CN105288594A - Growth factor porous micro-sphere compound system coated by injectable hydrogel - Google Patents

Abstract

The present invention relates to the preparation of a growth factor porous microsphere composite system wrapped by injectable hydrogel, which is characterized in that: it comprises hyaluronic acid (HA) and adipic acid dihydrazide (ADH) cross-linked HA-ADH sol is used as the first component, and polylactic acid (PLLA) porous microspheres loaded with growth factors are loaded in the first component. The complex system can degrade and slowly release growth factors under physiological conditions, realize the slow release of growth factors in the body, solve the problems of too fast release of growth factors and too short action period, and thus achieve the effect of nerve repair.

Description

A Composite System of Growth Factor Porous Microspheres Encapsulated by Injectable Hydrogel

technical field

The invention belongs to the field of biomedical materials for nerve tissue repair, in particular to a composite system of growth factor porous microspheres wrapped by injectable hydrogel.

Background technique

Peripheral nerve defect and repair has always been one of the diseases that plague many people in the world, mostly caused by transverse injury, burn or degenerative disease. Peripheral nerve injuries can be divided into two categories: non-defective stump injuries and stump-defective injuries. For the damage without defects between the broken ends, surgical methods such as adventitial anastomosis and perineural anastomosis can be used for anastomosis repair. However, the repair and functional reconstruction of long peripheral nerve defects is still one of the clinical problems, seriously affecting the quality of life of patients. Since the last century, the use of non-biological catheters to treat peripheral nerve defects has become a research hotspot. Non-degradable materials represented by silica gel can induce inflammation in surrounding tissues and lead to a large number of scars due to their long-term retention in place. Although the degradable nerve conduit materials represented by polylactic acid can achieve similar effects to autologous nerve transplantation, they cannot support nerve regeneration for long-segment nerve defects, and the implantation of foreign bodies inevitably produces inflammatory reactions, affecting Surgical success rate and postoperative recovery. This may be due to the lack of growth factors and other internal matrix as the reason for supporting tissues, so it is very necessary to build substances that promote nerve growth and repair in the nerve conduit.

Nerve growth factor (NGF) is a nerve cell growth regulator with dual biological functions of neuron nutrition and neurite growth. Studies have shown that NGF is highly expressed in nerve injury sites, and it can promote the proliferation of nerve cells, regeneration and differentiation of neurons to a certain extent. Basic fibroblast growth factor (bFGF), also known as dermal growth factor, has a wide range of biological activities on neuroectodermal cells, can improve the microenvironment of cell life, and promote the regeneration of damaged nerves. It is a nutrition and regeneration factor for nerve cells. , known as "cell activating factor". After people's long-term exploration, it is found that the joint application of nerve growth factor and basic fibroblast growth factor can promote the division and proliferation of neural stem cells, and increase the proportion of neural stem cells differentiated into neurons. However, compared with the half-life of bFGF10h, the biological half-life of NGF is too short, and it is easily inactivated by other substances, which limits its application range.

Contents of the invention

The technical problem to be solved by the present invention is to provide a composite system of growth factor microspheres wrapped by injectable hydrogel, a preparation method and an application. The complex system can degrade and slowly release growth factors under physiological conditions, realize the slow release of growth factors in the body, solve the problems of too fast release of growth factors and too short action period, and thus achieve the effect of nerve repair. , in order to solve the above technical problems, the technical solution adopted in the present invention is:

Preparation of a composite system of growth factor porous microspheres encapsulated by injectable hydrogel, which includes HA-ADH sol cross-linked with hyaluronic acid (HA) and adipate dihydrazide (ADH) as the first components, and polylactic acid (PLLA) porous microspheres loaded with growth factors in the first component.

According to the above scheme, the mass percentage of growth factors in the polylactic acid (PLLA) porous microspheres loaded with growth factors is 4wt%-12.5wt%.

According to the above scheme, the growth factors are nerve growth factor NGF and basic fibroblast growth factor bFGF, the mass ratio of which is 8.5-11.5:1.

According to the above scheme, the mixing mass ratio of HA-ADH sol and polylactic acid (PLLA) porous microspheres loaded with growth factors is 1:1.25-5.75.

According to the above scheme, the HA-ADH sol is obtained by reacting hyaluronic acid (HA) and adipic acid dihydrazide (ADH) under acidic conditions and the activation of EDC/NHS activator at 0-4°C owned.

According to the above scheme, the mass ratio of hyaluronic acid to adipic acid dihydrazide is 1:2-6, and the acidic condition is pH 4.5-5.75.

