Core-shell PVP-LbGp / PCL electrospun nanofiber as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug delivery systems, and particularly relates to a core-shell PVP-LbGp / PCL electrospun nanofiber as well as a preparation method and application thereof. Experimental results show that the core-shell PVP-LbGp / PCL electrospun nanofiber with the lycium barbarum glycopeptide (LbGp) loading function is successfully constructed by the aid of the core-shell PVP-LbGp / PCL electrospun nanofiber. The LbGp-loaded nanofiber has typical dual-phase drug release characteristics of rapid release in the initial stage and slow and continuous release in the later stage. The nanofibers maintain the phenotype and skeleton form of retinal pigment epithelial cells (RPE), and inhibit the excessive proliferation of two RPE cells derived from iPSCs of normal and diabetic patients. And good biocompatibility is achieved. The invention proves the feasibility of the core-shell PVP-LbGp / PCL electrospun nanofiber as a future promising DDSs, and the core-shell PVP-LbGp / PCL electrospun nanofiber can effectively carry and release LbGp, and is used for tissue engineering RPE cell regeneration and treatment of retina degenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery systems, and in particular relates to a core-shell PVP-LbGp / PCL electrospun nanofiber and a preparation method and use thereof. Background Art

[0002] Retinal pigment epithelial cells (RPE), a layer of cells that perform multiple functions at the back of the eye, are a promising target for the prevention and treatment of a variety of clinical diseases, including proliferative diabetes retinopathy (PDR), age-related macular degeneration (AMD), choroidal neovascularization (CNV), retinitis pigmentosa (RP), and Leber's congenital amaurosis (LCA). In recent decades, nano-drug delivery platforms and tissue-engineered RPE have been widely used to treat RPE-related diseases.

[0003] Lycium barbarum glycopeptide (LbGp) is a glycoconjugate and is considered to be the most promising monomer component in wolfberry fruit. It includes LbGp1 (88kDa), LbGp2 (68.2kDa), LbGp3 (92.5kDa), LbGp4 (214.8kDa), and LbGp5 (23.7kDa). It is mainly composed of sugar chains and covalently bound to polypeptides. Its molecular weight is 88kDa, and its monosaccharide composition is arabinose (Ara), galactose (Gal) and glucose (Glc), with a molar ratio of 2.5:1.0:1.0. Its protein content is 30%, and the linkage between the sugar group and the core protein skeleton is O-linked. It also contains 18 other natural amino acids, which can ensure the quantification and reproducibility of experimental drugs. Studies have shown that LbGp has the effects of lowering blood sugar, lowering blood lipids, reducing depression and anxiety behaviors, and preventing the occurrence and progression of acute colitis. In ophthalmology, it can promote the survival of photoreceptors and protect the optic nerve through anti-oxidation, anti-inflammation and anti-apoptosis (programmed cell death) mechanisms. However, the effect of LbGp encapsulated in nanofibers and sustained release on RPE has not been studied.

[0004] Through intravitreal drug delivery systems (DDSs), drugs can be delivered directly into the vitreous, preventing frequent injections. Therefore, intravitreal implant devices may replace intravitreal injections and become a new means of treating posterior segment diseases. For an efficient DDSs, some key issues need to be considered: high drug loading and encapsulation efficiency, simultaneous drug delivery, drug release time control, cost and operation friendliness. Compared with other DDSs, the electrospinning strategy allows the selection of materials and treatment methods from a wide range. In addition, it allows the manipulation of drug release rate by modifying the degradation rate of the fiber. It can also be easily placed in the defect area, making drug delivery easier than other methods.

[0005] Therefore, using coaxial electrospinning technology, natural product LbGp was selected as a model drug, polyvinylpyrrolidone (PVP) was selected as the spinning solution for the core, and polycaprolactone (PCL) was selected as the spinning solution for the shell to jointly construct drug-loaded core-shell nanofibers. Nanofibers were used to realize new DDSs for retinal diseases and provide the possibility for effective drug release. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art, a core-shell PVP-LbGp / PCL electrospun nanofiber and its preparation method and use. The present invention uses core-shell PVP-LbGp / PCL electrospun nanofibers to develop a new type of DDSs, which provides the possibility of delivering drugs into the vitreous body through a controlled release method to treat posterior segment diseases.

[0007] Specifically, the present invention is realized through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing core-shell PVP-LbGp / PCL electrospun nanofibers, the preparation method comprising the following steps:

[0009] (1) Preparation of shell spinning solution: 0.1-5 g of PCL was placed in an autoclaved glass conical flask, and then 5-10 mL of a mixed solvent of chloroform and N,N-dimethylformamide was added to dissolve it. After the mouth of the conical flask was sealed, it was stirred for 5-20 hours to prepare a shell spinning solution;

[0010] (2) Preparation of LbGp solution: 0.05-0.15 g of LbGp was placed in an autoclaved glass conical bottle, dissolved in 20-50 mL of N,N-dimethylformamide, the bottle mouth was sealed, and then 10-40 mL of anhydrous ethanol was added to prepare a LbGp solution;

[0011] (3) Preparation of a core layer spinning solution containing LbGp: dissolving an appropriate amount of PVP powder in the LbGp solution prepared in step (2), stirring at room temperature for 5-20 hours, and preparing a core layer spinning solution containing LbGp;

[0012] (4) Preparation of core-shell PVP-LbGp / PCL electrospun nanofibers: The shell layer spinning solution prepared in step (1) and the core layer spinning solution containing LbGp prepared in step (3) are respectively encapsulated into syringes, and the syringes are connected to the coaxial spinning needles with polytetrafluoroethylene pipes to perform coaxial electrospinning to prepare core-shell PVP-LbGp / PCL electrospun nanofibers.

