC culture medium is 30-60 μM, and the co-incubation time of MSC and Ac4ManNAz is 24-48 h.

[0020] Wherein, in step (2-1), Ac4ManNAz is first dissolved in DBCO and prepared into a 100 mM stock solution before co-incubation.

[0021] In step (2-1), the extrusion process is carried out in two steps: using a liposome extruder to extrude MSCs first through a 5 μm filter membrane, extruding back and forth 10 to 20 times, and then through a 0.4 μm filter membrane, extruding back and forth 10 to 20 times.

[0022] In step (2-2), the centrifugal purification process is carried out in two steps: first, ultracentrifuge at 8,000-10,000 g for 30 min, take the supernatant, and remove the cells and cell debris in the precipitate; then ultracentrifuge the supernatant at 100,000 g for 1-1.5 h; take the precipitate and resuspend it in PBS.

[0023] The present invention also provides a targeting system obtained by the above preparation method, comprising a hydrogel GelMA-DBCO and a nanoparticle NV-N3 modified with an azide group.

[0024] The present invention also provides the use of the above-mentioned targeting system in the preparation of a product for treating spinal cord injury. After the gel GelMA-DBCO is locally implanted, the subsequently administered nanoparticles NV-N3 achieve targeting to the region through specific group reactions.

[0025] The subsequent administration of the nanoparticle NV-N3 is nasal administration, intrathecal injection or intravenous injection.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention constructs an artificial targeting system based on bio-orthogonal click chemistry reaction, and its composition includes a gel (GelMA-DBCO) modified with a targeting receptor dibenzocyclooctene (DBCO) and a nanoparticle (NV-N3) modified with a targeting ligand azido (N3). On the one hand, after local gel implantation, the targeting system can improve the enrichment of subsequent drug administration in a specific area, which is used to enhance the targeting ability to a specific area, and effectively achieves the targeting of nanoparticles and the treatment of spinal cord injury. On the other hand, the system has high specificity and stability, and only nanoparticles carrying a specific group azido will target the specific area, and the implanted gel scaffold maintains for more than one week in the body, and its targeting enhancement effect also exceeds one week. Therefore, the present invention effectively combines local implantation and subsequent drug administration strategies in the treatment of spinal cord injury. Local implantation promotes the targeting ability of subsequent drug administration, and subsequent drug administration makes up for the deficiency of local drug administration, providing a new treatment plan for the treatment of spinal cord injury in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the H NMR spectrum of GelMA-DBCO in Example 1;

[0029] Figure 2 This is a photo of the GelMA-DBCO after UV crosslinking into gel in Example 1;

[0030] Figure 3 is a confocal scanning image of MSC modified with azido groups in Example 1;

[0031] Figure 4 is a transmission electron microscope image of NV-N3 in Example 1;

[0032] Figure 5 is the in vitro binding of GelMA-DBCO and NV-N3 in Example 1;

[0033] Figure 6 is the biocompatibility test of GelMA and GelMA-DBCO in Example 1;

[0034] Figure 7 is the degradation of GelMA-DBCO in rats in Example 2;

[0035] Figure 8 This is the result of a single administration of the targeted system in Application Example 1 in a rat spinal cord injury model;

[0036] Fig. 9 This is the result of continuous administration of the targeted system in Application Example 2 in a rat spinal cord injury model. DETAILED DESCRIPTION

[0037] In order to describe the present invention more specifically, the technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and specific implementation methods. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0038] Example 1

[0039] 1. Preparation of GelMA-DBCO Gel

[0040] 224 mg of methacryloyl gelatin GelMA was added to 10 mL of ultrapure water to obtain a GelMA solution. 14 mg of DBCO-NHS was weighed and added to 5 mL of N,N-dimethylformamide. After the above solutions were mixed, 9.8 μL of triethylamine was added and stirred at room temperature for reaction. The reactant was dialyzed and freeze-dried to obtain a white solid, which was GelMA-DBCO. 3 mg of GelMA-DBCO was weighed and dissolved in 500 μL of deuterated water to detect the hydrogen spectrum structure. Figure 1 The structure of GelMA-DBCO is shown in the H NMR spectrum. Weigh 50 mg of GelMA-DBCO and add it to 500 μL of a solution containing 0.25% LAP. After the solution is homogenized and stabilized at 37°C, 10 μL of the GelMA-DBCO solution is taken and UV crosslinked for 40 seconds to obtain a GelMA-DBCO gel. Figure 2 , the gel is a light yellow translucent solid.

[0041] 2. Modification of azido groups on the surface of MSC membrane

[0042] Ac4MAnNAz was added to the culture medium of MSC and co-incubated with MSC to make the concentration of Ac4MAnNAz 0, 15, 30, 60 μM, respectively. After 24 hours, the cells were stained with DBCO-Cy5 and the azido modification of the cell surface was observed under confocal microscope. Figure 3 It can be seen that when the concentration of Ac4MAnNAz is greater than or equal to 30μM, the azido modification level of the cells is the highest. The cells modified with azido groups were digested and collected, and extruded with a liposome extruder to pass through 5μm and 0.4μm filter membranes in turn; the suspension was ultracentrifuged at 10,000g to remove cell debris in the precipitate, the supernatant was taken, and then centrifuged at 100,000g, the precipitate was taken, and resuspended with PBS to obtain nanoparticles modified with azido groups. Figure 4 This is a transmission electron microscopy image of NV-N3, which shows that NV-N3 presents a vesicle structure.

