CN112138172B - Preparation method of antagonist functionalized L-polylactic acid porous microspheres - Google Patents

Preparation method of antagonist functionalized L-polylactic acid porous microspheres Download PDF

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CN112138172B
CN112138172B CN202011032266.6A CN202011032266A CN112138172B CN 112138172 B CN112138172 B CN 112138172B CN 202011032266 A CN202011032266 A CN 202011032266A CN 112138172 B CN112138172 B CN 112138172B
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陈亮
顾勇
崔文国
蔡峰
许眙昌
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First Affiliated Hospital of Suzhou University
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Abstract

The invention discloses a preparation method of antagonist functionalized L-polylactic acid porous microspheres, which comprises the following steps: (1) preparing BSA nanoparticles loaded with antagonists; (2) preparing porous levorotatory polylactic acid microspheres: preparing microspheres by using a microfluidic device by using levorotatory polylactic acid, gelatin and polyvinyl alcohol as raw materials; then, placing the microspheres in alkaline water for treatment, and then cleaning with deionized water to obtain the levorotatory polylactic acid porous microspheres; (3) preparing antagonist functionalized L-polylactic acid porous microspheres: and (2) coupling the BSA nanoparticles loaded with the antagonist and the L-polylactic acid porous microspheres by adopting an EDC/NHS method to prepare the antagonist functionalized L-polylactic acid porous microspheres. A series of material tests, cell experiments and in vivo experiments prove that the porous microsphere has good biocompatibility and anti-inflammatory performance, corrects the metabolic imbalance of extracellular matrix caused by inflammation, and promotes the recovery of tissue function.

Description

Preparation method of antagonist functionalized L-polylactic acid porous microspheres
Technical Field
The invention belongs to the technical field of functional materials, and particularly relates to a preparation method of antagonist functionalized L-polylactic acid porous microspheres.
Background
Inflammatory reactions have beenPlays an important role in chronic non-infectious diseases, triggers cascade reaction through a series of signal paths, and participates in the disease occurrence in a plurality of systems and organs[1-5]. In clinical practice, various therapeutic methods such as gene therapy, hormone therapy and the like have been developed for inflammatory responses[6]. However, the effect of gene therapy has yet to be examined because of the high number of genes involved in the inflammatory response and the low transfection efficiency[7]. The side effects of hormone therapy are a great concern for medical workers[8]. In inflammatory reaction, the increase of inflammatory factor concentration leads to the imbalance of tissue extracellular matrix metabolism, thereby causing functional disorder and accelerating disease process[9]. In recent years, antagonist therapy against inflammatory factors has received increasing attention. The antagonist can specifically bind to inflammatory factor, and effectively inhibit its activity, thereby correcting extracellular matrix (ECM) metabolic disorder and restoring tissue function[10,11]. Compared with other therapies, the antagonist therapy has definite action target and high action efficiency, and can form local high concentration by minimally invasive injection to a pathological part to play a therapeutic role.
Different antagonists can be used to specifically bind different inflammatory factors, so that different diseases can be treated. However, exogenous antagonists, as a protein, have a short half-life and cannot maintain activity, and are difficult to act by direct injection, and therefore, need a suitable carrier to encapsulate them. In recent years, researchers have prepared various drug carriers, such as liposomes, ceramic nanoparticles, polymer nanoparticles, and the like, which have respective advantages but have various disadvantages: the liposome has defects in the aspect of drug slow release[12](ii) a Due to the particularity of the material, the ceramic nanoparticles cannot effectively wrap protein; the polymer nanoparticles have poor hydrophilicity and poor wrapping efficiency[13]. The Bovine Serum Albumin (BSA) nanoparticles can be effectively combined with protein drugs because of the existence of various drug binding sites in albumin molecules. The preparation method is simple, nontoxic and non-antigenic, and has attracted wide attention in recent years[14-16]. Thus, BSA nanoparticles were used as antagonist carriers,can improve the wrapping efficiency, keep the activity of the medicine and achieve the aim of slow release. The nano-particle coated with the antagonist is injected to an inflammation part, so that the biological activity and the action time of the antagonist are prolonged, and the effect of inhibiting inflammation is better exerted.