According to the above scheme, the average diameter of the polylactic acid (PLLA) porous microspheres loaded with growth factors is 2±1 μm.

According to the above-mentioned scheme, the polylactic acid (PLLA) porous microspheres loaded with growth factors is that the solution containing growth factors added with porogens and emulsifiers is the inner water phase, PLLA is the oil phase, and the solution containing the emulsifier The solution is the external water phase, and the double emulsion is obtained by using the W/O/W double emulsion method, then stirred evenly, left to solidify, centrifuged to collect the precipitate, washed, and freeze-dried to obtain.

A preparation method of the growth factor porous microsphere composite system wrapped by injectable hydrogel is as follows:

(1) Hyaluronic acid (HA) and adipic acid dihydrazide (ADH) are obtained under acidic conditions and the activation of EDC/NHS activator to obtain HA-ADH sol;

(2) Use the solution containing NGF and bFGF components added with porogen and emulsifier as the inner water phase, polylactic acid (PLLA) as the oil phase, and the solution containing PVA as the outer water phase, using W/O/ Double emulsion is obtained by the double emulsion method, then stirred evenly, left to solidify, centrifuged to collect the precipitate, washed, freeze-dried to obtain polylactic acid (PLLA) porous microspheres loaded with growth factors;

(3) Stir the HA-ADH sol obtained in the above steps and the polylactic acid (PLLA) porous microspheres loaded with growth factors evenly.

According to the above scheme, the W/O/W double emulsion method of the step (2) is: PLLA is dissolved in methylene chloride to obtain an oil phase, and then added to the water containing growth factors added with porogens and emulsifiers In the phase, a W/O/W emulsion is formed under stirring or ultrasonic conditions, and then an external water phase containing an emulsifier is added to it, and the high-speed stirring is performed for a period of time to form a double emulsion. Using the W/O/W double emulsion method to obtain polylactic acid (PLLA) porous microspheres loaded with growth factors can avoid the leakage of hydrophilic growth factors into the continuous phase and adhere to the surface of the microspheres, causing the problem of burst release.

According to the above scheme, the encapsulation rate of the growth factor in the step (2) is 55-65wt%.

According to the above scheme, the reaction time of step (1) is 18-24h.

According to the above scheme, the freeze-drying time in step (2) is 24-48h, and the curing time is 80-120min.

According to the above scheme, the stirring temperature in step (3) is 0-4°C.

According to the above scheme, the dosage ratio of polylactic acid (PLLA) to porogen in the water phase of step (2) is 1:0.67-0.89, the dosage ratio of polylactic acid (PLLA) to emulsifier is 1:1-1.33, and the external water The dosage ratio of polylactic acid (PLLA) and emulsifier in the phase is 1:0.53-0.71.

According to the above scheme, the porogen in the step (2) is sodium carbonate (Na ₂ CO ₃ ); the emulsifier is polyvinyl alcohol (PVA), and the relative number average molecular weight of PVA is 35,000-50,000.

Beneficial effects of the present invention:

1. Slow release of growth factors

In the present invention, polylactic acid is made into porous microspheres loaded with growth factors, which improves the drug loading and stability of the growth factors, and then the drug-loaded porous microspheres are loaded in HA-ADH sol, and wrapped with HA-ADH sol Finally, the obtained growth factor microsphere composite system wrapped by injectable hydrogel, on the one hand: it can be transformed into a gel state in the physiological environment of the human body, thus, it has a good encapsulation and fixation effect on polylactic acid porous microspheres , on the other hand, under physiological conditions, the molecular chains of polylactic acid undergo chemical decomposition to become small molecules. During this process, the growth factor drugs contained in it are gradually released into body fluids. degraded, and the release rate increased continuously. In this way, uniform and slow drug release can be achieved, while adhesion and burst release caused by direct contact with tissues can be avoided.

2. Synergy between NGF and bFGF

Nerve growth factor (NGF) is one of the most important biologically active substances in the nervous system, which has important biological effects on the normal development of the nervous system and the promotion of regeneration. Basic fibroblast growth factor (bFGF), also known as dermal growth factor, has a wide range of biological activities on neuroectodermal cells, can improve the microenvironment of cell life, and promote the regeneration of damaged nerves. It is a nutrition and regeneration factor for nerve cells. , known as "cell activating factor". Studies have shown that the combined application of nerve growth factor and basic fibroblast growth factor can promote the division and proliferation of neural stem cells and increase the proportion of neural stem cells differentiated into neurons. However, compared with the half-life of bFGF10h, the biological half-life of NGF is too short, and it is easily inactivated by other substances, which limits its application range. The composite microsphere system of the present invention can realize the slow and stable release of the two growth factors, overcome the shortcomings of the short half-life of the NGF growth factor and be easily subjected to chemical damage, and can achieve a higher level based on the joint action of the nerve growth factor and the basic fibroblast growth factor. Better than using NGF alone to promote the repair of defective nerves.