[0043] 3. In vitro binding of GelMA-DBCO and NV-N3

[0044] Weigh 50 mg of GelMA-DBCO and add it to 500 μL of a solution containing 0.25% LAP to prepare a 10% GelMA-DBCO solution. After the solution is uniform and stable at 37°C, measure 25 μL of GelMA-DBCO solution and spread it on the bottom of the confocal dish. After UV crosslinking for 40 seconds, obtain the GelMA gel modified with DBCO. Add the DiD-stained NV-N3 suspension to the confocal dish. After 2 hours, discard the liquid in the confocal dish, wash it twice with PBS, and then use Cy5-N3 to fluorescently stain the GelMA-DBCO gel. After washing it twice with PBS, observe the fluorescence signals of the nanoparticles and gel under a confocal microscope. Figure 5 The fluorescence signal of the nanoparticles is closely aligned with that of the gel, indicating that the two can be connected to each other through a bioorthogonal reaction.

[0045] 4. Biocompatibility testing of GelMA and GelMA-DBCO

[0046] Weigh 50 mg of GelMA and GelMA-DBCO and add them to 500 μL of a solution containing 0.25% LAP to prepare 10% GelMA and GelMA-DBCO solutions. After the solution is uniform and stable at 37°C, measure 25 μL of GelMA or GelMA-DBCO solution and add it to 1 mL of cell culture medium. Use this culture medium to incubate MSCs. After 24 hours, use CCK-8 to detect the cell activity of each group. Figure 6 ,The experimental results showed that GelMA and GelMA-DBCO did not affect cell activity and had good ,biocompatibility.

[0047] Example 2

[0048] Degradation of GelMA-DBCO in rats

[0049] 200-220g SD rats were weighed and anesthetized, and the hair on the back was shaved with a shaver. The skin at the T9-T10 vertebral segment was fully disinfected with iodine. The skin was cut open with a scalpel, and the fascia under the skin was separated with forceps. The muscles close to the vertebrae on both sides were cut open with a scalpel, and the wound was opened with a spreader. The cone plate was removed with a rongeur to expose the spinal cord. The spinal cord was completely cut with ophthalmic scissors to form a 3mm injury gap.

[0050] After sufficient hemostasis with cotton balls, GelMA-DBCO was implanted at the injury site. The rat's back muscles and skin were sutured in turn, disinfected with iodine, injected with penicillin, and placed in an incubator to wait for awakening. Rats are relatively weak after spinal cord injury modeling, and need to urinate, inject penicillin, and change water, food, and bedding regularly.

[0051] On the 7th, 14th, 21st and 28th days after modeling, a group of rats were killed, and the spinal cord tissues were removed to detect the degradation of GelMA-DBCO in the spinal cord injury site. Figure 7 It can be seen that GelMA-DBCO still had residues after 14 days and was completely degraded on the 21st day, which showed that it had certain stability and biodegradability.

[0052] Application Example 1

[0053] Results of single administration of the targeted system in a rat spinal cord injury model

[0054] 200-220g SD rats were weighed and anesthetized, and the hair on the back was shaved with a shaver. The skin at the T9-T10 vertebral segment was fully disinfected with iodine. The skin was cut open with a scalpel, and the fascia under the skin was separated with forceps. The muscles close to the vertebrae on both sides were cut open with a scalpel, and the wound was opened with a spreader. The cone plate was removed with a rongeur to expose the spinal cord. The spinal cord was completely cut with ophthalmic scissors to form a 3mm injury gap.

[0055] After sufficient hemostasis with cotton balls, GelMA-DBCO was implanted at the injury site in the targeted gel group, and GelMA was implanted at the injury site in the blank gel group. The back muscles and skin of the rats were sutured in turn, disinfected with iodine, injected with penicillin, and placed in an incubator to wait for awakening. Rats were relatively weak after spinal cord injury modeling, and needed to urinate, inject penicillin, and change water, food, and bedding regularly.

[0056] On the third day after modeling, 20 μg of DiD-labeled NV or NV-N3 was administered intranasally. The rats were killed 12 hours after administration, and the spinal cord tissue was removed to detect the fluorescence signal of the spinal cord injury site. Figure 8 It can be seen that the modification of the targeting group promoted the enrichment and retention of nanoparticles in the injured area. Only in the group modified with both DBCO and N3, the targeting of nanoparticles in the spinal cord injury area after nasal administration was enhanced, reflecting the high specificity of the system.