[0013] As an optional manner, in the above preparation method, in step (1), 1-2 g of PCL is placed in a high-pressure sterilized glass conical flask, and then a mixed solvent of 6-8 mL of chloroform and N,N-dimethylformamide is added to dissolve it. After the mouth of the conical flask is sealed, it is stirred for 10-15 hours to prepare a shell spinning solution with a mass volume concentration of 10-15%.

[0014] Preferably, in step (1), 1.32 g of PCL is placed in a high-pressure sterilized glass conical flask, and then a mixed solvent of 7 mL of chloroform and N,N-dimethylformamide is added to dissolve it. After the mouth of the conical flask is sealed, it is stirred for 12 hours to prepare a shell spinning solution with a mass volume concentration of 12%.

[0015] As an optional manner, in the above preparation method, in step (2), 0.08-0.10 g of LbGp is ​​placed in a high-pressure sterilized glass conical bottle, dissolved in 20-40 mL of N,N-dimethylformamide, the bottle mouth is sealed, and ultrasonic oscillation is performed in an ultrasonic cleaner for 20 minutes, and then placed on a magnetic stirrer and stirred for 12 hours, and then 20-40 mL of anhydrous ethanol is added to prepare a 500-1500 μg / mL LbGp solution.

[0016] Preferably, in step (2), 0.09 g of LbGp is ​​placed in a high-pressure sterilized glass conical bottle, dissolved in 30 mL of N,N-dimethylformamide, the bottle mouth is sealed, ultrasonic oscillation is performed in an ultrasonic cleaner for 20 minutes, and then placed on a magnetic stirrer and stirred for 12 hours, followed by adding 30 mL of anhydrous ethanol to prepare a 1500 μg / mL LbGp solution.

[0017] Preferably, the remaining low concentration (1000 μg / mL, 500 μg / mL) LbGp solutions are prepared by diluting the 1500 μg / mL LbGp solution with a mixed solution of N,N-dimethylformamide and anhydrous ethanol (v:v=1:1).

[0018] As an optional manner, in the above preparation method, in step (3), an appropriate amount of PVP powder is dissolved in the LbGp solution prepared in step (2), and the mixture is stirred at room temperature for 10-15 hours to prepare a core layer spinning solution containing LbGp with a PVP mass concentration of 12-16%.

[0019] Preferably, in step (3), a proper amount of PVP powder is dissolved in the LbGp solution prepared in step (2), and the mixture is stirred at room temperature for 12 hours to prepare a core layer spinning solution containing LbGp with a PVP mass concentration of 15%.

[0020] As an optional manner, in the above preparation method, in step (4), the shell layer spinning solution prepared in step (1) and the core layer spinning solution containing LbGp prepared in step (3) are respectively encapsulated into a 20 mL syringe, and the syringe is connected to the coaxial spinning needle with a polytetrafluoroethylene pipe. The distance between the roller collector covered with tin foil and the tip of the coaxial needle is 10 cm, the flow rate of the core layer spinning solution is 0.5 mL / h, and the flow rate of the shell layer spinning solution is 1.5 mL / h. The entire spinning process is carried out under the following conditions: temperature: 25°C; humidity: 40%; voltage: 15 kV; X-axis scanning speed: 8 mm / s; drum collector speed: 1697~1701 rpm, and coaxial electrospinning is carried out to prepare core-shell PVP-LbGp / PCL electrospun nanofibers.

[0021] As an optional manner, in the above preparation method, in step (1), the volume ratio of chloroform to N,N-dimethylformamide is 1:1, in step (2), the volume ratio of N,N-dimethylformamide to anhydrous ethanol is 1:1, and in step (4), the prepared electrospun nanofibers are dried in a vacuum drying oven for 72 hours and then stored in a vacuum desiccator.

[0022] In a second aspect, the present invention provides core-shell PVP-LbGp / PCL electrospun nanofibers prepared by the preparation method described in the first aspect.

[0023] Preferably, the diameter of the core-shell PVP-LbGp / PCL electrospun nanofibers is distributed between 330 and 580 nm.

[0024] Preferably, the core-shell PVP-LbGp / PCL electrospun nanofibers exhibit typical biphasic drug release characteristics: rapid release in the early stage and slow and sustained release in the later stage.

[0025] Preferably, the core-shell PVP-LbGp / PCL electrospun nanofibers inhibit the activity of RPE cells.

[0026] In a third aspect, the present invention provides use of the core-shell PVP-LbGp / PCL electrospun nanofibers described in the second aspect in the preparation of a drug for treating retinal degenerative diseases.

[0027] As an option, in the above use, the retinal degenerative disease is selected from one or more of the following: proliferative diabetic retinopathy, age-related macular degeneration, choroidal neovascularization, retinitis pigmentosa or Leber congenital amaurosis.