3. Protective properties of nerve repair process

Hyaluronic acid hydrogel can be used as an adhesive surgical instrument during surgery, protecting tissue during surgery, and providing surgery with a safe tool to make space and move tissue; after surgery, the hydrogel changes at the surgical site Adhesive surgical implants that keep damaged nerves separated from other tissue surfaces and provide a barrier to prevent postoperative adhesions. As an ideal natural moisturizing factor, hyaluronic acid can firmly maintain 98% of water after surgery, providing a good microenvironment for nerve repair. The encapsulation of hyaluronic acid makes the polylactic acid drug-loaded porous microspheres evenly dispersed, avoiding the direct contact between the microspheres and the damaged nerves, and allowing the drug to be released evenly and slowly.

4. Good biocompatibility

Hyaluronic acid (HA), as a mucopolysaccharide widely present in body fluids and tissues, is considered to be a polysaccharide that fills spaces, stabilizes structures, coats cells, and protects cells. Its main role is to stabilize intercellular fibers and membranes. Protein structure, so highly purified HA can be applied to sensitive parts such as nerves and eyes. After cross-linking with ADH, compared with the HA fluid applied alone, the viscoelasticity and better rheological properties of HA are increased, the residence time in tissues is prolonged, and good biocompatibility can be maintained. Avoid the problem that the simple HA hydrogel has low mechanical properties, is relatively fragile, and is prone to collapse in the body.

Polylactic acid (PLLA), as a high molecular polymer, has no immunogenicity, is also a kind of relatively good biocompatibility and biodegradability, and finally degrades into carbon dioxide and water in the body. Biodegradable material for the human body.

5. Ease and feasibility of system preparation

The preparation process of the composite system of hyaluronic acid hydrogel and polylactic acid drug-loaded porous microspheres is relatively simple, the reaction temperature is around room temperature, the reaction conditions are relatively mature, and it has strong operability. The use of W/O/W double emulsion method avoids the leakage of hydrophilic growth factors to the continuous phase and adheres to the surface of microspheres, causing the problem of burst release.

6. Wide applicability of the system

The hyaluronic acid-coated growth factor porous microsphere composite system can not only be applied to the repair of peripheral nerve defects, but also can be loaded with different drugs through polylactic acid porous microspheres, and is widely used in ophthalmology, ENT diseases, etc. The drug delivery system is expected to solve the problems of short drug action time, unstable action system, postoperative tissue adhesion and slow recovery in the process of peripheral nerve injury repair.

Description of drawings

Fig. 1 is the HA-ADH sol NMR spectrum of the embodiment of the present invention 1;

Fig. 2 is the schematic diagram of the preparation of the growth factor porous microsphere composite system wrapped by injectable hydrogel of the present invention;

Fig. 3 is a graph of the average in vitro weight loss rate of a growth factor porous microsphere composite system wrapped by injectable hydrogel provided by Examples 1-5 of the present invention;

Fig. 4 is a graph of the average cumulative release rate of NGF of a composite system of growth factor porous microspheres wrapped in injectable hydrogel in Examples 1-5 provided by the present invention.

detailed description

In order to better understand the present invention, the content of the present invention is further described below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

Example 1

Preparation of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

(1) Dissolve 0.5g HA in 100ml of ultrapure water at 0-4°C, stir magnetically to fully dissolve, then add 2gADH, then add 0.8gEDC and 0.7gNHS, 1mol/lHCl to adjust the pH to 5.0, react for 20h, and dialyze That is to get HA-ADH sol;

(2) 1.5g of PLLA was dissolved in 20mL of dichloromethane, then added to NGF, bFGF (NGF4.5mg/ml; bFGF was 0.5mg/ml) aqueous phase containing 1g of sodium carbonate and 1.5g of PVA, and stirred Or form an O/W emulsion under ultrasonic conditions, then add 3ml of 26.7% PVA external water phase to it, stir at high speed for a period of time to form a double emulsion, transfer the double emulsion to a beaker, and stir at 300rpm for 80min to solidify. Collect the precipitate by centrifugation and keep the supernatant aqueous phase, wash three times and keep the washing liquid, then freeze-dry the precipitate for 24 hours;

(3) At 0-4°C, 0.35 g of the HA-ADH sol prepared in the above steps and 1.25 g of PLLA porous microspheres loaded with growth factors were evenly stirred to obtain the product.