However, the single direct injection of nanoparticles wrapped with antagonists has the problems of small injection dosage and leakage of liquid along the needle passage after injection, and greatly influences the treatment effect. Therefore, it is necessary to load nanoparticles into an ideal biomaterial, which not only realizes high-efficiency loading but also improves injection efficiency. The current injectable biological materials comprise hydrogel and derivatives thereof, organic polymer materials and the like[17]. Although the hydrogel material has good biocompatibility, the hydrogel material has low mechanical strength and is easy to crack into fragments under larger biological stress, thereby influencing the uniform distribution of the drug[18]. Although the organic polymer material has high mechanical strength, the organic polymer material has poor hydrophilicity and cannot be combined with water-soluble BSA nanoparticles, and the loading of the nanoparticles has a serious problem. Meanwhile, in most cases, the two materials are physically loaded, and the loading efficiency and the uniform dispersion of the nanoparticles are unsatisfactory[19]. While the inflammation part is swollen and has a narrow space, the volume of the material entering the inflammation part by minimally invasive injection is limited, and the physical load is difficult to ensure the concentration of the medicine in unit volume. Moreover, the failure of synchronous release of the drug on the surface and inside of the material can seriously affect the persistence of the drug effect[20]. Therefore, an injectable biomaterial with a certain mechanical strength is needed, which can realize efficient and uniform carrying of nanoparticles through chemical bonds and simultaneously realize synchronous release of drugs inside and outside.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a preparation method of antagonist functionalized L-polylactic acid (PLLA) porous microspheres, and the PLLA porous microspheres are prepared by using a microfluidic device. Aiming at the defect of poor hydrophilicity of PLLA, surface modification is carried out through alkaline hydrolysis, active carboxyl groups on the surface of the microsphere are exposed, amino groups are rich on the surface of BSA nanoparticles wrapping the antagonist due to the chitosan, and amide bonds are formed through a carbodiimide method to complete grafting, so that the antagonist functionalized injectable PLLA porous microsphere is constructed. A series of material science tests, cell experiments and in vivo experiments prove that the PLLA porous microspheres functionalized by the antagonist have good biocompatibility and anti-inflammatory performance, correct the metabolic imbalance of extracellular matrix caused by inflammation and promote the recovery of tissue function.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of antagonist functionalized L-polylactic acid porous microspheres comprises the following steps:
(1) preparation of antagonist-loaded BSA nanoparticles
(2) Preparation of porous levorotatory polylactic acid microspheres
Preparing microspheres by using a microfluidic device by using levorotatory polylactic acid, gelatin and polyvinyl alcohol as raw materials; then, placing the microspheres in alkaline water for treatment, and then cleaning with deionized water to obtain the levorotatory polylactic acid porous microspheres;
(3) preparation of antagonist functionalized L-polylactic acid porous microspheres
And (3) coupling the BSA nanoparticle loaded with the antagonist with the L-polylactic acid porous microsphere by adopting an EDC method to prepare the antagonist functionalized L-polylactic acid porous microsphere.
Further, the specific preparation steps of the step (1) are as follows: preparing a BSA aqueous solution, and adding an antagonist into the BSA aqueous solution; pumping ethanol into BSA solution, and stirring at room temperature overnight; adding chitosan solution dissolved in acetic acid into the mixed solution, and mixing; then pumping the ethanol solution, and stirring and mixing at room temperature; and centrifuging the mixed solution at 12000rpm for 20min, and washing the mixed solution for multiple times by using a 50% ethanol solution to obtain the antagonist-loaded BSA nanoparticle.
Further, in the step (1), the flow rate of the ethanol pumped into the BSA solution for the first time in the step (1) is 2 mL/min; the flow rate of the BSA solution pumped in by the ethanol for the second time is 0.5mL/min, and the time for stirring and mixing the BSA solution pumped in by the ethanol for the second time at room temperature is 8 h.