[0028] In a fourth aspect, the present invention provides use of the core-shell PVP-LbGp / PCL electrospun nanofibers described in the second aspect in the construction of bionic RPE tissue engineering.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention successfully constructs PVP-LbGp / PCL electrospun nanofibers loaded with LbGp. The LbGp-loaded nanofibers exhibit typical biphasic drug release characteristics: rapid release in the early stage and slow and continuous release in the later stage. The nanofibers maintain the activity and skeleton morphology of RPE cells and have good biocompatibility. LbGp inhibits the excessive proliferation of two types of RPE cells derived from iPSCs of normal and diabetic patients. The present invention confirms the feasibility of core-shell PVP-LbGp / PCL electrospun nanofibers as promising DDSs in the future, which can effectively carry and release LbGp for tissue engineering RPE cell regeneration and the treatment of retinal degenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Schematic diagram of the coaxial electrospinning setup and parameters used to fabricate LbGp-loaded PVP / PCL core-shell nanofibers.

[0032] Figure 2 : Flowchart of hiPSC differentiation into RPE.

[0033] Figure 3 : Laser confocal scanning image of coaxial nanofibers. Figure 3 A PCL shell containing 7-hydroxycoumarin-3-carboxylic acid exhibits green fluorescence. Figure 3 B contains the PVP core of Rose Red B, which shows red fluorescence. Figure 3 C is Figure 3 A and Figure 3 Merged image of B. Scale bar is 10 μm.

[0034] Figure 4 :SEM images of PVP / PCL coaxial electrospun nanofibers without or with different concentrations of LbGp ( Figure 4 A) and diameter relative frequency distribution diagram ( Figure 4 B).

[0035] Figure 5 : FTIR of PVP / PCL coaxial electrospun nanofibers without or loaded with different concentrations of LbGp shows the chemical bond between LbGp and nanofibers.

[0036] Figure 6 : TG curves of PVP / PCL coaxial electrospun nanofibers without or with different concentrations of LbGp ( Figure 6 A) and DSC curve ( Figure 6 B).

[0037] Figure 7 : Figure 7 A Schematic diagram of the water contact angle of PVP / PCL coaxial electrospun nanofibers without or loaded with different concentrations of LbGp. Figure 7 B. Statistical bar graph of water contact angle.

[0038] Figure 8 : Cumulative release percentage curve of PVP / PCL coaxial electrospun nanofibers loaded with different concentrations of LbGp.

[0039] Fig. 9 : Bright field images of Normal-hiPSCs-RPE cells after treatment with different concentrations of LbGp (A: Control; B: 250μg / mL; C: 500μg / mL; D: 1000μg / mL; E: 2000μg / mL; F: 4000μg / mL; G: 8000μg / mL) and CCK-8 method to detect cell activity (H). ** P < 0.01; **** P<0.001.

[0040] Fig.10 : Bright field images of DM-hiPSCs-RPE cells after treatment with different concentrations of LbGp (A: Control; B: 250μg / mL; C: 500μg / mL; D: 1000μg / mL; E: 2000μg / mL; F: 4000μg / mL; G: 8000μg / mL) and CCK-8 method to detect cell activity (H). * P < 0.05; *** P < 0.001; **** P<0.0001.

[0041] Fig.11: Effect of PVP / PCL coaxial electrospun nanofibers loaded with different concentrations of LbGp on the cell activity of N-hiPSC-RPE and DM-hiPSCs-RPE. Fig.11 A: N-hiPSC-RPE; Fig.11 B: DM-hiPSCs-RPE. **** p<0.0001.

[0042] Fig.12 :Flow cytometry detection of RPE cell cycle. Fig.12 A Flow cytometry detection of the cycle of N-hiPSC-RPE. (a, b, c, d) The cycle of N-hiPSC-RPE after treatment with different concentrations of LbGp. (e) The bar graph counts the total number of N-hiPSC-RPE cells entering the G2 phase and S phase. Fig.12 B Flow cytometry detection of the cycle of DM-hiPSCs-RPE. (a, b, c, d) CCK-8 analysis of the cycle of DM-hiPSCs-RPE after treatment with different concentrations of LbGp. (e) Bar graph statistics of the total number of cells in DM-hiPSCs-RPE entering the G2 phase and S phase. a: 0μg / mL; b: 500μg / mL; c: 1000μg / mL; d: 1500μg / mL. . ( **** ),( *** ),( ** ),( * ) represent p<0.0001, p<0.001, p<0.01, p<0.05 respectively.

[0043] Fig.13 : F-actin / DAPI immunofluorescence staining of Normal-hiPSCs-RPE cultured on nanofibers loaded with different concentrations of LbGp. 0 :0μg / mL; M 1 :500μg / mL; M 2 :1000μg / mL; M 3 :1500μg / mL. Scale bar:100μm.

[0044] Fig.14 : F-actin / DAPI immunofluorescence staining of DM-hiPSCs-RPE cultured on nanofibers loaded with different concentrations of LbGp. 0 :0μg / mL; M 1 :500μg / mL; M 2 :1000μg / mL; M 3 :1500μg / mL. Scale bar:100μm. DETAILED DESCRIPTION

[0045] The present invention first selects a hydrophobic synthetic polymer PCL with biodegradability and biocompatibility as the spinning solution of the shell, selects a hydrophilic synthetic polymer PVP with biocompatibility, non-cytotoxicity, film-forming and adhesion properties as the spinning solution of the core, selects the extract component LbGp of the Chinese medicine wolfberry as the model drug of the nuclear layer PVP, and uses coaxial electrospinning technology to construct core-shell PVP-LbGp / PCL electrospun nanofibers, and preliminarily explains the effect of the nanofiber membrane of the PVP / PCL core-shell structure on the release of LbGp drugs. Then, Normal-hiPSCs-RPE and DM-hiPSCs-RPE are combined with PVP-LbGp / PCL electrospun nanofibers to construct a bionic tissue engineering RPE sheet, and after 7 days of culture, the adhesion, proliferation and morphological characteristics of the two RPEs are observed and analyzed. In short, the present invention provides an important reference for the construction of drug delivery and bionic RPE tissue engineering for retinal degenerative diseases.