Structural Characterization of HA-ADH Sol

Adjust the pH value of the hydrogel obtained in the above step (1) to 7.4, place it in a constant temperature water bath at 37°C (simulating the physiological environment of the human body) until it completely transforms into a gel state, and freeze-dry it for 24-48 hours for later use.

Take 6-10 mg of freeze-dried hydrogel sample and dissolve it in 500 μl of deuterated deuterium water, and carry out H NMR detection. The nuclear magnetic spectrum is shown in Figure 1. The nuclear magnetic spectrum shows that: the peak at δ1.70 is attributed to the amino hydrogen at a in Figure 1, the peaks at δ1.40 (at Figure 1c) and 1.90 (at Figure 1b) are respectively assigned to the hydrogen on the methylene, and δ2. 90-δ3.60 is the structural peak containing hyaluronic acid, which is a sign of hyaluronic acid, which proves that ADH and HA are successfully cross-linked to form amides.

Characterization of Encapsulation Efficiency of PLA Drug-loaded Microspheres

The supernatant aqueous phase and the washing solution were combined, and the content of the growth factor was determined by enzyme-linked immunosorbent assay. After analysis, the encapsulation rate of the sample of the present invention was 58%.

TEM and DOS detection of polylactic acid drug-loaded microspheres

Take 2ml of the above reaction solution (before centrifugation) for TEM and DOS detection. The diameter of the polylactic acid drug-loaded microspheres of the present invention is 2.5 μm.

Drug Release Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

On the ultra-clean workbench, freeze-dry the sample prepared above and put it into a sterile 5ml centrifuge tube, add 3ml of phosphate buffer (pH=7.4), and culture in a constant temperature shaking incubator at 37°C with a rotation speed of 60r/min. The extracts were taken at 1d, 3d, 6d, 9d, 12d, and 15d respectively, and an equal amount of PBS buffer was added, and the cumulative release of growth factors was detected by enzyme-linked immunosorbent assay. The cumulative release of growth factors within 15 days was 68%.

Degradation Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

Take 1ml of the system prepared in (3) above and put it into a 15ml centrifuge tube, add 3ml of PBS buffer solution (pH=7.4), and degrade it in a water bath shaker at constant temperature (37°C, 60r/min). The degradation time is 1d, 3d, 6d, 9d, 12d, 15d and so on. After the corresponding degradation time, the sample was taken out, washed with ultrapure water, and the weight loss rate was measured. After testing, the sample of the present invention has a weight loss rate of 71% within 15 days.

Cytotoxicity assay of a composite system of growth factor porous microspheres encapsulated by injectable hydrogel (PC12 cells)

(1) The experiment was divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group. The above-mentioned materials were made into a 96-well plate with a hole size by a puncher, sterilized by ultraviolet light, and put into a 96-well plate for later use, with 5 parallel plates in each group.

(2) Take PC12 cells grown in logarithmic phase, adjust the concentration to 2×10 ⁴ cells/ml, add 100 μl per well into the 96-well plate in (1), and use empty cell culture medium as a control. 5% CO ₂ , cultured at 37°C for 72 hours.

(3) Measure at 24h, 48h, and 72h respectively. The specific method is as follows: add 10 μl CCK-8 solution to each well, continue to culture in the incubator for 4 hours, measure the OD value of each well at 450 nm, and calculate the cell survival rate.

CCK-8 experimental results

Example 2

Preparation of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

(1) Dissolve 1.0g HA in 100ml of ultrapure water at 0-4°C, stir magnetically to fully dissolve, then add 2.0gADH, then add 1.6gEDC and 1.4gNHS, 1mol/lHCl to adjust the pH to 4.5, react for 19h, Dialysis to obtain HA-ADH sol;

(2) 2.25g of PLA was dissolved in 20mL of dichloromethane, then added to NGF, bFGF (NGF7.875mg/ml; bFGF is 0.75mg/ml) aqueous phase containing 2.0g sodium carbonate and 3.0gPVA, in Form an O/W emulsion under stirring or ultrasonic conditions, then add 6ml of 26.7% PVA external water phase to it, stir at high speed for a period of time to form a double emulsion, transfer the double emulsion to a beaker, stir at 300rpm for 100min to solidify. Collect the precipitate by centrifugation and keep the supernatant aqueous phase, wash three times and keep the washing liquid, then freeze-dry the precipitate for 36 hours;

(3) At 0-4°C, 0.85 g of the HA-ADH sol obtained in the above steps and 1.98 g of PLLA porous microspheres loaded with growth factors were evenly stirred.