Further, the specific preparation steps of the step (2) are as follows: dissolving levorotatory polylactic acid in dichloromethane, dissolving gelatin and polyvinyl alcohol in deionized water, and then ultrasonically and uniformly mixing the three raw materials to obtain emulsion; taking the emulsion as a discontinuous phase and the polyvinyl alcohol solution as a continuous phase, preparing microspheres by using a micro-fluidic device with two channels and a coaxial needle, collecting the microspheres in ice water at 4 ℃, and slowly stirring overnight; then placing the mixture into warm water at 45 ℃ and stirring the mixture for 3 hours, washing the mixture with deionized water for three times, and storing the mixture after freeze-drying.
Further, in the step (2), the mass fraction of the levorotatory polylactic acid dissolved in the dichloromethane is 2wt%, and the mass fractions of the gelatin and the polyvinyl alcohol dissolved in the deionized water are 7.5 wt% and 1wt%, respectively.
Further, the specific preparation steps of the step (3) are as follows: ultrasonically dispersing the microspheres prepared in the step (2) in MES buffer solution with the pH value of 6.0, respectively adding EDC and NHS with the mass ratio of 2:3, reacting for 15min at 37 ℃ after ultrasonic dispersion, centrifuging and removing supernatant; dispersing the BSA nanoparticles loaded with the antagonist prepared in the step (1) in deionized water, adding the centrifuged microspheres into the deionized water, ultrasonically mixing, and reacting at 37 ℃ overnight; finally, centrifuging again, and repeatedly cleaning with deionized water to obtain the antagonist functionalized L-polylactic acid porous microsphere.
Has the advantages that: the invention provides a preparation method of antagonist functionalized L-polylactic acid porous microspheres, which adopts L-polylactic acid with good biocompatibility and certain physical strength as a material, and firstly, the prepared PLLA microspheres are placed in a sodium hydroxide solution for surface modification, ester bonds on the surface are broken, and hydrophilic groups (carboxyl and alcohol groups) are exposed, so that the material is multifunctional; as the chitosan on the surface of the BSA nanoparticle contains rich amino groups, the BSA nanoparticle and the carboxyl on the surface of the microsphere form an amido bond under an EDC/NHS system, and the amido bond is grafted on the surface of the PLLA porous microsphere, so that the efficient and uniform loading of the nanoparticle is realized, and the drug concentration of unit volume is improved. Physical characterization of the system shows that a plurality of pores with different sizes and communicated inside and outside exist on the surface of the porous microsphere, and compared with a non-porous microsphere, the porous structure increases the attachable area of the nanoparticles and improves the loading capacity. And the BSA nanoparticles have regular shapes and relatively consistent sizes. Through chemical grafting, the nanoparticles are distributed on the surface of the microsphere in a large amount and uniformly. By measuring the drug sustained release curve, the antagonist can be continuously and effectively released due to the stability of the BSA nanoparticles, the duration is about 35 days, and the total release amount is about 79.85% + -2.12%. In vitro experiments, compared with a control group, the microsphere loaded with the nanoparticles effectively inhibits the gene expression of cell inflammation and factor secretion, and meanwhile, in vivo verification is carried out in a rat intervertebral disc degeneration model, and the microsphere with the functionalized antagonist can effectively inhibit inflammatory reaction, correct extracellular matrix disorder and obviously delay intervertebral disc degeneration. Therefore, the microsphere prepared by the invention has important application prospect.
Drawings
FIG. 1A is a flow diagram of the preparation of PLLA microspheres and alkaline hydrolysis using a microfluidic device; b is a schematic diagram of preparation of BSA nanoparticles and grafting of PLLA microspheres; c is an electron microscope picture of the nano-particles (BNP), the Microspheres (MS) and the grafted microspheres (MS-BNP); d is an FTIR picture of PLLA Microspheres (MS), alkali treated microspheres (Modified MS), BSA Nanoparticles (BNP) and nanoparticles grafted (MS-BNP).
FIG. 2A is a distribution diagram of the particle size of nanoparticles; b is a particle size distribution diagram of the PLLA microspheres; c is a microsphere aperture analysis chart; d is an EDS elemental analysis chart of the pure PLLA microspheres and the microspheres grafted by the nanoparticles; e is antagonist sustained-release diagram.
FIG. 3A is a graph showing degradation of PLLA microspheres before and after alkaline hydrolysis; b is a quantitative analysis line graph of the degradation condition of the microspheres; c is a histogram of quantitative analysis of the degradation of the microspheres.