[0046] Design: (1) Construction, performance analysis and drug release behavior of core-shell PVP-LbGp / PCL electrospun nanofibers. The coaxiality of the nanofibers was verified using a laser confocal microscope. The morphological characteristics of the nanofiber membrane were observed using a field emission scanning electron microscope (SEM). The composition and chemical interactions of the nanofibers loaded with different concentrations of LbGp were analyzed using Fourier transform infrared spectroscopy (FTIR). The thermal behavior of electrospun nanofibers loaded with different concentrations of LbGp was analyzed using differential scanning calorimetry (DSC) and thermogravimetry (TG). The changes in the hydrophilicity of coaxial electrospun nanofibers without or with different concentrations of LbGp were analyzed using the water contact angle (WCA). The spectral characteristics of LbGp were analyzed using a fluorescence spectrometer. The standard curve of LbGp solutions with known concentrations was determined using a UV-visible spectrophotometer. According to the standard curve of LbGp solution, the drug release behavior of electrospun nanofibers loaded with different concentrations of LbGp was further analyzed by UV-visible spectrophotometer. (2) Construction of biomimetic RPE tissue engineering and study of the interaction mechanism between cells and PVP-LbGp / PCL electrospun nanofibers. The CCK8 method was used to detect the activity and biocompatibility of LbGp and nanofiber membrane on the two RPEs. Flow cytometry was used to detect the cell cycle, and F-actin / DAPI immunofluorescence staining was used to observe the effect of nanofibers on the adhesion and skeleton formation of the two cells.

[0047] Results: (1) PVP-LbGp / PCL electrospun nanofibers with core-shell structure can be successfully constructed by coaxial electrospinning technology. The nanofibers can show uniform, randomly arranged, smooth surface morphology, and the diameter distribution is between 330 and 580 nm. When the concentration of LbGp is ​​less than 1000 μg / mL, as the drug concentration increases, the fiber diameter gradually decreases, and vice versa, the diameter begins to gradually increase. In the unloaded or drug-loaded nanofibers, the same is that PCL at 1160 cm -1 and PVP at 2945cm -1 and 1723cm -1 The characteristic peaks of infrared spectra appear at 3376 and 3399 cm -1 A characteristic broad peak appears. LbGp can be successfully loaded into the nanofiber membrane, and there is an exothermic effect. PVP / PCL core-shell coaxial electrospun nanofibers are hydrophilic, and the hydrophilicity increases with the loading of LbGp. LbGp can excite light, and the excitation peak of LbGp is ​​located at around 456nm. The ultraviolet absorbance of LbGp shows a good linear relationship with the mass concentration, and the correlation coefficient R 2 =0.99998. LbGp showed a typical biphasic drug release characteristic in the nanofibers, with rapid release in the early stage and slow and continuous release in the later stage. (2) CCK8 results suggest that when the concentration of LbGp is ​​between 500 and 1500 μg / mL, it has an inhibitory effect on the proliferation of both RPE and shows a certain concentration dependence. Nanofibers without or with different concentrations of LbGp have good biocompatibility for both cells, and the cells adhere well and maintain a regular hexagonal shape.

[0048] Conclusion: PVP-LbGp / PCL electrospun nanofibers loaded with LbGp can be successfully constructed. The LbGp-loaded nanofibers exhibit a typical biphasic drug release profile: rapid release in the early stage and slow sustained release in the later stage. The nanofibers can maintain the activity and skeletal morphology of the two types of RPE cells and have good biocompatibility. LbGp can inhibit the activity of the two types of cells. The present invention confirms the feasibility of core-shell PVP-LbGp / PCL electrospun nanofibers as promising DDSs in the future, which can effectively carry and release LbGp for the treatment of RPE tissue regeneration and retinal degenerative diseases.

[0049] The present invention will be further described with reference to specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0050] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0052] Example:

[0053] 1. Test materials

[0054] Test drug: Lycium Barbarum Glycopeptide (LbGp) powder was provided by Ningxia Tianren Lycium Biotechnology Co., Ltd. The preparation method of the raw material LbGp powder used in the present invention has obtained invention patent CN107021995B, which uses a heating flocculation method to remove some impurities, replacing the step of using a large amount of ethanol precipitation in the traditional extraction method, thereby improving production safety and the yield of the final glycopeptide product.

[0055] Experimental cells: Normal-hiPSCs were kindly donated by Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences; DM-hiPSCs were kindly donated by Hunan Aier Eye Research Institute.

[0056] 2. Test methods

[0057] 2.1 Flowchart of the construction of core-shell PVP-LbGp / PCL electrospun nanofibers

[0058] See also Figure 1 .

[0059] 2.2 Flowchart of hiPSC differentiation into RPE

[0060] See also Figure 2 .

[0061] 2.3 Preparation of drugs

[0062] (1) 1.32 g of PCL was placed in an autoclaved glass conical bottle, and then 7 mL of a mixed solvent of chloroform and N,N-dimethylformamide (v:v=1:1) was added to dissolve it. After the bottle mouth was sealed, it was stirred at room temperature for 12 h to prepare a solution with a concentration of 12% (w / v) as the shell spinning solution.

