Preparation method and application of metformin grafted polycaprolactone nanoscaffold

Abstract

The application discloses a preparation method and application of a metformin grafted polycaprolactone nanoscaffold, and the polycaprolactone nanofiber membrane is coated with a layer of polydopamine in a weak alkaline Tri-HCl buffer solution, is blended with metformin in the weak alkaline Tri-HCl buffer solution, and is incubated on a shaking table at 37 DEG C, so that a product is obtained; after washing and freeze-drying, the metformin grafted polycaprolactone nanoscaffold is obtained. The METG-PCLN developed in the application can continuously release MET into the fibrotic microenvironment in sensory neurons and EFs. In vitro cell experiments and animal model experiments prove that the METG-PCLN can significantly slow down the development of EFs by finely adjusting the release of sensory nerve CGRP. Therefore, the application provides a promising proof of concept for the potential clinical treatment of EFs, and shows considerable conversion value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a preparation method of metformin grafted polycaprolactone nanoscaffold and application of the metformin grafted polycaprolactone nanoscaffold in preparation of a medicine for preventing or treating epidural fibrosis, and further provides a medicine composition for preventing or treating epidural fibrosis. BACKGROUND

[0002] Organ fibrosis is a complex process that can lead to serious clinical conditions such as liver cirrhosis, kidney fibrosis, cardiac fibrosis and epidural fibrosis (EF), which severely damages organ function and can even be life-threatening. Despite extensive research, the underlying mechanisms of fibrosis are still not fully understood. Existing research shows that EF is characterized by scar tissue formation, mainly derived from excessive proliferation of fibroblasts, and is associated with decreased tissue cell density and increased extracellular matrix components such as collagen 1 (Col1), fibronectin and dermatan sulfate. Recent studies have highlighted the involvement of sensory neural networks originating from the dorsal root ganglion (DRG) in fibrosis. Calcitonin gene-related peptide (CGRP) positive nerves are ubiquitous in adhesions, which represent primary sensory nerves, and after neuronal depolarization, CGRP is released from nerve terminals and mediates various biological functions, which suggests that sensory nerves play a role in the fibrosis process. In addition, sensory axons innervating muscle help maintain muscle integrity, thereby reducing fibrosis and collagenization. CGRP has been shown to reduce myocardial fibrosis, so it is reasonable to hypothesize that sensory nerves can utilize CGRP to exert a potential anti-fibrotic effect. To date, no studies have confirmed the role of sensory nerves in regulating EF progression.

[0003] Laminectomy is a common spinal surgery performed to relieve pressure on the spinal cord and nerve roots caused by conditions such as herniated discs and spinal stenosis. However, approximately 8-40% of patients experience failed back surgery syndrome (FBSS) after this procedure, which is mainly attributed to epidural fibrosis (EF). FBSS is mainly characterized by persistent low back or leg pain caused by EF compressing the dura mater or nerve roots. About 4-9% of patients require revision surgery, which is to expose the same surgical area again. Due to the lack of bony structures as markers, these revision surgeries face more technical challenges and are at higher risk due to the increased likelihood of dural tear and nerve root lesions caused by fibrous adhesions. Despite significant advances in surgical techniques and equipment, symptomatic fibrotic revision surgeries often result in poor clinical outcomes. Currently, surgeons cannot predict which patients will develop symptomatic EF, and once it occurs, there is no effective treatment. To avoid EF, preventive measures should be implemented. However, due to limited understanding of its underlying mechanisms, there is currently no effective treatment. Our study first found that sensory nerve-derived CGRP can inhibit the progression of EF. This finding suggests that sensory nerves could become a new target for preventing EF, but specific strategies targeting this process have not yet been developed.

[0004] Currently, many strategies have been employed to prevent EF and reduce dural adhesions, including local or systemic administration and physical barriers based on biomaterials. In recent years, nanofibrous polymeric materials such as polycaprolactone (PCL) and polylactide (PLA) have been widely used in tissue engineering due to their excellent printability, adjustable mechanical properties, and biodegradability. In particular, PCL, due to its high porosity and large surface-to-volume ratio, ensures long-term stable release of drugs. In addition, PCL degradation can be customized according to the service life of the loaded drugs, thus eliminating the need to remove the device. These characteristics make PCL an ideal choice for physical barriers and drug delivery systems.