Structural Characterization of HA-ADH Sol

Adjust the pH value of the hydrogel obtained in the above step (1) to 7.4, place it in a constant temperature water bath at 37°C until it completely transforms into a gel state, and freeze-dry it for 24-48 hours for later use.

Referring to the characterization method of Example 1, the structure of the HA-ADH sol was characterized. NMR results showed that amides were successfully formed.

Characterization of Encapsulation Efficiency of PLA Drug-loaded Microspheres

The supernatant aqueous phase and the washing solution were combined, and the content of the growth factor was determined by enzyme-linked immunosorbent assay, and the encapsulation efficiency of the sample of the present invention was 60%.

TEM and DOS detection of polylactic acid drug-loaded microspheres

Take 2ml of the above reaction solution (before centrifugation) for TEM and DOS detection. The diameter of the polylactic acid drug-loaded microspheres of the present invention is 2.7 μm.

Drug Release Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

On the ultra-clean workbench, freeze-dry the sample prepared above and put it into a sterile 5ml centrifuge tube, add 3ml of phosphate buffer (pH=7.4), and culture in a constant temperature shaking incubator at 37°C with a rotation speed of 60r/min. The extracts were taken at 1d, 3d, 6d, 9d, 12d, and 15d respectively, and an equal amount of PBS buffer was added, and the cumulative release of growth factors was detected by enzyme-linked immunosorbent assay. The cumulative release of growth factors within 15 days was 64%.

Degradation Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

Take 1ml of the system prepared in (3) above and put it into a 15ml centrifuge tube, add 3ml of PBS buffer solution (pH=7.4), and degrade it in a water bath shaker at constant temperature (37°C, 60r/min). The degradation time is 1d, 3d, 6d, 9d, 12d, 15d and so on. After the corresponding degradation time, the sample was taken out, washed with ultrapure water, and the weight loss rate was measured. After testing, the sample of the present invention has a weight loss rate of 69% within 15 days.

Cytotoxicity assay of a composite system of growth factor porous microspheres encapsulated by injectable hydrogel (PC12 cells)

(1) The experiment was divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group. The above-mentioned materials were made into a 96-well plate with a hole size by a puncher, sterilized by ultraviolet light, and put into a 96-well plate for later use, with 5 parallel plates in each group.

(2) Take PC12 cells grown in logarithmic phase, adjust the concentration to 2×10 ⁴ cells/ml, add 100 μl per well into the 96-well plate in (1), and use empty cell culture medium as a control. 5% CO ₂ , cultured at 37°C for 72h.

(3) Measure at 24h, 48h, and 72h respectively. The specific method is as follows: add 10 μl CCK-8 solution to each well, continue to culture in the incubator for 4 hours, measure the OD value of each well at 450 nm, and calculate the cell survival rate.

CCK-8 experimental results

Example 3

Preparation of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

(1) Dissolve 1.0g HA in 100ml ultrapure water under the condition of 0-4℃, magnetically stir until fully dissolved, then add 3.0gADH, then add 1.6gEDC and 1.4gNHS, 1mol/lHCl to adjust the pH to 5.5, react for 21h, Dialysis to obtain HA-ADH sol;

(2) 1.5g of PLA was dissolved in 20mL of dichloromethane, then added to NGF, bFGF (NGF9.5mg/ml; bFGF was 1mg/ml) aqueous phase containing 1g of sodium carbonate and 1.5g of PVA, stirring or Form an O/W emulsion under ultrasonic conditions, then add 3ml of 26.7% PVA external water phase to it, stir at high speed for a period of time to form a double emulsion, transfer the double emulsion to a beaker, and stir at 300rpm for 110min to solidify. Collect the precipitate by centrifugation and keep the supernatant aqueous phase, wash three times and keep the washing liquid, and then freeze-dry the precipitate for 30 hours;

(3) At 0-4°C, 0.87g of HA-ADH hydrogel obtained in the above steps and 1.13g of PLLA porous microspheres loaded with growth factors were uniformly stirred.

Structural Characterization of HA-ADH Sol

Adjust the pH value of the hydrogel obtained in the above step (1) to 7.4, place it in a constant temperature water bath at 37°C until it completely transforms into a gel state, and freeze-dry it for 24-48 hours for later use.

Referring to the characterization method of Example 1, the structure of the HA-ADH sol was characterized. NMR results showed that amides were successfully formed.

Characterization of Encapsulation Efficiency of PLA Drug-loaded Microspheres

The supernatant aqueous phase and the washing solution were combined, and the content of growth factors was determined by enzyme-linked immunosorbent assay. The encapsulation efficiency of the sample of the present invention was 65%.