[0063] (2) Take 0.09g of LbGp and place it in a high-pressure sterilized glass conical bottle, dissolve it with 30mL of N,N-dimethylformamide, and seal the bottle mouth. In order to fully dissolve the drug, first use an ultrasonic cleaner to ultrasonically oscillate and crush it for 20 minutes, and then place it on a magnetic stirrer and stir for 12 hours. Then add 30mL of anhydrous ethanol to prepare a solution with an LbGp concentration of 1500μg / mL. On this basis, the remaining low-concentration (1000μg / mL, 500μg / mL) LbGp solutions are diluted with a mixed solution of N,N-dimethylformamide and anhydrous ethanol (v:v=1:1). The drug is dissolved in the core layer spinning solution for encapsulation to prepare different nanofiber membranes (M 0 、M 1 、M 2 、M 3 ) as shown in Table 1.

[0064] Table 1 Carrier concentration and drug concentration of core layer spinning solution

[0065]

[0066] (3) 1.8423 g of PVP powder was dissolved in 13 mL of a certain concentration of LbGp solution, and stirred at room temperature for 12 h to prepare a core layer co-spinning solution with a PVP concentration of 15% (wt). A certain amount of PVP was dissolved in anhydrous ethanol and N, N-dimethylformamide (v:v=1:1), and then stirred at room temperature for 12 h to prepare a core layer spinning solution with a concentration of 15% (wt).

[0067] 2.4 Setting of spinning parameters:

[0068] For the prepared coaxial spinning solution, first encapsulate the core layer spinning solution and the shell layer spinning solution into 20mL syringes respectively, and connect the syringe to the coaxial spinning needle with a certain length of polytetrafluoroethylene pipe. The distance between the roller collector covered with tin foil and the tip of the coaxial needle is about 10cm. The flow rate of the core layer spinning solution is 0.5mL / h, and the flow rate of the shell layer spinning solution is 1.5mL / h. The entire spinning process is carried out under the following conditions: temperature: 25℃; humidity: 40%; voltage: 15kV; X-axis scanning speed: 8mm / s; drum collector speed: 1697~1701rpm. Finally, the prepared nanofibers are dried in a vacuum drying oven for 72h and then stored in a vacuum desiccator for the next step.

[0069] In the process of preparing nanofibers, the operations described in the previous paragraph can be carried out as needed using the blank core layer spinning solution prepared in Section 2.3 (3) or the core layer spinning solution containing LbGp or the core layer spinning solution containing other active ingredients.

[0070] 2.5 RPE differentiation:

[0071] The two types of hiPSCs were revived and RPE differentiation was started after the cells recovered well. 4 Pieces / cm 2 The cells were inoculated into the cell culture plate and then cultured with ncTarget-hPSC complete medium until 80% fusion was reached, and differentiation medium was used for RPE differentiation. From day 0 to day 10, day 0-10 medium was used. From day 10 to day 20, day 10-20 medium was used. From day 20 to day 42, day 20-42 medium was used. On day 42 of differentiation, the cells were passaged once. The specific passage process is as follows: 1mL 1% Matrigel was added to each well of the six-well culture plate for coating, shaken left and right, and then placed in the incubator for 40 minutes. Remove the cells to be passaged from the incubator, discard the spent medium, add 1mL Dpbs to each well to wash twice, shake gently, and aspirate. Then, add about 1 mL of TrypLE Express weak enzyme to each well of the six-well culture plate, put the culture plate into the incubator and contact it with the partition of the incubator to heat it evenly, digest it at 37°C for a suitable time (40 minutes for RPE differentiated from Normal-hiPSCs, 30 minutes for RPE differentiated from DM-hiPSCs), gently place the digested cell culture plate in the clean bench, discard the TrypLE Express weak enzyme, add about 2 mL of 42-day culture medium along the bottom of the culture plate, and shake the cell culture plate horizontally to separate the cells from the cell matrix. Finally, subculture to a new six-well plate pre-coated with 1% Matrigel at a ratio of 1:3 to 1:4 for continued culture. When the cells are cultured to the 63rd or 84th day, they can be subcultured again and frozen. Subculture once every 2 to 3 days during the entire differentiation process.

[0072] 3. Test results

[0073] 3.1 Verification of the coaxiality of PVP / PCL coaxial electrospun nanofiber membrane

[0074] In order to verify the coaxiality of the electrospun nanofibers, laser confocal ultra-high resolution microscopy was used to take pictures. The present invention uses PCL containing 7-hydroxy-coumarin-3-carboxylic acid as the shell electrospinning liquid and PVP containing Rose Bengal B as the core electrospinning liquid to co-spin to form nanofibers. Figure 3 Results Tips: Figure 3 The PCL shell wall in A showed green fluorescence; Figure 3 The PVP core exhibits red fluorescence in B. The above results show that the core-shell structure of nanofibers can be successfully prepared when the concentration of PCL is 12% (w / v) and the distance between the coaxial needle and the collector is 10 cm.