[0005] Considering the enormous costs associated with new drug development, repurposing existing drugs is a viable alternative. Preliminary validation found that metformin (MET) significantly promotes DRG cell secretion of CGRP at both mRNA and protein levels. MET is a widely used drug for the treatment of type 2 diabetes associated with obesity and has also shown analgesic effects in neuropathic, inflammatory, and visceral pain. It is well-tolerated, and new applications are emerging. However, if MET is used directly locally, the short duration of drug action will significantly weaken its therapeutic effect, so appropriate local administration methods need to be adopted to achieve local continuous drug delivery and fully exert the effect of MET. SUMMARY

[0006] The present application aims at the above-mentioned problems, and provides a metformin grafted PCL nanoscaffold (METG-PCLN), which is constructed by using a copolymer grafting technology, so as to have the functions of slow release of MET, physical barrier performance, biocompatibility and biodegradability. The innovative method targets sensory nerve regulation fibrogenesis, and can solve the EF progression.

[0007] The technical scheme of the present application is as follows: after a polydopamine layer is coated on the surface of the polycaprolactone nanofiber membrane in a weak alkaline Tri-HCl buffer, the polycaprolactone nanofiber membrane is blended with metformin in the weak alkaline Tri-HCl buffer and incubated on a shaking table at 37 DEG C, and the product is washed and freeze-dried to obtain a metformin grafted polycaprolactone nanoscaffold.

[0008] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0009] In the first aspect of the present application, a preparation method of a metformin grafted polycaprolactone nanoscaffold is provided, comprising the following steps:

[0010] A. Preparation of polycaprolactone nanofiber membrane

[0011] A polycaprolactone solution with a final concentration of 10% to 20% is electrospun under the voltage conditions of-4kv and +12kv to form a polycaprolactone nanofiber membrane. Preferably, the final concentration of the polycaprolactone solution is 18%, the distance between the spinning nozzle and the nanofiber membrane receiving plate is 15cm, the outflow speed of the spinning solution is 0.05 to 0.3mm / min, and preferably 0.15mm / min.

[0012] B. Polydopamine coating

[0013] The polycaprolactone nanofiber membrane is placed in a 10mM, pH=8.5 Tri-HCl buffer, dopamine hydrochloride is added, and then incubated in a 37 DEG C shaker at 120rpm / min for 4-24h (preferably 15h), so as to cover a layer of polydopamine on the surface of the PCL nanofiber;

[0014] Preferably, the shape of the polycaprolactone nanofiber membrane is rectangular or square; the mass ratio between the polycaprolactone nanofiber membrane and dopamine hydrochloride is 2.5:1, and the mass-volume ratio between the polycaprolactone nanofiber membrane and the Tri-HCl buffer is 5:1.

[0015] C. Metformin grafting

[0016] The PCL coated with polydopamine is washed with deionized water for 3 times, then 10 mM, pH=0.5 Tri-HCl buffer and metformin are added, and the obtained product is incubated at 37 DEG C in a shaking table at 100 rpm / min for 12-48 h (preferably 24 h), and then the obtained product is washed with deionized water for 3 times, and then freeze-dried to obtain the metformin grafted polycaprolactone nanoscaffold.

[0017] Specifically, the volume of the Tri-HCl buffer is 60% of the volume of the Tri-HCl buffer in step B, and the mass of the metformin is 4% of the mass of the polycaprolactone nanofiber membrane before coating.

[0018] The freeze-drying conditions of the metformin grafted polycaprolactone nanoscaffold are as follows: cold trap temperature is -50 DEG C, and vacuum degree is less than 0.34 mbar.

[0019] In the second aspect of the present application, the metformin grafted polycaprolactone nanoscaffold prepared by the above method is provided.

[0020] In the third aspect of the present application, the application of the metformin grafted polycaprolactone nanoscaffold is provided, and specifically, the application in the preparation of a drug for preventing or treating epidural fibrosis.

[0021] Specifically, the drug for preventing or treating epidural fibrosis is a drug for promoting the secretion of calcitonin gene-related peptide of sensory nerves.

[0022] In the fourth aspect of the present application, a drug composition for preventing or treating epidural fibrosis is provided, which comprises an active ingredient and a pharmaceutically acceptable adjuvant, wherein the active ingredient is the metformin grafted polycaprolactone nanoscaffold.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] In the preparation aspect, the raw materials polycaprolactone, dopamine hydrochloride and metformin used in the present application all belong to common materials in the medical field and can be obtained by purchase; meanwhile, the preparation process condition is mild and simple to master, which is helpful for industrialized popularization and application. Experimental results show that after metformin grafting, the average fiber diameter, mechanical property and hydrophilic property of the PCL fiber are not affected, but the release of MET can be effectively delayed, and 80% of the MET is released only at the 6th week after administration.