TEM and DOS detection of polylactic acid drug-loaded microspheres

Take 2ml of the above reaction solution (before centrifugation) for TEM and DOS detection. The diameter of the polylactic acid drug-loaded microspheres of the present invention is 1.9 μm.

Drug Release Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

On the ultra-clean workbench, freeze-dry the sample prepared above and put it into a sterile 5ml centrifuge tube, add 3ml of phosphate buffer (pH=7.4), and culture in a constant temperature shaking incubator at 37°C with a rotation speed of 60r/min. The extracts were taken at 1d, 3d, 6d, 9d, 12d, and 15d respectively, and an equal amount of PBS buffer was added, and the cumulative release of growth factors was detected by enzyme-linked immunosorbent assay. The cumulative release of growth factors within 15 days was 69%.

Degradation Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

Take 1ml of the system prepared in (3) above and put it into a 15ml centrifuge tube, add 3ml of PBS buffer solution (PH=7.4), and degrade it in a water-bath shaker with constant temperature shaking (37°C, 60r/min). The degradation time is 1d, 3d, 6d, 9d, 12d, 15d and so on. After the corresponding degradation time, the sample was taken out, washed with ultrapure water, and the weight loss rate was measured. After testing, the sample of the present invention has a weight loss rate of 75% within 15 days.

Cytotoxicity assay of a composite system of growth factor porous microspheres encapsulated by injectable hydrogel (PC12 cells)

(1) The experiment was divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group. The above-mentioned materials were made into a 96-well plate with a hole size by a puncher, sterilized by ultraviolet light, and put into a 96-well plate for later use, with 5 parallel plates in each group.

(2) Take PC12 cells grown in logarithmic phase, adjust the concentration to 2×10 ⁴ cells/ml, add 100 μl per well into the 96-well plate in (1), and use empty cell culture medium as a control. 5% CO ₂ , cultured at 37°C for 72 hours.

(3) Measure at 24h, 48h, and 72h respectively. The specific method is as follows: add 10 μl CCK-8 solution to each well, continue to culture in the incubator for 4 hours, measure the OD value of each well at 450 nm, and calculate the cell survival rate.

CCK-8 experimental results

Example 4

Preparation of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

(1) Dissolve 0.5g HA in 100ml ultrapure water at 0-4°C, stir magnetically to fully dissolve, then add 3gADH, then add 1.2gEDC and 1.35gNHS, 1mol/lHCl to adjust the pH to 5.0, react for 23h, dialyze That is to get HA-ADH sol;

(2) 2.25g of PLLA was dissolved in 20mL of dichloromethane solution, and then added to the aqueous phase of NGF and bFGF (NGF14.375mg/ml; bFGF was 1.25mg/ml) containing 2.0g of sodium carbonate and 2.25g of PVA, Form an O/W emulsion under stirring or ultrasonic conditions, then add 4.5ml of 26.7% PVA external water phase to it, stir at high speed for a period of time to form a double emulsion, transfer the double emulsion to a beaker, and stir at 300rpm for 120min to solidify . Collect the precipitate by centrifugation and keep the supernatant aqueous phase, wash three times and keep the washing liquid, and then freeze-dry for 48 hours to complete;

(3) At 0-4°C, 0.39 g of the HA-ADH sol obtained in the above steps and 2.05 g of PLLA porous microspheres loaded with growth factors were evenly stirred.

Structural Characterization of HA-ADH Sol

Adjust the pH value of the hydrogel obtained in the above step (1) to 7.4, place it in a constant temperature water bath at 37°C until it completely transforms into a gel state, and freeze-dry it for 24-48 hours for later use.

Referring to the characterization method of Example 1, the structure of the HA-ADH sol was characterized. NMR results showed that amides were successfully formed.

Characterization of Encapsulation Efficiency of PLA Drug-loaded Microspheres

The above supernatant aqueous phase was combined with the washing solution, and the content of growth factors was determined by enzyme-linked immunosorbent assay. The encapsulation efficiency of the sample of the present invention was 62%.

Detection of Polylactic Acid Drug-loaded Microspheres by TEM and DOS

Take 2ml of the above reaction solution (before centrifugation) for TEM and DOS detection. The diameter of the polylactic acid drug-loaded microspheres of the present invention is 1.5 μm.