[0075] 3.2 Analysis of the morphology, structure and diameter of nanofibers

[0076] In order to observe the morphological characteristics of coaxial electrospun nanofibers loaded with different concentrations of LbGp, SEM was used to study their morphology. Figure 4 The SEM morphology and diameter relative frequency distribution of coaxial electrospun nanofibers without or with different concentrations of LbGp are shown. In general, all coaxial electrospun nanofibers showed a uniform nanofiber structure. Figure 4 The SEM image of A shows that the prepared nanofibers have a smooth surface, no beads, droplets or obvious drug agglomeration, and the fibers are randomly arranged. The diameters of the coaxial electrospun nanofibers loaded with different concentrations of LbGp are slightly different. 2 ) coaxial electrospun nanofiber membrane has the smallest diameter. 3 ) and then began to increase gradually. Figure 4 B shows that the diameter of the coaxial electrospun nanofiber membrane is distributed between 330 and 580 nm. When the drug concentration is lower than 1000 μg / mL, the fiber diameter gradually decreases with the increase of drug loading. When the drug concentration is 1000 μg / mL, the diameter begins to increase again. These results show that the addition of different concentrations of LbGp has an effect on the diameter of the nanofibers.

[0077] 3.3 Characteristic FTIR spectra of coaxial electrospun nanofibers loaded with different concentrations of LbGp

[0078] In order to identify the characteristic functional groups of coaxial electrospun nanofibers without or loaded with different concentrations of LbGp, FTIR analysis was performed. Figure 5 The FTIR results show that PCL is at 1160cm -1 (C=O) and PVP at 2945cm -1 and 1723cm -1 In addition, in the drug-free group (M 0 ) and drug loading group (M 1 、M 2 、M 3 ) of the coaxial electrospun nanofibers showed that the nanofiber membranes of the drug-loaded group and the non-drug-loaded group had higher peaks at 3376 and 3399 cm -1 A characteristic broad peak appeared, which indicated that the polycomplex PVP and PCL coexisted in the nanofibers in a mixed manner and the drug LbGp was successfully encapsulated.

[0079] 3.4TG and DSC analysis

[0080] Thermogravimetry (TG) was used to analyze the relationship between the mass and temperature of coaxial electrospun nanofibers under program-controlled temperature. TG results ( Figure 6 A) Display: M 0 、M 1 、M 2 and M 3 The first stage of cooling was from 330℃ to 429℃, 432℃, 442℃, and 439℃, respectively. In this stage, the weight percentages of coaxial electrospun nanofibers with different concentrations of LbGp lost 58.50%, 63.72%, 80.83%, and 83.96%, respectively. 0 、M 1 、M 2 and M 3 The temperature of the second stage ended at 527℃, 477℃, 482℃ and 486℃, respectively. In this stage, the weight percentage of coaxial electrospun nanofibers with different concentrations of LbGp lost 29.33%, 24.56%, 10.18% and 8.20%, respectively. 0 、M 1 、M 2 and M 3 There are 12.17%, 11.72%, 8.99% and 7.84% by weight. These results indicate that LbGp was successfully loaded into the nanofibers.

[0081] Differential scanning calorimetry (DSC) was used to analyze the material state of each component in the coaxial electrospun nanofibers. DSC curve ( Figure 6 B) Display: M 0 、M 1 、M 2 and M 3 The crystallization temperature (Tc) of M is around 60℃. 0 The melting points (Tm) of M are 120.16°C, 149.50°C, 182.83°C and 232.66°C respectively. 0 、M 1 、M 2 and M 3 There is an obvious exothermic behavior between 360℃-480℃, which indicates that there is an exothermic effect in coaxial electrospun nanofibers.

[0082] 3.5WCA

[0083] In order to investigate the wettability of PVP / PCL coaxial electrospun nanofibers without or with different concentrations of LbGp, water contact angle tests were performed. Figure 7 The results showed that the water contact angle of the unloaded nanofibers was 64.60±0.45 degrees. The water contact angle of the nanofibers loaded with 500μg / mL LbGp was 55.90±0.63 degrees. The water contact angle of the nanofibers loaded with 1000μg / mL LbGp was 54.10±0.36 degrees. The water contact angle of the nanofibers loaded with 1500μg / mL LbGp was 46.80±0.43 degrees. There were statistical differences between the drug-loaded group and the unloaded group. The above results show that PVP / PCL core-shell coaxial electrospun nanofibers are hydrophilic, and the hydrophilicity increases with the loading of the hydrophilic drug LbGp.

[0084] 3.6 LbGp in vitro release test

[0085] In order to explore the in vitro release behavior of LbGp, the cumulative release percentage of LbGp in different samples at different time points was measured ( Figure 8 ). The results showed that LbGp was released rapidly after 0.5h and completely released after 7 days. Electrospun nanofibers loaded with different concentrations of LbGp were prepared by coaxial electrospinning under the same core spinning solution concentration, shell spinning solution and electrospinning parameters, and their drug release behaviors were different. At 0.5h, the cumulative release percentages of LbGp in nanofibers loaded with LbGp concentrations of 500μg / mL, 1000μg / mL and 1500μg / mL reached 24.71%, 25.21% and 51.30%, respectively, and the nanofibers loaded with a drug concentration of 1500μg / mL had the fastest release rate in the first 0.5h. At 72h, the cumulative release percentages of LbGp in nanofibers loaded with LbGp concentrations of 500μg / mL, 1000μg / mL and 1500μg / mL reached 75.10%, 76.61% and 80.48%, respectively. The above results showed that the release characteristics of LbGp in vitro were initially rapid release, followed by slow sustained release.