FIG. 4A is a graph of cell dying and vital staining; b is an activity evaluation and quantitative analysis graph of cells in the microspheres; c is a cell mass analysis chart of unit area in different time; d is a diagram of the effect of the microspheres on cell proliferation.
FIG. 5A is a graph of PCR evaluation of the effect of antagonist functionalized microspheres on inflammatory gene expression; and B is a graph for detecting the influence of the antagonist functionalized microspheres on the concentration of the cell supernatant inflammatory factors by ELISA.
FIG. 6A is a graph showing the results of X-ray assessment of intervertebral space height for different treatment groups; b, evaluating intervertebral disc signal intensity graphs of different treatment groups by MRI; c is an X-ray and MRI result quantitative analysis chart; d is the experimental schematic diagram in the antagonist functional microsphere.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 preparation of antagonist-loaded BSA nanoparticles
Adding 100mg BSA into 10mL deionized water, stirring uniformly, and then adding 10 mu g of antagonist dissolved in phosphate buffer; 40mL of ethanol was pumped into the antagonist-containing BSA solution at a flow rate of 2mL/min and stirred at room temperature overnight; adding 40mL of chitosan solution dissolved in 1% acetic acid solution and having a concentration of 1mg/mL into the mixed solution; pumping ethanol at the flow rate of 0.5mL/min, and stirring at room temperature for 8 h; and centrifuging the obtained nanoparticles at 12000rpm for 20min, and washing the nanoparticles with 50% ethanol solution for three times to obtain the antagonist-loaded BSA nanoparticles.
Example 2 preparation of L-polylactic acid porous microspheres
Dissolving PLLA in dichloromethane, and dissolving gelatin and polyvinyl alcohol PVA in deionized water to prepare solutions with final concentrations of 2wt%, 7.5 wt% and 1wt%, respectively; adding 1g of mixed solution of gelatin and PVA into 3g of PLLA solution, and ultrasonically mixing uniformly (power is 10%, ultrasound is carried out for 2s, and interval is 1s) to obtain emulsion.
The micro-fluidic device for preparing the microspheres is a coaxial needle head and has two channels, the inner diameter and the outer diameter are respectively (0.26mm i.dx0.51mm o.d,0.84mm i.d x1.27 mm o.d and 25G/18G), and the tail end of the needle head is connected with a PVC (polyvinyl chloride) tube for collection; the syringe loaded with the emulsion as the discontinuous phase was connected to the inner diameter of the device; the continuous phase is 1% PVA solution, and is connected with the outer diameter of the device, the flow rate of the continuous phase and the outer diameter of the device is set to be 20:1, and the continuous phase and the device are continuously operated until the emulsion is used up; collecting the microspheres in a 500mL beaker containing ice water at 4 ℃, and stirring gently overnight; then placing the mixture into warm water at 45 ℃ and stirring for 3 hours, washing the mixture with deionized water for three times, and freeze-drying and storing the mixture. And (3) uniformly mixing 25mL of 0.25M NaOH solution with ethanol with the same volume, putting the freeze-dried microspheres into alkaline water for 3 hours, and then washing the microspheres with deionized water for three times to obtain the L-polylactic acid porous microspheres.
EXAMPLE 3 preparation of antagonist functionalized L-polylactic acid porous microspheres
Ultrasonically dispersing the levorotatory polylactic acid porous microspheres prepared in example 2 into 5mL MES (pH 6.0) buffer solution, respectively adding 40mg of EDC and 60mg of NHS, ultrasonically reacting for 15s at 37 ℃ for 15min, and centrifuging to remove supernatant; dispersing the antagonist-loaded BSA nanoparticles prepared in example 1 in 10mL of deionized water, adding the centrifuged EDC-activated microspheres, and ultrasonically mixing uniformly; reacting overnight at 37 ℃, finally centrifuging again, and repeatedly washing with deionized water for three times to obtain the antagonist functionalized L-polylactic acid porous microsphere.
FIG. 1A, B is a flow chart of the preparation of the microsphere of the present invention, in which, in FIG. 1A, the surface carboxyl groups are exposed after the PLLA microsphere is treated with alkali solution, and in FIG. 1B, the amino groups on the surface of the BSA nanoparticle and the carboxyl groups on the surface of the PLLA microsphere are coupled to prepare the antagonist functionalized L-polylactic acid porous microsphere.