Drug Release Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

On the ultra-clean workbench, freeze-dry the sample prepared above and put it into a sterile 5ml centrifuge tube, add 3ml of phosphate buffer (pH=7.4), and culture in a constant temperature shaking incubator at 37°C with a rotation speed of 60r/min. The extracts were taken at 1d, 3d, 6d, 9d, 12d, and 15d respectively, and an equal amount of PBS buffer was added, and the cumulative release of growth factors was detected by enzyme-linked immunosorbent assay. The cumulative release of growth factors within 15 days was 71%.

Degradation Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

Take 1ml of the system prepared in (3) above and put it into a 15ml centrifuge tube, add 3ml of PBS buffer solution (pH=7.4), and degrade it in a water bath shaker at constant temperature (37°C, 60r/min). The degradation time is 1d, 3d, 6d, 9d, 12d, 15d and so on. After the corresponding degradation time, the sample was taken out, washed with ultrapure water, and the weight loss rate was measured. After testing, the sample of the present invention has a weight loss rate of 69% within 15 days.

Cytotoxicity assay of a composite system of growth factor porous microspheres encapsulated by injectable hydrogel (PC12 cells)

(1) The experiment was divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group. The above-mentioned materials were made into a 96-well plate with a hole size by a puncher, sterilized by ultraviolet light, and put into a 96-well plate for later use, with 5 parallel plates in each group.

(2) Take PC12 cells grown in logarithmic phase, adjust the concentration to 2×10 ⁴ cells/ml, add 100 μl per well into the 96-well plate in (1), and use empty cell culture medium as a control. 5% CO ₂ , cultured at 37°C for 72h.

(3) Measure at 24h, 48h, and 72h respectively. The specific method is as follows: add 10 μl CCK-8 solution to each well, continue to culture in the incubator for 4 hours, measure the OD value of each well at 450 nm, and calculate the cell survival rate.

CCK-8 experimental results

Example 5

Preparation of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

(1) Dissolve 0.25g HA in 100ml of ultrapure water at 0-4°C, stir magnetically to fully dissolve, then add 1.0gADH, then add 0.4gEDC and 0.35gNHS, 1mol/lHCl to adjust the pH to 5.0, react for 24h, Dialysis to obtain HA-ADH sol;

(2) 0.75g of PLLA was dissolved in 20mL of dichloromethane, then added to NGF, bFGF (NGF2.125mg/ml; bFGF is 0.25mg/ml) aqueous phase containing 0.5g sodium carbonate and 0.75gPVA, in Form an O/W emulsion under stirring or ultrasonic conditions, then add 1.5ml of 26.7% PVA external water phase to it, stir at high speed for a period of time to form a double emulsion, transfer the double emulsion to a beaker, and stir at 300rpm for 90min to solidify. Collect the precipitate by centrifugation and keep the supernatant aqueous phase, wash three times and keep the washing liquid, then freeze-dry the precipitate for 40 hours to complete;

(3) At 0-4° C., 0.19 g of the HA-ADH sol obtained in the above steps and 0.65 g of PLLA porous microspheres loaded with growth factors were uniformly stirred.

Structural Characterization of HA-ADH Sol

Adjust the pH value of the hydrogel obtained in the above step (1) to 7.4, place it in a constant temperature water bath at 37°C until it completely transforms into a gel state, and freeze-dry it for 24-48 hours for later use.

Referring to the characterization method of Example 1, the structure of the HA-ADH sol was characterized. NMR results showed that amides were successfully formed.

Characterization of Encapsulation Efficiency of PLA Drug-loaded Microspheres

The above supernatant aqueous phase was combined with the washing solution, and the content of growth factors was determined by enzyme-linked immunosorbent assay. The encapsulation efficiency of the sample of the present invention was 59%.

Detection of Polylactic Acid Drug-loaded Microspheres by TEM and DOS

Take 2ml of the above reaction solution (before centrifugation) for TEM and DOS detection. The diameter of the polylactic acid drug-loaded microspheres of the present invention is 2.9 μm.

Drug Release Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

On the ultra-clean workbench, freeze-dry the sample prepared above and put it into a sterile 5ml centrifuge tube, add 3ml of phosphate buffer (pH=7.4), and culture in a constant temperature shaking incubator at 37°C with a rotation speed of 60r/min. The extracts were taken at 1d, 3d, 6d, 9d, 12d, and 15d respectively, and an equal amount of PBS buffer was added, and the cumulative release of growth factors was detected by enzyme-linked immunosorbent assay. The cumulative release of growth factors within 15 days was 65%.