[0086] 3.7 Effects of different concentrations of LbGp on the activity of Normal-hiPSCs-RPE and DM-hiPSCs-RPE

[0087] In order to explore the concentration range of LbGp on the activity of Normal-hiPSCs-RPE and DM-hiPSCs-RPE, this experiment selected 0μg / mL, 500μg / mL, 1000μg / mL, 2000μg / mL, 4000μg / mL, and 8000μg / mL LbGp solutions to treat cells for 7 days. The CCK8 experiment was used to detect the toxicity of the drug to the cells, and the concentration of the drug when the "reaction" was inhibited by half (Median inhibitory concentration, IC 50 ). The morphology of cells was observed using an inverted fluorescence microscope. Fig. 9 and Fig.10 These are the experimental results of Normal-hiPSCs-RPE and DM-hiPSCs-RPE. As can be seen from the figure: when the drug concentration exceeds 2000μg / mL, the viability of both cells is less than 50%. When the drug concentration is lower than 500μg / mL, there is no statistically significant difference compared with the control group. Compared with the control group, the cell viability of the experimental group decreased, indicating that LbGp can inhibit the proliferation of Normal-hiPSCs-RPE and DM-hiPSCs-RPE. IC of Normal-hiPSCs-RPE 50 The value was 1017 μg / mL, and the IC 50 The value is 1048 μg / mL, therefore, the concentrations observed in the present invention are 500, 1000, and 1500 μg / mL, three different concentrations. **** ),( *** ),( ** ),( * ) represent p<0.0001, p<0.001, p<0.01, p<0.05 respectively.

[0088] 3.8 Effects of different concentrations of LbGp nanofiber membranes on the activity of Normal-hiPSCs-RPE and DM-hiPSCs-RPE

[0089] N-hiPSC-RPE and DM-hiPSCs-RPE were used to evaluate the effect of PVP / PCL coaxial electrospun nanofibers without or with different concentrations of LbGp on the growth of two types of RPE. N-hiPSC-RPE and DM-hiPSCs-RPE were seeded on the coated coaxial electrospun nanofiber membrane. CCK-8 method was used to detect cell survival rate. Fig.11 The coaxial electrospun nanofibers loaded with different concentrations of LbGp show an inhibitory effect on the growth of RPE. It can be seen from the figure that the non-drug-loaded nanofibers have good biocompatibility for both cells. On the 7th day, the cell survival rates of N-hiPSCs-RPE and DM-hiPSCs-RPE on non-drug-loaded nanofibers reached 99%. The drug-loaded nanofibers showed an inhibitory effect on the proliferation of both cells. And with the increase in the concentration of added drugs, the survival rates of both cells gradually decreased. It shows that within a certain range of drug concentrations, the inhibitory effect of cells is concentration-dependent. On the 7th day, the survival rate of N-hiPSCs-RPE dropped to 79%, and the survival rate of DM-hiPSCs-RPE dropped to 81%, indicating that LbGp has an inhibitory effect on both cells.

[0090] 3.9 Effects of different concentrations of LbGp on the cell cycle of N-hiPSCs-RPE and DM-hiPSCs-RPE

[0091] The flow cytometry results of N-hiPSCs-RPE showed that compared with the N-hiPSCs-RPE group without drug treatment, the activity of N-hiPSCs-RPE treated with different concentrations of LbGp was reduced ( Fig.12 A) In the cell proliferation cycle, the proportion of cells entering the G2 phase (nucleic acid synthesis phase) and the S phase (protein synthesis phase) in the entire cell cycle can reflect the activity of the cell. Fig.12 The results of A showed that the ratio of cells entering the S phase and G2 phase in the N-hiPSCs-RPE group without drug treatment was (32.96±1.91)%, the ratio of cells entering the S phase and G2 phase in the N-hiPSCs-RPE group treated with 500μg / mL LbGp medium was (27.75±0.05)%, the ratio of cells entering the S phase and G2 phase in the N-hiPSCs-RPE group treated with 1000μg / mL LbGp medium was (23.89±0.14)%, and the ratio of cells entering the S phase and G2 phase in the N-hiPSCs-RPE group treated with 1500μg / mL LbGp medium was (16.25±0.14)%. There were statistical differences between the drug-treated groups and the non-drug-treated groups, but there was no statistical difference between the drug-treated groups when compared pairwise. ( **** ),( *** ),( ** ),( * ) represent p<0.0001, p<0.001, p<0.01, p<0.05, respectively. The above results indicate that LbGp can inhibit the proliferation of N-hiPSCs-RPE.

[0092] The flow cytometry results of DM-hiPSCs-RPE showed that compared with the DM-hiPSCs-RPE group without drug treatment, the proliferation activity of DM-hiPSCs-RPE treated with different concentrations of LbGp was reduced ( Fig.12 B) In the cell proliferation cycle, the proportion of cells entering the G2 phase (nucleic acid synthesis phase) and the S phase (protein synthesis phase) in the entire cell cycle can reflect the cell proliferation activity. Fig.12B The results showed that the ratio of cells entering the S phase and G2 phase in the DM-hiPSCs-RPE group without drug treatment was (25.46±0.84)%, the ratio of cells entering the S phase and G2 phase in the DM-hiPSCs-RPE group treated with 500μg / mL LbGp medium was (16.98±0.40)%, the ratio of cells entering the S phase and G2 phase in the DM-hiPSCs-RPE group treated with 1000μg / mL LbGp medium was (18.63±0.20)%, and the ratio of cells entering the S phase and G2 phase in the DM-hiPSCs-RPE group treated with 1500μg / mL LbGp medium was (22.59±3.18)%. Compared with the group without drug treatment, the group with drug treatment inhibited the proliferation of DM-hiPSCs-RPE. There was a statistical difference between the 500μg / mL group and the 1000μg / mL group and the group without drug treatment. There was no statistical difference between the 500μg / mL group and the 1000μg / mL group. ** ),( * ) represent p<0.01, p<0.05, respectively. The above results indicate that LbGp can inhibit the proliferation of DM-hiPSCs-RPE.

