Targeting system based on bio-orthogonal click chemistry reaction and preparation method and application thereof

Abstract

The application discloses a preparation method of a targeting system based on bio-orthogonal chemistry, and the targeting system comprises a gel GelMA-DBCO modified with a targeting receptor DBCO and a nanoparticle NV-N3 modified with a targeting ligand azido, and the preparation method comprises the following steps: mixing GelMA and DBCO-NHS after being respectively dissolved, adding triethylamine, stirring and reacting at room temperature to obtain GelMA-DBCO; adding the GelMA-DBCO into a solution containing a photoinitiator, and crosslinking under ultraviolet light to obtain a GelMA-DBCO gel; adding Ac4ManNAz into a culture medium of MSC to obtain mesenchymal stem cells MSC modified with N3 on the surface, digesting and collecting the cells, extruding the MSC through a filter membrane by using a liposome extruder to obtain the nanoparticle modified with azido on the surface; and then purifying by centrifugation and resuspending to obtain NV-N3. The application also provides application of the above-mentioned targeting system in preparation of a product for treating spinal cord injury. The targeting system provided by the application has the advantages of high specificity and stability, good biocompatibility, biodegradability, and effective improvement of the targeting capacity of the nanoparticle in a spinal cord injury area.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of preparation of formulation materials and regenerative medicine, and particularly relates to a targeting system based on bio-orthogonal click chemistry reaction and a preparation method and application thereof. BACKGROUND

[0002] Spinal cord injury is a serious central nervous system disease, which is usually caused by external impact or other risk factors. After suffering from spinal cord injury, the patient's spinal cord function is temporarily or permanently abnormal, and the motor and sensory functions are lost, which can even lead to death. Based on the pathological characteristics of spinal cord injury, the active oxygen and inflammatory factors in the microenvironment of the damaged site significantly increase in the early stage of injury, which further triggers more serious secondary injury, including necrosis of neural tissue, formation of glial scar and occurrence of neural cavity. Therefore, the local microenvironment can be relieved by delivering drugs to the injury site to achieve a therapeutic effect.

[0003] At present, the drug treatment of spinal cord injury can be divided into two categories. One is to achieve drug delivery through local implantation system, that is, to wrap the drug in a high molecular biological material such as hyaluronic acid, gelatin, etc. to form a semi-solid with a three-dimensional network structure, and then to implant 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 drug delivery method requires opening the vertebral plate around the spinal cord for drug delivery, it causes great damage to the body, so it is generally given only once. And the cavity at the injury site is between a few microliters and tens of microliters. Therefore, the number and space of local implantation drug delivery are limited, and the amount of drug delivery is greatly limited. The other is to achieve drug delivery through non-local implantation system, including intravenous injection, intrathecal injection and nasal administration. Although these drug delivery methods have less invasiveness and can achieve continuous drug delivery at different time points, the drugs lack targeting to the injury site after entering the body through the above-mentioned methods, and the enrichment in the injury area is low, so the therapeutic effect of drug delivery is still limited. Therefore, how to combine the advantages of the two may become a solution to the problem of drug delivery.

[0004] Bio-orthogonal chemistry refers to a chemical reaction 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 azido group and dibenzocyclooctene (DBCO) are mild, rapid and specific, which can be used as targeting in the body. SUMMARY

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

[0006] The application adopts the technical scheme as follows:

[0007] A preparation method of a targeting system based on a bio-orthogonal click chemistry reaction, the targeting system comprising a gel (GelMA-DBCO) modified with a targeting receptor dibenzocyclooctyne (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) Methyl methacrylate gelatin (GelMA) and dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS) are dissolved and mixed, triethylamine is added, and the reaction is stirred at room temperature to obtain a gel modified with a targeting receptor GelMA-DBCO;

[0010] (1-2) GelMA-DBCO is added to a solution containing a photoinitiator to obtain a GelMA-DBCO solution, and a GelMA-DBCO gel is obtained by ultraviolet crosslinking;

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

[0012] (2-1) Acetylene-modified mannose (Ac4ManNAz) is added to the culture medium of MSC to obtain mesenchymal stem cells (MSC) modified with N3 on the surface, the cells are digested and collected, and the MSC modified with azido on the surface is obtained by extruding the MSC through a filter membrane using a liposome extruder;

[0013] (2-2) The nanoparticle modified with azido on the surface is purified by centrifugation, and NV-N3 is obtained by resuspension.

