Polycaprolactone nerve scaffold loaded with DNSP-11 polypeptide PEG microspheres and preparation method of polycaprolactone nerve scaffold

Abstract

The invention discloses a polycaprolactone nerve scaffold loaded with DNSP-11 polypeptide PEG microspheres and a preparation method of the polycaprolactone nerve scaffold. The preparation method comprises the following steps: step 1, synthesizing a DNSP-11 polypeptide sequence by using a solid-phase synthesis method; step 2, adding a polyvinyl alcohol solution into the PEG solution to form a PEG emulsion; adding the polypeptide solution into the PEG emulsion, and hardening by using glutaraldehyde as a cross-linking agent; 3, dissolving polycaprolactone in a mixed solvent of chloroform and methanol, then dispersing the PEG microspheres in a PCL solution, and then adding a nonionic surfactant; and preparing a fiber membrane by using an electrostatic spinning method, and rolling the fiber membrane into the nerve scaffold. According to the polycaprolactone nerve scaffold disclosed by the invention, the PEG microspheres provide a slow release effect of DNSP-11, and the PCL fibers provide mechanical support and an additional microsphere slow release carrier, so that long-term controllable release of DNSP-11 polypeptide is realized; in addition, according to the polycaprolactone nerve scaffold, the bioavailability of DNSP-11 polypeptide can be improved, inactivation of the polypeptide in an extreme environment is avoided, and the stability of the polypeptide is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres and a preparation method thereof. Background Art

[0002] Peripheral nerve injury is usually caused by trauma, iatrogenic injury or congenital defects, resulting in partial or complete loss of sensory and motor function. The incidence of peripheral nerve injury is approximately 2% to 8% worldwide, which has a significant impact on the quality of life of patients. Peripheral nerve injury not only affects individual patients, but also imposes a heavy economic burden on society because patients require long-term rehabilitation and care.

[0003] Traditionally, neurotrophic factors such as nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) are believed to promote peripheral nerve regeneration. However, direct delivery of these protein neurotrophic factors has many drawbacks, such as low bioavailability, high requirements for storage environment, easy burst release, and inability to maintain long-term activity in vivo. These drawbacks limit the effectiveness of neurotrophic factors in clinical applications.

[0004] In contrast, peptide drugs have advantages such as good stability, high purity, low production cost and low immunogenicity. DNSP-11 peptide is an 11-amino acid peptide derived from the precursor protein of glial cell-derived neurotrophic factor (GDNF), with the sequence of PPEAPAEDRSL, which can exhibit effects similar to neurotrophic factors and has the ability to promote axon regeneration. Compared with larger neurotrophic factors, DNSP-11 has the advantages of easy synthesis, long-term storage stability, improved biodistribution and potentially higher bioavailability due to its smaller molecular weight and simple structure.

[0005] Therefore, how to deliver the DNSP-11 polypeptide to the site of peripheral nerve injury, increase its sustained release performance and improve its stability, and enhance its application in nerve repair is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0006] The purpose of the present invention is to provide a polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres and a preparation method thereof in view of the deficiencies in the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect is to provide a method for preparing a polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres, comprising the following steps:

[0009] Step 1: synthesize the DNSP-11 polypeptide sequence on RinkAmide MBHA resin using solid phase synthesis, and cut the polypeptide using a TFA / water / triisopropylsilane mixture; then purify it by HPLC using a C18 reverse phase chromatography column, and characterize it by MALDI-TOF mass spectrometry and analytical HPLC; then dissolve the purified DNSP-11 polypeptide in a sterile citric acid buffer at a pH of 4.5-5.0 to prepare a solution with a concentration of 1-2 mg / mL for standby use;

[0010] Step 2, dissolving PEG with a molecular weight of 4000-500Da in dichloromethane to prepare a solution with a concentration of 8-10w / v%; then adding 0.5-1w / v% polyvinyl alcohol solution to form a PEG emulsion; adding the DNSP-11 polypeptide solution obtained in step 1 to the PEG emulsion, using 0.3-0.5v / v% glutaraldehyde solution as a cross-linking agent for hardening, washing and collecting the PEG microspheres after the reaction is completed;

[0011] Step three, dissolving polycaprolactone in a mixed solvent of chloroform and methanol to prepare a PCL solution with a concentration of 8-10w / v%; then dispersing the PEG microspheres obtained in step two in the PCL solution, and the mass ratio of PEG microspheres to the PCL solution is 1:(8-10); then adding 0.07-0.1w / v% of a non-ionic surfactant; using an electrospinning method to prepare a fiber membrane, freezing and drying it, and rolling the fiber membrane into a neural scaffold.