[0025] In the technical effect aspect, the METG-PCLN developed in the present application can release MET into the fibrotic microenvironment in sensory neurons and EFs. Both in vitro cell experiments and animal model experiments prove that the METG-PCLN can significantly slow down the development of EF by finely regulating the release of CGRP of sensory nerves.

[0026] Thus, the present application provides a promising proof-of-concept for the potential clinical treatment of EF and shows considerable translational value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Process schematic for the preparation of MET grafted PCL nanofibers for Example 1 ;

[0028] Figure 2 SEM images of PCL, MET-PCLN (MET and PCL blend), METG-PCLN (MET grafted PCL) nanofibers for Example 1 ;

[0029] Figure 3 Diameter distribution of PCL, MET-PCLN, METG-PCLN nanofibers for Example 1 ;

[0030] Figure 4 Mechanical curves (A), Modulus (B) and Tensile stress (C) of PCL, MET-PCLN, METG-PCLN nanofibers for Example 1 ;

[0031] Figure 5 Water contact angle images of MET-PCLN, METG-PCLN nanofibers for Example 1 ;

[0032] Figure 6 Percentage of MET released from MET-PCLN and METG-PCLN nanofibers for Example 1 ;

[0033] Figure 7 Schematic of transwell co-culture system for Example 2;

[0034] Figure 8 Expression of a-SMA and Coll was significantly reduced in MET-PCLN and METG-PCLN groups in Example 2;

[0035] Figure 9 Schematic of metformin grafted polycaprolactone nanofiber scaffolds implanted in mice in Example 3;

[0036] Figure 10 MET release results after 4 and 8 weeks of metformin grafted polycaprolactone nanofiber scaffolds implanted in mice in Example 3;

[0037] Figure 11 Metformin grafted polycaprolactone nanofiber scaffolds significantly promoted DRG release of CGRP in Example 3;

[0038] Figure 12 NMR suggests that metformin grafted polycaprolactone nanofiber scaffolds significantly inhibited the progression of epidural fibrosis;

[0039] Figure 13 The metformin grafted polycaprolactone nanofiber scaffold significantly inhibits peridural fibrosis as indicated by HE staining. DETAILED DESCRIPTION

[0040] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the described embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without any inventive effort fall within the protection scope of the present application.

[0041] The ranges disclosed herein are presented in terms of both a lower limit and an upper limit. Each lower limit can have one or more upper limits, and each upper limit can have one or more lower limits. Ranges created by the selection of a lower limit and an upper limit are inclusive and combinable, that is, any lower limit can be combined with any upper limit to form a range. For example, the ranges 100-140 and 500-900 are presented for a particular parameter, it is understood that the ranges 100-140 and 500-900 are also contemplated. Furthermore, if a minimum range value of 1 and 2 is presented, and if a maximum range value of 3, 4, and 5 is presented, then the following ranges are all contemplated: 1-2, 1-4, 1-5, 2-3, 2-4, and 2-5.

[0042] In the present application, unless otherwise specified, the numerical range "a-b" represents a shorthand for any real combination of numbers between a and b, where a and b are both real numbers. For example, the numerical range "0-5" represents that all real numbers between "0-5" have been listed herein, and "0-5" is just a shorthand for these combinations of numbers.

[0043] In the present application, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined with each other to form new technical solutions.

[0044] Example 1 Preparation and detection of metformin grafted polycaprolactone nanofiber scaffold

[0045] I. Preparation method

[0046] The synthesis process is shown in Figure 1 The specific preparation method is as follows:

[0047] PCL was dissolved to a final concentration of 18%, and the PCL solution was transferred into a 10 ml syringe with a 21G needle for electrospinning; the distance between the nozzle and the receiving plate was 15 cm, the voltage was -4kv and +12kv, and the injection speed was 0.15 mm / min; then the obtained nanofiber membrane was cut into a rectangle (10*5mm).

[0048] 539mg of PCL rectangle was placed in 108ml of Tri-HCl buffer (10mM, pH=8.5), 215.6mg of dopamine (DA) hydrochloride was added, and then incubated in a 37℃ shaker at 120rpm / min for 15h to cover the surface of the PCL nanofiber with a layer of polydopamine.

[0049] The polydopamine-coated PCL was washed with deionized water 3 times, then 65ml of Tri-HCl buffer (10mM pH=0.5) and 22mg of metformin (MET) were added, and incubated in a 37℃ shaker at 100rpm / min for 24h. The resulting product was washed with deionized water 3 times, and freeze-dried to obtain metformin-grafted PCL nanofiber.