[0057] Application Example 2

[0058] Results of continuous drug delivery of targeted system in rat spinal cord injury model

[0059] 200-220g SD rats were weighed and anesthetized, and the hair on the back was shaved with a shaver. The skin at the T9-T10 vertebral segment was fully disinfected with iodine. The skin was cut open with a scalpel, and the fascia under the skin was separated with forceps. The muscles close to the vertebrae on both sides were cut open with a scalpel, and the wound was opened with a spreader. The cone plate was removed with a rongeur to expose the spinal cord. The spinal cord was completely cut with ophthalmic scissors to form a 3mm injury gap.

[0060] After sufficient hemostasis with cotton balls, GelMA-DBCO was implanted at the injury site in the targeted gel group, and GelMA was implanted at the injury site in the blank gel group. The back muscles and skin of the rats were sutured in turn, disinfected with iodine, injected with penicillin, and placed in an incubator to wait for awakening. Rats were relatively weak after spinal cord injury modeling, and needed to urinate, inject penicillin, and change water, food, and bedding regularly.

[0061] On the 3rd, 5th and 7th day after modeling, the rats were anesthetized and 20 μg of DiD-labeled NV-N3 was administered intranasally. A group of rats were killed before and 12 hours after each administration, and the spinal cord tissues were dissected to detect the fluorescence signal of the spinal cord injury site. Fig. 9 It can be seen that the modification of the targeting group promotes the enrichment and retention of nanoparticles in the damaged area, and such a targeting effect can be maintained for at least three nasal administrations.

[0062] The above description of implementation and application is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to the above embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a targeting system based on bioorthogonal click chemistry reaction, characterized in that: The targeting system comprises a gel GelMA-DBCO modified with a targeting receptor dibenzocyclooctene DBCO and a nanoparticle NV-N3 modified with a targeting ligand azido N3, and the preparation method comprises: (1) Preparation of GelMA-DBCO (1-1) Methacrylated gelatin GelMA and DBCO-NHS are dissolved separately and then mixed, triethylamine is added, and the mixture is stirred at room temperature to obtain a gel GelMA-DBCO modified with a targeting receptor; (1-2) adding GelMA-DBCO to a solution containing a photoinitiator to obtain a GelMA-DBCO solution, and subjecting the solution to UV crosslinking to obtain a GelMA-DBCO gel; (2) Preparation method of NV-N3 (2-1) adding azide-modified mannose Ac4ManNAz to the culture medium of MSC to obtain mesenchymal stem cells MSC with surface modification of N3, digesting and collecting the cells, and squeezing the MSC through a filter membrane using a liposome extruder to obtain nanoparticles with surface modification of azide groups; (2-2) The nanoparticles modified with azide groups on the surface are purified by centrifugation and resuspended to obtain NV-N3.

2. The method for preparing a targeting system based on bioorthogonal click chemistry reaction according to claim 1, characterized in that: In step (1-1), the mass ratio of GelMA to DBCO-NHS is 16-24:1, and the reaction is carried out at 10-30° C. for 16-24 hours.

3. The method for preparing a targeting system based on bioorthogonal click chemistry reaction according to claim 1, characterized in that: In step (1-2), the mass concentration of GelMA-DBCO in the GelMA-DBCO solution is 5 to 15%.

4. The method for preparing a targeting system based on bioorthogonal click chemistry reaction according to claim 1, characterized in that: The preparation method comprises: dialysis purification of the reaction product obtained in step (1-1), freeze-drying to obtain a white solid, and then adding the solid to a solution containing a photoinitiator.

5. The method for preparing a targeting system based on bioorthogonal click chemistry reaction according to claim 1, characterized in that: In step (2-1), the final concentration of Ac4ManNAz in the MSC culture medium is 30-60 μM, and the co-incubation time of MSC and Ac4ManNAz is 24-48 h.

6. The method for preparing a targeting system based on bioorthogonal click chemistry reaction according to claim 1, characterized in that: In step (2-1), the extrusion process is carried out in two steps: using a liposome extruder to extrude MSCs first through a 5 μm filter membrane, extruding back and forth 10 to 20 times, and then through a 0.4 μm filter membrane, extruding back and forth 10 to 20 times.

7. The method for preparing a targeting system based on bioorthogonal click chemistry reaction according to claim 1, characterized in that: In step (2-2), the centrifugal purification process is carried out in two steps: first, ultracentrifuge at 8,000-10,000 g for 30 min, take the supernatant, and remove the cells and cell debris in the precipitate; then ultracentrifuge the supernatant at 100,000 g for 1-1.5 h; take the precipitate and resuspend it in PBS.

8. A targeting system obtained by the preparation method according to any one of claims 1 to 7, comprising a hydrogel GelMA-DBCO and nanoparticles NV-N3 modified with an azide group.

9. Use of the targeting system according to claim 8 in preparing a product for treating spinal cord injury.

10. The use according to claim 9, characterized in that: After local implantation of the gel GelMA-DBCO, the subsequently administered nanoparticles NV-N3 were targeted to the area through specific group reactions.

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