[0012] Furthermore, the amino acid sequence of the DNSP-11 polypeptide is shown in SEQ ID NO: 1, which is PPEAPAEDRSL.

[0013] Furthermore, in the step 2, the PEG microspheres are prepared by using a high shear mixer and stirring at 8000-10000 rpm for 10-15 minutes.

[0014] Furthermore, in the step three, the parameters of the electrospinning are: voltage, 15 kV; flow rate, 0.8 mL / h; collection distance, 18 cm; spinning time, 4 hours; ambient temperature, 25° C.; and relative humidity, 40%.

[0015] Furthermore, the dimensions of the nerve stent are: wall thickness 0.7-0.9 mm, length 1.0-1.2 cm.

[0016] The second aspect is to provide a polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres prepared by the above-mentioned preparation method.

[0017] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0018] The polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres of the present invention has the PEG microspheres providing a sustained release effect of DNSP-11, and the PCL fibers provide mechanical support and additional microsphere sustained release carriers (with the gradual degradation of the PCL fibers, the loaded PEG microspheres can be gradually released), thereby achieving long-term controlled release of the DNSP-11 polypeptide.

[0019] The polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres of the present invention can improve the bioavailability of the DNSP-11 polypeptide, prevent the polypeptide from being inactivated in extreme environments, and increase the stability of the polypeptide. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0021] Example 1

[0022] This embodiment provides a method for preparing a polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres, comprising the following steps:

[0023] Step 1: Preparation and purification of DNSP-11 peptide

[0024] 1. Resin preparation:

[0025] RinkAmide MBHA resin (0.5 mmol / g loading) was used;

[0026] Wash with DCM and DMF three times, 5 mL each time, 5 minutes;

[0027] Treat with 20% piperidine / DMF solution for 20 min at room temperature;

[0028] The mixture was washed with DMF 5 times, 5 mL each time, for 2 minutes.

[0029] 2. Amino acid condensation cycle:

[0030] a) weighing 3 equivalents of Fmoc-amino acid (1.5 mmol);

[0031] b) dissolved in 5 mL of 0.5 M HBTU / HOBt solution;

[0032] c) Add 6 equivalents of DIEA (3 mmol, 522 μL);

[0033] d) adding the mixture to the resin;

[0034] e) reacting at room temperature for 60 minutes;

[0035] f) Wash with DMF 5 times, 5 mL each time, 2 minutes;

[0036] g) 20% piperidine / DMF solution treatment for 10 minutes at room temperature;

[0037] h) Wash with DMF 5 times, 5 mL each time, 2 minutes;

[0038] i) Repeat steps ah until the entire sequence (PPEAPAEDRSL) is completed.

[0039] 3. Final Fmoc removal:

[0040] 20% piperidine / DMF solution for 20 min at room temperature;

[0041] Wash with DMF 5 times, 5 mL each time, 2 minutes;

[0042] Wash with DCM 3 times, 5 mL each time, 2 minutes.

[0043] 4. Peptide cleavage:

[0044] Prepare cleavage mixture: TFA / water / triisopropylsilane (95:2.5:2.5 v / v / v), 10 mL;

[0045] Stir at room temperature for 3 hours, and collect the filtrate by filtration;

[0046] The peptides were precipitated with 50 mL of ice-cold ether and centrifuged at 3000 rpm for 10 min at 4 °C;

[0047] Wash with ether twice, 50 mL each time.

[0048] 5. Purification of crude peptides to remove by-products and incomplete sequences produced during the synthesis:

[0049] Dissolved in 10 mL of water / acetonitrile (80:20 v / v) mixed solvent;

[0050] HPLC purification was performed using a C18 reverse phase column;

[0051] Mobile phase A: 0.1% trifluoroacetic acid (TFA) in H2O; Mobile phase B: 0.1% TFA in acetonitrile (ACN);

[0052] Gradient elution: 5-50% B (0.1% TFA in CAN) in 30 min, flow rate 10 mL / min;

[0053] Detection wavelength: 220nm;

[0054] The main peak was collected and freeze-dried.