[0050] II. Performance test

[0051] Pure PCL and MET-blended PCL electrospun fibers were used as controls. To obtain MET-blended nanofibers, 1% (w / v) of MET was added to the PCL solution, and electrospinning was carried out under the same conditions.

[0052] The pure PCL, MET-blended PCL and MET-grafted PCL nanofibers were respectively named PCL, MET-PCLN or METG-PCLN, and the following tests were carried out: electron microscopy (SEM, FEI Quanta 200, Netherlands) was used to observe the nanofibers; Image J software was used to measure and calculate the fiber diameter distribution; the tensile sample was processed into a 20mm*10mm size sample, and tensile test was carried out on a universal testing machine (Instron 5943, Instron, USA); the release kinetics of MET was studied in a constant temperature shaking water bath (37℃, Jiangsu Taichang Medical Instrument Co., Ltd.) at a frequency of 100rpm / min; at predetermined time points, an equal amount of 5mL release buffer was taken, washed with 5mL PBS solution, and the release of MET was determined by ultraviolet-2550 spectrophotometer (Japan Shimadzu).

[0053] III. Experimental results

[0054] Compared with electrospun PCL fibers made from pure PCL and MET blends, metformin-grafted PCL nanofibers exhibit a dense, random nanofiber configuration. Figure 2 The average fiber diameters of PCL, MET-PCLN, and METG-PCLN were 392.8±248.8 nm, 317.4±272.7 nm, and 331.6±231.3 nm, respectively. Figure 3 The effect of MET composition on the mechanical properties of PCL / MET nanofibers was evaluated using stress-strain measurements. The results showed that the grafted drug had little effect on the mechanical properties of the material, and the mechanical curves, moduli, and tensile stresses of the three types of nanofibers were not significantly different. Figure 4 Contact angle testing showed that the water contact angles of MET-PCLN and METG-PCLN were 66.3°±0.9° and 64.8°±1.1°, respectively, indicating that the grafted materials are hydrophilic. Figure 5 These results indicate that the incorporation of MET into PCL nanofibers via blending or covalent grafting has no effect on the electrospinning process.

[0055] The cumulative release of MET from MET-PCLN and METG-PCLN nanofibers was investigated by incubation in a release medium. Results showed that MET-PCLN released approximately 80% of its drug load within 4 weeks; while METG-PCLN experienced a burst release of approximately 50% of the drug in week 1 and approximately 80% in week 6. Figure 6 This indicates that METG-PCLN can delay the release of MET.

[0056] Example 2: Metformin-grafted polycaprolactone nanofiber scaffold supports sensory nerve function and inhibits fibroblast activation. I. Experimental Methods

[0057] To further investigate whether metformin can promote the secretion of CGRP by dorsal root ganglion (DRG) cells, thereby inhibiting H2O2-induced fibroblast activation, a transwell co-culture system was used. Figure 7 The cells were cultured with fibroblasts in the upper layer and DRG cells in the lower layer, and treated with the corresponding drugs. The CON group received no intervention, while the PCL, MET-PCLN, and METG-PCN groups received the corresponding nanofiber materials.

[0058] II. Experimental Results

[0059] Western blot results showed that, compared with the control group and the PCL group, the expression of α-SMA and Col1 was significantly reduced in the MET-PCLN group and the METG-PCLN group. Figure 8 This indicates that fibroblast activation is inhibited.

[0060] Example 3 Metformin grafted poly-caprolactone nanofiber scaffolds promote sensory nerve secretion of anti-fibrosis neuropeptide CGRP to inhibit peridural fibrosis

[0061] I. Experimental methods

[0062] Sprague-Dawley (SD) female rats, 8 weeks old, were selected for laminectomy surgery. According to different postoperative treatments, the rats were divided into four groups: surgery control (CON) group, PCL group, MET-PCLN group and METG-PCLN group. The surgery control group was not treated with any drug and material, and was directly sutured in sequence. The PCL, MET-PCLN and METG-PCLN groups were treated according to the schematic diagram shown in the figure. After laminectomy, the nanofiber was precisely implanted on the surface of the spinal dura mater, and then the incision was sutured. Figure 9

[0063] II. Experimental results

[0064] Evaluation of the therapeutic effect of metformin grafted poly-caprolactone nanofiber scaffolds by targeting DRGs: At 4 and 8 weeks after surgery, the concentration of MET in DRGs in the MET-PCLN and METG-PCLN groups was detected by LC-MS / MS. The results showed that at 4 weeks, the concentration of MET in DRGs in the two groups was similar, and at 8 weeks, the concentration of MET in the MET-PCLN group was significantly higher than that in the MET-PCLN group ( Figure 10 ), confirming that PCL grafted with metformin has a significant drug release effect.