Degradation Characterization of a Growth Factor Porous Microsphere Composite System Encapsulated by Injectable Hydrogel

Take 1ml of the system prepared in (3) above and put it into a 15ml centrifuge tube, add 3ml of PBS buffer solution (pH=7.4), and degrade it in a water bath shaker at constant temperature (37°C, 60r/min). The degradation time is 1d, 3d, 6d, 9d, 12d, 15d and so on. After the corresponding degradation time, the sample was taken out, washed with ultrapure water, and the weight loss rate was measured. After testing, the sample of the present invention has a weight loss rate of 72% within 15 days.

Cytotoxicity assay of a composite system of growth factor porous microspheres encapsulated by injectable hydrogel (PC12 cells)

(1) The experiment was divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group. The above-mentioned materials were made into a 96-well plate with a hole size by a puncher, sterilized by ultraviolet light, and put into a 96-well plate for later use, with 5 parallel plates in each group.

(2) Take PC12 cells grown in logarithmic phase, adjust the concentration to 2×10 ⁴ cells/ml, add 100 μl per well into the 96-well plate in (1), and use empty cell culture medium as a control. 5% CO ₂ , cultured at 37°C for 72h.

(3) Measure at 24h, 48h, and 72h respectively. The specific method is as follows: add 10 μl CCK-8 solution to each well, continue to culture in the incubator for 4 hours, measure the OD value of each well at 450 nm, and calculate the cell survival rate.

CCK-8 experimental results

Claims (10)

1. the preparation of a somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel, it is characterized in that: it comprises and is cross-linked with hyaluronic acid and adipic dihydrazide the HA-ADH colloidal sol obtained and makes the first component, and the porous polylactic acid microball that be loaded with somatomedin of load in the first component.

2. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, it is characterized in that: described somatomedin is nerve growth factor and basic fibroblast growth factor bFGF, and its mass ratio is 8.5-11.5:1; The described mass percent being loaded with somatomedin in the porous polylactic acid microball of somatomedin is 4wt%-12.5wt%.

3. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: HA-ADH colloidal sol is 1:1.25-5.75 with the mass ratio of the porous polylactic acid microball being loaded with somatomedin.

4. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: described HA-ADH colloidal sol be by hyaluronic acid and adipic dihydrazide under the activation of acid condition and EDC/NHS activator 0-4 DEG C be obtained by reacting; Described hyaluronic acid and the mass ratio of adipic dihydrazide are 1:2-6, and described acid condition is pH is 4.5-5.75.

5. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: described in be loaded with the porous polylactic acid microball of somatomedin average diameter be 2 ± 1 μm.

6. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, it is characterized in that: the described porous polylactic acid microball being loaded with somatomedin is the solution containing somatomedin being added with porogen and emulsifying agent is interior aqueous phase, take PLLA as oil phase, be outer aqueous phase with the solution containing emulsifying agent, use W/O/W multi-emulsion method to obtain emulsion, then stir, leave standstill solidification, centrifugal collecting precipitation, washing, lyophilizing obtains.

7. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: step is as follows:

(1) by hyaluronic acid and adipic dihydrazide in acid condition and under the activation of EDC/NHS activator 0-4 DEG C be obtained by reacting HA-ADH colloidal sol;

(2) be interior aqueous phase to be added with the solution containing NGF and bFGF component of porogen and emulsifying agent, take polylactic acid as oil phase, be outer aqueous phase with the solution containing PVA, W/O/W multi-emulsion method is used to obtain emulsion, then stir, leave standstill solidification, centrifugal collecting precipitation, washing, lyophilizing obtains growth factor-loaded porous polylactic acid microball;

(3) HA-ADH colloidal sol above-mentioned steps obtained and growth factor-loaded porous polylactic acid microball stir.

8. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, it is characterized in that: described W/O/W multi-emulsion method is: be dissolved in by PLLA in dichloromethane, obtain oil phase, then join be added with porogen and emulsifying agent containing in the aqueous phase of somatomedin, stirring or forming W/O/W type emulsion under ultrasonic condition, and then the outer aqueous phase added wherein containing emulsifying agent, high-speed stirring mixes emulsion;

In described step (2), the envelop rate of somatomedin is 55-65wt%.

9. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: the response time of described step (1) is reaction 18-24h;

Freeze-drying time described in step (2) is 24-48h, and hardening time is 80-120min;

Step (3) whipping temp is 0-4 DEG C;

In step (2) aqueous phase, the amount ratio of polylactic acid and porogen is 1:0.67-0.89, and the amount ratio of polylactic acid and emulsifying agent is 1:1-1.33, and in outer aqueous phase, the amount ratio of polylactic acid and emulsifying agent is 1:0.53-0.71.

10. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: described step (2) porogen is sodium carbonate; Emulsifying agent is polyvinyl alcohol, and PVA relative number average molecular weight is 35000-50000.