[0055] 6. Peptide characterization:

[0056] The molecular weight was confirmed by MALDI-TOF mass spectrometry and the purity was >98% by analytical HPLC.

[0057] 7. Final preparation:

[0058] Dissolve in sterile citric acid buffer at pH 5.0 to a concentration of 1 mg / mL; sterilize by filtration with a 0.22 μm filter membrane; aliquot and store at -80°C for future use.

[0059] Step 2: Preparation and purification of PEG microspheres loaded with DNSP-11 peptide

[0060] 1. Preparation of PEG microspheres:

[0061] Polyethylene glycol (PEG) with a molecular weight of 4000Da was dissolved in dichloromethane (DCM) to prepare a 10% (w / v) solution (100ml), and then a 1% (w / v) polyvinyl alcohol (PVA) aqueous solution (100ml) was added and stirred at 10000rpm for 10 minutes using a high shear mixer to form a PEG emulsion.

[0062] 2. Loading of DNSP-11 peptide:

[0063] The DNSP-11 solution (20 mL) prepared in step 1 was slowly added to the PEG emulsion (200 mL) and gently stirred for 5 minutes; DCM was removed by evaporation under reduced pressure at 40°C, and 0.5% (v / v) glutaraldehyde solution (20 mL) was slowly added as a cross-linking agent for hardening, and incubated at room temperature for 4 hours to form stable PEG microspheres;

[0064] Wash the microspheres three times with 10 mL of PBS each time and centrifuge at 4000 rpm for 10 minutes; repeat the wash three times to ensure that the unencapsulated peptides are removed;

[0065] The PEG microspheres were resuspended in 5 mL of sterile PBS, aliquoted and stored at -80°C until use.

[0066] Step 3: Preparation of polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres

[0067] Dissolve polycaprolactone (PCL, Mw = 80000) in a chloroform / methanol (v / v = 3:1) mixed solvent to prepare a 10 w / v% PCL solution; then disperse the PEG microspheres prepared in the above step 2 in the PCL solution, wherein the mass ratio of the PEG microspheres to the PCL solution is 1:10; then add 0.1 w / v% nonionic surfactant (10 mL, such as Tween 80), and disperse using a magnetic stirrer at room temperature for 4 hours to ensure uniform dispersion;

[0068] The electrospinning was carried out using a uniaxial electrospinning device, and the parameters were as follows: voltage, 15 kV; flow rate, 0.8 mL / h; collection distance, 18 cm; collection plate, flat collector covered with aluminum foil; spinning time, 4 hours; ambient temperature, 25°C; relative humidity, 40%; immediately after spinning, the fiber membrane was frozen in a -20°C refrigerator for 1 hour;

[0069] The collected fiber membranes were dried in a vacuum drying oven at 37 °C for 12 h to remove the residual solvent;

[0070] Subsequently, the electrospun membrane was rolled into a neural scaffold with a wall thickness of 0.9 mm and a length of 1.2 cm using a tubular mold with an inner diameter of 2 mm.

[0071] Example 2

[0072] This embodiment provides a method for preparing a polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres, comprising the following steps:

[0073] Step 1: Preparation and purification of DNSP-11 polypeptide (same as Example 1)

[0074] Final preparation: dissolved in sterile citric acid buffer at pH = 4.5, concentration 2 mg / mL; sterilized by filtration with a 0.22 μm filter membrane; aliquoted and stored at -80°C for future use.

[0075] Step 2: Preparation and purification of PEG microspheres loaded with DNSP-11 polypeptide

[0076] 1. Preparation of PEG microspheres:

[0077] Polyethylene glycol (PEG) with a molecular weight of 5000Da was dissolved in dichloromethane (DCM) to prepare a solution (100ml) with a concentration of 8% (w / v), and then a 0.5% (w / v) polyvinyl alcohol (PVA) aqueous solution (100ml) was added and stirred at 8000rpm for 15 minutes using a high shear mixer to form a PEG emulsion.