[0065] The immunofluorescence results of DRGs showed that the CGRP + neurons in the MET-PCLN and METG-PCLN groups were more than those in the control and PCL groups ( Figure 11 ).NMR and HE staining showed that metformin grafted poly-caprolactone nanofiber scaffolds significantly inhibited peridural fibrosis ( Figure 12 and Figure 13 ).

[0066] The above has specifically described the preferred embodiments of the present application, but the present application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or replacements without deviating from the spirit of the present application. These equivalent modifications or replacements are all included in the scope defined by the claims of the present application.

Claims

1. A method for preparing metformin grafted poly-caprolactone nanoscaffold, characterized in that, It comprises the following steps: after the polycaprolactone nanofiber membrane is coated with a layer of polydopamine in weak alkaline Tri-HCl buffer, it is blended with metformin in weak alkaline Tri-HCl buffer and incubated at 37 DEG C on a shaking table, and the product is washed and freeze-dried to obtain a metformin grafted polycaprolactone nanoscaffold, and the specific steps are as follows: A, preparation of polycaprolactone nanofiber membrane The polycaprolactone solution with a final concentration of 10% to 20% is electrospun into a polycaprolactone nanofiber membrane under the voltage condition of-4kv and +12kv; B, polydopamine coating The polycaprolactone nanofiber membrane is placed in 10mM, pH=8.5 Tri-HCl buffer, dopamine hydrochloride is added, and then incubated in a 37 DEG C shaker at 120rpm / min for 4-24h to cover the surface of the PCL nanofiber with a layer of polydopamine; C, metformin grafting The polydopamine-coated polycaprolactone nanofiber membrane is washed with deionized water several times, then 10mM, pH=8.5 Tri-HCl buffer and metformin are added, and incubated at 37 DEG C on a shaking table at 100rpm / min for 12-48h, then the obtained product is washed with deionized water several times, and freeze-dried to obtain a metformin grafted polycaprolactone nanoscaffold.

2. The preparation method of the metformin grafted polycaprolactone nanoscaffold according to claim 1, characterized in that: wherein In step A, the final concentration of the polycaprolactone solution is 18%, the distance between the spinning nozzle and the nanofiber membrane receiving plate is 15cm, and the outflow speed of the spinning solution is 0.05-0.3mm / min.

3. The preparation method of the metformin grafted polycaprolactone nanoscaffold according to claim 1, characterized in that: wherein, In step B, the shape of the polycaprolactone nanofiber membrane is rectangular or square; The mass ratio between the polycaprolactone nanofiber membrane and dopamine hydrochloride is 2.5:1, and the mass-volume ratio between the polycaprolactone nanofiber membrane and Tri-HCl buffer is 5:1, The co-incubation time of the polycaprolactone nanofiber membrane and dopamine hydrochloride under shaking condition is 15h.

4. The preparation method of the metformin grafted polycaprolactone nanoscaffold according to claim 1, characterized in that: wherein In step C, the volume of Tri-HCl buffer is 60% of the volume of Tri-HCl buffer in step B, and the mass of metformin is 4% of the mass of the polycaprolactone nanofiber membrane before coating, The co-incubation time of the polydopamine-coated polycaprolactone nanofiber membrane and metformin under shaking condition is 24h.

5. The preparation method of the metformin grafted polycaprolactone nanoscaffold according to claim 1, characterized in that: wherein In step C, after washing with deionized water for 3 times, the freeze-drying conditions of the metformin grafted polycaprolactone nanoscaffold are: cold trap temperature-50 DEG C, vacuum degree less than 0.34mbar.

6. A metformin grafted poly-caprolactone nanoscaffold characterized in that, Prepared by the method of any one of claims 1-5.

7. The use of the metformin grafted polycaprolactone nanoscaffold of claim 6 in the preparation of a drug for preventing or treating epidural fibrosis.

8. The use according to claim 7, characterized in that: wherein, The drug for preventing or treating epidural fibrosis is a drug for promoting sensory nerves to secrete calcitonin gene-related peptide.

9. A pharmaceutical composition for preventing or treating epidural fibrosis, characterized by, The active component is the metformin grafted polycaprolactone nanoscaffold of claim 6, and the pharmaceutically acceptable adjuvant.

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