[0014] In step (1-1), the mass ratio of GelMA and 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 5-15%.

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

[0018] The preparation method further includes dialysis purification of the reaction product obtained in step (1-1), followed by freeze-drying to obtain a white solid, which is the GelMA-DBCO gel modified with a targeting receptor, which is then added to a solution containing a photoinitiator. The specific process is as follows: the product is placed in a dialysis bag and dialyzed in pure water for 72 hours, with the pure water 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 MSC and Ac4ManNAz are co-incubated for 24-48 hours.

[0020] Wherein, in step (2-1), Ac4ManNAz is first dissolved in DBCO to prepare 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, ultracentrifugation is performed at 8,000-10,000 g for 30 minutes, the supernatant is taken, and cells and cell debris in the precipitate are removed; the supernatant is then ultracentrifuged at 100,000 g for 1-1.5 hours; the precipitate is taken and resuspended in PBS.

[0023] The present invention also provides a targeting system obtained by the above preparation method, comprising a hydrogel GelMA-DBCO and nanoparticles 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 application is based on a bio-orthogonal click chemistry reaction to construct an artificial targeting system, which is composed of a gel modified with a targeting receptor, dibenzocyclooctyne (DBCO) (GelMA-DBCO) and a nanoparticle modified with a targeting ligand, azido group (N3) (NV-N3). On the one hand, after local gel implantation, the targeting system can improve the enrichment of subsequent administration in a specific area, for enhancing the targeting ability of the specific area, effectively realizing the targeting of the nanoparticles and the treatment of spinal cord injury. On the other hand, the system has high specificity and stability, only the nanoparticles carrying the specific group, azido group, can target the specific area, and the gel scaffold after implantation can maintain in the body for more than one week, and the targeting enhancement effect also lasts for more than one week. Therefore, the application effectively combines the local implantation and subsequent administration strategy in the treatment of spinal cord injury. Local implantation promotes the targeting ability of subsequent administration, and subsequent administration makes up for the lack of local administration, providing a new treatment scheme for the future treatment of spinal cord injury. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the nuclear magnetic hydrogen spectrum of GelMA-DBCO in Example 1;

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

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

[0031] Figure 4 is the 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 detection 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 is the single administration result of the targeting system in the rat spinal cord injury model in Application Example 1;

[0036] Figure 9 is the continuous administration result of the targeting system in the rat spinal cord injury model in Application Example 2. DETAILED DESCRIPTION

[0037] In order to describe the present invention in more detail, the technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the claims of the present invention.

[0038] Example 1

[0039] 1. Preparation of GelMA-DBCO Gel

[0040] Add 224 mg of methacrylated gelatin GelMA to 10 mL of ultrapure water to obtain a GelMA solution. Then weigh 14 mg of DBCO-NHS and add it to 5 mL of N,N-dimethylformamide. After mixing the above solutions, add 9.8 μL of triethylamine and stir at room temperature to react. The reactant is dialyzed and lyophilized to obtain a white solid, GelMA-DBCO. Weigh 3 mg of GelMA-DBCO and dissolve it in 500 μL of deuterated water. Detect the structure by hydrogen spectrum. Figure 1 The structure of GelMA-DBCO is shown in the H NMR spectrum. 50 mg of GelMA-DBCO was added to 500 μL of a solution containing 0.25% LAP. After the solution was homogenized and stabilized at 37°C, 10 μL of the GelMA-DBCO solution was 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 MSC membrane surface with azide groups

[0042] Ac4MAnNAz was added to the culture medium of MSC and incubated with MSC at the concentrations of 0, 15, 30, and 60 μM. After 24 h, the cells were stained with DBCO-Cy5 and the azide modification of the cell surface was observed under confocal microscopy. Figure 3 It can be seen that when the concentration of Ac4MAnNAz is greater than or equal to 30μM, the level of cell azide modification is the highest. The azide-modified cells were digested and collected, and squeezed with a liposome extruder, passing the cells through 5μm and 0.4μm filter membranes in sequence. The suspension was ultracentrifuged at 10,000g to remove cell debris in the precipitate. The supernatant was then centrifuged at 100,000g again, and the precipitate was collected and resuspended in PBS to obtain azide-modified nanoparticles. 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] Take 50 mg GelMA-DBCO into 500 μL solution containing 0.25% LAP, prepare 10% GelMA-DBCO solution, after the solution is uniform and stable at 37°C, take 25 μL GelMA-DBCO solution, spread on the bottom of the confocal dish, after UV crosslinking for 40 s, get the DBCO modified GelMA gel. Add DiD stained NV-N3 suspension to the confocal dish, discard the liquid in the confocal dish after 2 h, wash twice with PBS, then use Cy5-N3 to fluorescently stain the GelMA-DBCO gel, wash twice with PBS, then observe the fluorescence signal of nanoparticles and gel under confocal microscope, see Figure 5 . It can be seen that the fluorescence signal of nanoparticles closely matches the fluorescence signal of gel, which shows that the two can be connected by bio-orthogonal reaction.