[0093] 3.12 Nanofibers promote RPE adhesion and skeleton formation

[0094] In order to explore the effect of nanofiber membranes loaded with different concentrations of LbGp on the skeleton structure of Normal-hiPSCs-RPE and DM-hiPSCs-RPE, F-actin / DAPI fluorescence staining was used to observe the morphological characteristics of the two cells. Fig.13 and Fig.14 As shown in the figure, both RPE cells can adhere well to the nanofibers loaded with different concentrations of LbGp and have a complete hexagonal morphological structure. These results indicate that nanofibers loaded with different concentrations of LbGp can promote the adhesion and cytoskeleton formation of Normal-hiPSCs-RPE and DM-hiPSCs-RPE.

[0095] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing core-shell PVP-LbGp / PCL electrospun nanofibers, characterized in that: The preparation method comprises the following steps: (1) Preparation of shell spinning solution: 0.1-5 g of PCL was placed in an autoclaved glass conical flask, and then 5-10 mL of a mixed solvent of chloroform and N,N-dimethylformamide was added to dissolve it. After the mouth of the conical flask was sealed, it was stirred for 5-20 hours to prepare a shell spinning solution; (2) Preparation of LbGp solution: 0.05-0.15 g of LbGp was placed in an autoclaved glass conical bottle, dissolved in 20-50 mL of N,N-dimethylformamide, the bottle mouth was sealed, and then 10-40 mL of anhydrous ethanol was added to prepare a LbGp solution; (3) Preparation of a core layer spinning solution containing LbGp: dissolving an appropriate amount of PVP powder in the LbGp solution prepared in step (2), stirring at room temperature for 5-20 hours, and preparing a core layer spinning solution containing LbGp; (4) Preparation of core-shell PVP-LbGp / PCL electrospun nanofibers: The shell layer spinning solution prepared in step (1) and the core layer spinning solution containing LbGp prepared in step (3) are respectively encapsulated into syringes, and the syringes are connected to the coaxial spinning needles with polytetrafluoroethylene pipes to perform coaxial electrospinning to prepare core-shell PVP-LbGp / PCL electrospun nanofibers.

2. The preparation method according to claim 1, characterized in that: In step (1), 1-2 g of PCL is placed in a high-pressure sterilized glass conical flask, and then a mixed solvent of 6-8 mL of chloroform and N,N-dimethylformamide is added to dissolve it. After the mouth of the conical flask is sealed, it is stirred for 10-15 hours to prepare a shell spinning solution with a mass volume concentration of 10-15%.

3. The preparation method according to claim 1, characterized in that: In step (2), 0.08-0.10 g of LbGp is ​​placed in a high-pressure sterilized glass conical bottle, dissolved in 20-40 mL of N,N-dimethylformamide, the bottle mouth is sealed, ultrasonically oscillated and crushed in an ultrasonic cleaner for 20 min, then placed on a magnetic stirrer and stirred for 12 h, and then 20-40 mL of anhydrous ethanol is added to prepare a 500-1500 μg / mL LbGp solution.

4. The preparation method according to claim 1, characterized in that: In step (3), a proper amount of PVP powder is dissolved in the LbGp solution prepared in step (2), and the mixture is stirred at room temperature for 10-15 hours to prepare a core layer spinning solution containing LbGp with a PVP mass concentration of 12-16%.

5. The preparation method according to claim 1, characterized in that: In step (4), the shell layer spinning solution prepared in step (1) and the core layer spinning solution containing LbGp prepared in step (3) are respectively encapsulated into a 20 mL syringe, and the syringe is connected to the coaxial spinning needle with a polytetrafluoroethylene pipe. The distance between the roller collector covered with tin foil and the tip of the coaxial needle is 10 cm, the flow rate of the core layer spinning solution is 0.5 mL / h, and the flow rate of the shell layer spinning solution is 1.5 mL / h. The entire spinning process is carried out under the following conditions: temperature: 25°C; humidity: 40%; voltage: 15 kV; X-axis scanning speed: 8 mm / s; drum collector speed: 1697~1701 rpm, and coaxial electrospinning is carried out to prepare core-shell PVP-LbGp / PCL electrospun nanofibers.

6. The preparation method according to claim 2 or claim 3 or claim 5, characterized in that: In step (1), the volume ratio of chloroform to N,N-dimethylformamide is 1:1, in step (2), the volume ratio of N,N-dimethylformamide to anhydrous ethanol is 1:1, and in step (4), the prepared electrospun nanofibers are dried in a vacuum drying oven for 72 hours and then stored in a vacuum desiccator.

7. Core-shell PVP-LbGp / PCL electrospun nanofibers prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the core-shell PVP-LbGp / PCL electrospun nanofibers according to claim 7 in the preparation of drugs for treating retinal degenerative diseases.

9. The use according to claim 8, characterized in that: The retinal degenerative disease is selected from one or more of the following: proliferative diabetic retinopathy, age-related macular degeneration, choroidal neovascularization, retinitis pigmentosa or Leber congenital amaurosis.

10. Use of the core-shell PVP-LbGp / PCL electrospun nanofibers according to claim 7 in the construction of bionic RPE tissue engineering.

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