[0078] 2. Loading of DNSP-11 peptide:

[0079] The DNSP-11 solution (20 mL) prepared in step 1 was slowly added to the PEG emulsion (200 mL) and gently stirred for 5 minutes; DCM was removed by evaporation under reduced pressure at 40°C, and 0.3% (v / v) glutaraldehyde solution (20 mL) was slowly added as a cross-linking agent for hardening, and incubated at room temperature for 4 hours to form stable PEG microspheres;

[0080] Wash the microspheres three times with 10 mL of PBS each time and centrifuge at 4000 rpm for 10 minutes; repeat the wash three times to ensure that the unencapsulated peptides are removed;

[0081] The PEG microspheres were resuspended in 5 mL of sterile PBS, aliquoted and stored at -80°C until use.

[0082] Step 3: Preparation of polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres

[0083] Dissolve polycaprolactone (PCL, Mw = 80000) in a chloroform / methanol (v / v = 3:1) mixed solvent to prepare a PCL solution with a concentration of 8 w / v%; then disperse the PEG microspheres prepared in the above step 2 in the PCL solution, wherein the mass ratio of the PEG microspheres to the PCL solution is 1:8; then add 0.07 w / v% of a nonionic surfactant (10 mL, such as Tween 80), and disperse using a magnetic stirrer at room temperature for 4 hours to ensure uniform dispersion;

[0084] The electrospinning was carried out using a uniaxial electrospinning device, and the parameters were as follows: voltage, 15 kV; flow rate, 0.8 mL / h; collection distance, 18 cm; collection plate, flat collector covered with aluminum foil; spinning time, 4 hours; ambient temperature, 25°C; relative humidity, 40%; immediately after spinning, the fiber membrane was frozen in a -20°C refrigerator for 1 hour;

[0085] The collected fiber membranes were dried in a vacuum drying oven at 37 °C for 12 h to remove the residual solvent;

[0086] Subsequently, the electrospun membrane was rolled into a neural scaffold with a wall thickness of 0.7 mm and a length of 1.0 cm using a tubular mold with an inner diameter of 2 mm.

[0087] Verification Example 1

[0088] 1. Experimental methods:

[0089] The preparation method of Example 1 was used to prepare a neural scaffold, and neural scaffold samples were taken, each containing 10 mg DNSP-11;

[0090] The sample was immersed in phosphate buffered saline (PBS, pH = 7.4) at 37 °C, with a total volume of 50 mL;

[0091] At predetermined time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h), 1 mL of supernatant was removed and supplemented with 1 mL of fresh PBS;

[0092] The concentration of DNSP-11 in the supernatant was determined using high performance liquid chromatography (HPLC);

[0093] Calculate the cumulative release percentage;

[0094] The experiment was repeated 3 times and the average value was taken.

[0095] 2. The experimental data are shown in Table 1 below:

[0096] Table 1

[0097] Time (h) Cumulative release percentage (%) 0.5 5.2±0.3 1 8.7±0.5 2 13.5±0.7 4 19.8±1.1 8 27.3±1.4 12 33.6±1.8 24 45.2±2.3 48 58.7±2.9 72 68.4±3.4 96 75.9±3.8 120 81.6±4.1 144 85.8±4.3 168 88.9±4.4

[0098] It can be seen that within the first 12 hours, about 33.6% of DNSP-11 was released, indicating an initial burst release phase. After 24 hours, the cumulative release reached 45.2%, indicating a sustained slow release. After 7 days (168 hours), the cumulative release reached 88.9%, close to complete release. The release curve showed a typical sustained release pattern: rapid release in the early stage and slow release in the later stage.

[0099] These data prove that the neural scaffold of the present invention can achieve a sustained release effect of the DNSP-11 polypeptide, providing sufficient therapeutic concentration in the initial stage and then maintaining a stable release rate, which is beneficial for long-term treatment.

[0100] Verification Example 2

[0101] 1. Sample:

[0102] a) PCL fibers directly loaded with DNSP-11 polypeptide (control group);

[0103] b) PCL fibers loaded with DNSP-11 polypeptide PEG microspheres (Example 1, experimental group).

[0104] 2. In vitro release experiment:

[0105] The two groups of samples were immersed in phosphate buffered saline (PBS, pH = 7.4) at 37°C;

[0106] Samples were taken at predetermined time points (0, 1, 3, 6, 12, 24, 48, 72, 96, 120, 168 hours); the concentration of released DNSP-11 was determined using high performance liquid chromatography (HPLC).

[0107] 3. Determination of peptide activity:

[0108] The bioactivity of the released DNSP-11 was evaluated using PC12 cell viability assay;

[0109] Experiments were performed at 0, 24, 72, and 168 h time points.