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

[0046] Take 50 mg GelMA and GelMA-DBCO into 500 μL solution containing 0.25% LAP, prepare 10% GelMA and GelMA-DBCO solution, after the solution is uniform and stable at 37°C, take 25 μL GelMA or GelMA-DBCO solution, add to 1 mL cell culture medium. Incubate MSC with the medium. After 24 h, detect the cell activity of each group by CCK-8. See Figure 6 , the experimental results show that GelMA and GelMA-DBCO do not affect cell activity and have good biocompatibility.

[0047] Example 2

[0048] Degradation of GelMA-DBCO in rats in vivo

[0049] Weigh 200-220 g of SD rats, anesthetize, and shave the back hair with a hair clipper. Disinfect the skin at the T9-T10 spinal segment with iodophor. Cut the skin with a scalpel, and separate the fascia under the skin with forceps. Cut the muscle next to the spine with a scalpel, and open the wound with a spreader. Cut off the cone plate with bone rongeur to expose the spinal cord. Cut the spinal cord completely with ophthalmic scissors to form a 3 mm injury gap.

[0050] After stopping bleeding with cotton balls, implant GelMA-DBCO at the injury site. Suture the back muscles and skin of the rats in turn, disinfect with iodophor, inject penicillin, and place in an incubator to wait for recovery. Rats are relatively weak after spinal cord injury modeling, and need to urinate daily, inject penicillin, and change water, food and bedding regularly.

[0051] On the 7th, 14th, 21st and 28th day after modeling, a batch of rats were sacrificed, and spinal cord tissues were dissected to detect the degradation of GelMA-DBCO at the spinal cord injury site, as shown in Figure 7 It can be seen that GelMA-DBCO still has residues after 14 days, and is completely degraded on the 21st day, having certain stability and biodegradability.

[0052] Application Example 1

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

[0054] A 200-220g SD rat was weighed and anesthetized, the back hair was shaved with a hair clipper, and the skin position at the T9-T10 vertebral segment was disinfected with iodophor. The skin was incised with a scalpel, and the fascia under the skin was separated with forceps. The muscle close to the two sides of the vertebrae was incised with a scalpel, and the wound was opened with a spreader. The lamina was removed with a rongeur to expose the spinal cord. The spinal cord was completely cut with an ophthalmic scissors to form an injury gap of about 3mm.

[0055] After the cotton ball was fully hemostatic, the targeting gel group implanted GelMA-DBCO at the injury site; the blank gel group implanted GelMA at the injury site. The rat back muscles and skin were sutured in turn, iodophor was disinfected, penicillin was injected, and the rat was placed in an incubator to wait for recovery. The rat was relatively weak after spinal cord injury modeling, and needed to urinate daily, inject penicillin, and regularly change water, food and bedding.

[0056] On the 3rd day after modeling, 20μg of DiD-labeled NV or NV-N3 was administered by nasal administration, and the rats were sacrificed at 12h after administration, and the spinal cord tissues were dissected to detect the fluorescence signal at the spinal cord injury site, as shown in Figure 8 It can be seen that the modification of the targeting group promotes the enrichment and retention of nanoparticles in the injury area. Only the group with simultaneous modification of DBCO and N3 has enhanced targeting of nanoparticles in the spinal cord injury area after nasal administration, reflecting the high specificity of the system.

[0057] Application Example 2

[0058] Results of continuous administration of the targeting system in a rat spinal cord injury model

[0059] A 200-220g SD rat was weighed and anesthetized, the back hair was shaved with a hair clipper, and the skin position at the T9-T10 vertebral segment was disinfected with iodophor. The skin was incised with a scalpel, and the fascia under the skin was separated with forceps. The muscle close to the two sides of the vertebrae was incised with a scalpel, and the wound was opened with a spreader. The lamina was removed with a rongeur to expose the spinal cord. The spinal cord was completely cut with an ophthalmic scissors to form an injury gap of about 3mm.