[0110] 4. Peptide stability analysis:

[0111] The structural integrity of the released DNSP-11 was analyzed using mass spectrometry (MS);

[0112] Measurements were performed at 0, 72, and 168 hours.

[0113] 5. Experimental results (Table 2-4)

[0114] Table 2 Cumulative release percentage

[0115] Time (h) Direct load (%) PEG microsphere loading (%) 0 0 0 1 15.2±1.8 8.7±1.2 3 28.6±2.5 16.3±1.7 6 39.4±3.1 23.5±2.1 12 52.7±3.8 32.6±2.6 24 68.3±4.2 44.2±3.1 48 79.5±4.7 57.8±3.5 72 86.2±5.1 68.4±3.9 96 90.8±5.3 76.9±4.2 120 93.7±5.5 83.6±4.5 168 95.9±5.6 90.9±4.8

[0116] Table 3 PC12 cell survival rate

[0117]

[0118] Table 4 Peptide structural integrity

[0119] Time (h) Direct load (%) PEG microsphere loading (%) 0 100±2 100±2 72 76±5 91±3 168 58±7 84±4

[0120] The above data shows that:

[0121] 1) The PEG microsphere-loaded group had a slower release rate, which was beneficial for long-term administration.

[0122] 2) The PEG microsphere-loaded group maintained a higher neuronal cell survival rate at each time point, indicating that it retained better biological activity.

[0123] 3) The structural integrity of DNSP-11 in the PEG microsphere-loaded group was better, indicating that this method can better protect the peptide from degradation.

[0124] These results demonstrate that the method of loading DNSP-11 peptides via PEG microspheres can improve the bioavailability and activity retention of the peptides, which has obvious advantages over the method of directly loading the peptides.

[0125] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres, characterized in that: The steps include: Step 1: synthesize the DNSP-11 polypeptide sequence on RinkAmide MBHA resin using solid phase synthesis, and cut the polypeptide using a TFA / water / triisopropylsilane mixture; then purify it by HPLC using a C18 reverse phase chromatography column, and characterize it by MALDI-TOF mass spectrometry and analytical HPLC; then dissolve the purified DNSP-11 polypeptide in a sterile citric acid buffer at a pH of 4.5-5.0 to prepare a solution with a concentration of 1-2 mg / mL for standby use; Step 2, dissolving PEG with a molecular weight of 4000-500Da in dichloromethane to prepare a solution with a concentration of 8-10w / v%; then adding 0.5-1w / v% polyvinyl alcohol solution to form a PEG emulsion; The DNSP-11 polypeptide solution obtained in step 1 is added to the PEG emulsion, and 0.3-0.5 v / v% glutaraldehyde solution is used as a cross-linking agent for hardening. After the reaction is completed, the PEG microspheres are washed and collected; Step 3, dissolving polycaprolactone in a mixed solvent of chloroform and methanol to prepare a PCL solution with a concentration of 8-10 w / v%; then dispersing the PEG microspheres obtained in step 2 in the PCL solution, and the mass ratio of the PEG microspheres to the PCL solution is 1:(8-10); Then, 0.07-0.1 w / v% of a nonionic surfactant is added; a fiber membrane is prepared by an electrospinning method, and the fiber membrane is frozen and dried, and then rolled into a neural scaffold.

2. The preparation method according to claim 1, characterized in that: The amino acid sequence of the DNSP-11 polypeptide is shown in SEQ ID NO: 1, which is PPEAPAEDRSL.

3. The preparation method according to claim 1, characterized in that: In the step 2, the PEG microspheres are prepared by using a high shear mixer and stirring at 8000-10000 rpm for 10-15 minutes.

4. The preparation method according to claim 1, characterized in that: In the step 3, the electrospinning parameters are: voltage, 15 kV; flow rate, 0.8 mL / h; collection distance, 18 cm; spinning time, 4 hours; ambient temperature, 25° C.; and relative humidity, 40%.

5. The preparation method according to claim 1, characterized in that: The dimensions of the nerve stent are: wall thickness 0.7-0.9 mm, length 1.0-1.2 cm.

6. A polycaprolactone neural scaffold loaded with DNSP-11 polypeptide PEG microspheres prepared by the preparation method according to any one of claims 1 to 5.

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