[0060] After the cotton ball is fully hemostatic, the target gel group is implanted with GelMA-DBCO at the injury site; the blank gel group is implanted with GelMA at the injury site. The back muscles and skin of the rats are sutured in turn, iodophor is sterilized, penicillin is injected, and the rats are placed in an incubator to wait for recovery. The rats are relatively weak after spinal cord injury modeling, and the rats need to be urinated daily, injected with penicillin, and regularly changed with water, food and bedding.

[0061] On the 3rd, 5th and 7th day after modeling, the rats are anesthetized, 20 μg of DiD-labeled NV-N3 is given by nasal administration, a batch of rats is sacrificed at 12 h before and after each administration, and spinal cord tissue is dissected to detect the fluorescence signal of the spinal cord injury site, as shown in Figure 9 It can be seen that the modification of the targeting group promotes the enrichment and retention of nanoparticles in the injury area, and such targeting effect can be maintained at least three times of nasal administration.

[0062] The above description of implementation and application is for the convenience of ordinary skilled persons in the art to understand and apply the present application. Those skilled in the art can obviously easily make various modifications to the above embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art to the present application according to the disclosure of the present application should be within the scope of protection of the present application.

Claims

1. A method for preparing a targeting system based on bio-orthogonal click chemistry reaction, characterized in that, The targeting system comprises a gel GelMA-DBCO modified with a targeting receptor dibenzocyclooctyne DBCO and a nanoparticle NV-N3 modified with a targeting ligand azido N3, and the preparation method comprises the following steps: (1) Preparation of GelMA-DBCO (1-1) After methyl methacrylate gelatin GelMA and DBCO-NHS are respectively dissolved and mixed, triethylamine is added, and the mixture is stirred and reacted at room temperature to obtain a gel GelMA-DBCO modified with a targeting receptor; (1-2) GelMA-DBCO is added to a solution containing a photoinitiator to obtain a GelMA-DBCO solution, and a GelMA-DBCO gel is obtained by ultraviolet crosslinking; (2) Preparation method of NV-N3 (2-1) Azido-modified mannose Ac4ManNAz is added to the culture medium of MSC to obtain mesenchymal stem cells MSC modified with N3 on the surface, the cells are digested and collected, and the MSC modified with azido on the surface is extruded through a filter membrane by using a liposome extruder to obtain nanoparticles modified with azido on the surface; (2-2) The nanoparticles modified with azido on the surface are purified by centrifugation, and resuspended to obtain NV-N3.

2. The method for preparing a bio-orthogonal click chemistry reaction-based targeting system according to claim 1, characterized by, 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.

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

4. The method for preparing a bio-orthogonal click chemistry reaction-based targeting system according to claim 1, wherein, The preparation method comprises the following steps: the reaction product obtained in step (1-1) is dialyzed and purified, and then freeze-dried to obtain a white solid, which is then added to a solution containing a photoinitiator.

5. The method for preparing a bio-orthogonal click chemistry reaction-based targeting system according to claim 1, wherein, In step (2-1), the final concentration of Ac4ManNAz in the MSC culture medium is 30-60μM, and the MSC is co-incubated with Ac4ManNAz for 24-48h.

6. The method for preparing a bio-orthogonal click chemistry reaction-based targeting system according to claim 1, wherein, In step (2-1), the extrusion process is carried out in two steps: the MSC is extruded through a 5μm filter membrane using a liposome extruder, and the extrusion is repeated for 10-20 times; and then the MSC is extruded through a 0.4μm filter membrane, and the extrusion is repeated for 10-20 times.

7. The method of preparing a bio-orthogonal click chemistry reaction-based targeting system according to claim 1, wherein, In step (2-2), the centrifugal purification process is carried out in two steps: first, ultracentrifugation is carried out at 8,000-10,000g for 30min, and the supernatant is taken to remove the cells and cell fragments in the precipitate; and then, ultracentrifugation is carried out at 100,000g for 1-1.5h; and the precipitate is resuspended with PBS.

8. A targeting system obtained by the preparation method of any one of claims 1-7, comprising a hydrogel GelMA-DBCO and a nanoparticle NV-N3 modified with azido.

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

10. Use according to claim 9, characterized in that, After the local implantation of the gel GelMA-DBCO, the subsequent administration of the nanoparticle NV-N3 realizes the targeting of the region through specific group reaction.

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