Example 4 physical characterization of antagonist functionalized L-polylactic acid porous microspheres
1. Analysis of nanoparticle size
Firstly, a particle size analyzer is used for analyzing the particle size distribution of nanoparticles, and different types of microspheres are subjected to particle size analysis, the result is shown in fig. 2, fig. 2A is a particle size distribution diagram of BSA nanoparticles, and the particle sizes of the BSA nanoparticles are mostly distributed at 200 nm; FIG. 2B is a plot of the particle size distribution of the PLLA microspheres, the average particle size of which is about 100 μm; FIG. 2C is a pore size distribution diagram of the porous microspheres, with an average pore size of about 10 μm.
2. Observation of nanoparticle and microsphere morphology (SEM)
After freeze-drying the microspheres, observing the shapes of the microspheres under an optical microscope, observing the microspheres under an SEM, and sticking a proper amount of microspheres, nanoparticles and grafted microspheres on a tray through conductive adhesive. Spraying gold by using an ion sputtering instrument, then carrying out the gold spraying under the condition that 10KV is used as an accelerating voltage, and randomly selecting 200 microspheres to calculate the diameter and the aperture.
An electron microscope image of the BSA Nanoparticles (BNP), the L-PLA porous Microspheres (MS) and the L-PLA porous microspheres (MS-BNP) grafted by the BSA nanoparticles is shown in FIG. 1C, and the grafted BSA nanoparticles are uniformly distributed on the surfaces of the microspheres (the arrow points to the BSA nanoparticles).
3. Energy spectrometer (EDS) elemental analysis
To compare the elemental differences before and after PLLA microsphere grafting, different groups of samples were specifically analyzed using EDS. Fig. 2D is an EDS elemental analysis chart of the microspheres grafted with pure PLLA microspheres and BSA nanoparticles, where N, S element content is increased after grafting, which indirectly indicates that BSA nanoparticles are successfully grafted.
4. Fourier transform Infrared Spectroscopy (FTIR)
For the difference of different groups of materials and the formation of chemical bonds, we used FTIR for analytical comparison at 4cm-1The resolution of (2) performs 128 scans with a scan wavenumber range of 400-4000cm-1。
FTIR graphs of BSA Nanoparticles (BNP), L-PLA porous Microspheres (MS) not treated with alkali solution, L-PLA porous microspheres (Modified MS) treated with alkali solution, and L-PLA porous microspheres (MS-BNP) grafted with BSA nanoparticles are shown in FIG. 1D, which shows that 1310cm of L-PLA porous microspheres treated with alkali solution appears-1Peak, 1400cm of MS-BNP, indicating successful exposure of the carboxyl group-1The peaks indicate successful grafting.
5. In vitro release of antagonists
To evaluate the in vitro release of the drug, 30mg of grafted microspheres were immersed in 50ml centrifuge tubes containing 10ml of PBS. The centrifuge tubes were placed on a 37 ℃ shaker. At the corresponding time points (1, 2, 3, 5, 7, 9, 12, 15, 18, 23, 28, 35d), the drug-containing buffer was collected and fresh buffer was added and stored at-20 ℃ prior to assay. The antagonist released from the nanoparticles was measured using an ELISA kit. Release curves were plotted based on the original content in the nanoparticles.
Fig. 2E is a graph of sustained release of the antagonist, from which it can be seen that the antagonist is released continuously for about 35 days.
6. Biodegradability of porous levorotatory polylactic acid microsphere
The 15mg L-polylactic acid porous microspheres were equally divided into three portions, 10ml of Phosphate Buffered Saline (PBS) was added to each portion, and incubation was performed at 37 ℃ with slow stirring. The microspheres were dried at 2W, 4W and 6W respectively and weighed (Wt). Residual weight (%) was calculated according to the formula Wt/W0. The experiment was repeated three times for each time point.
FIG. 3A is a graph showing the degradation of porous L-polylactic acid microspheres before and after alkaline hydrolysis, wherein the hydrophilicity of the microspheres is improved after the alkaline hydrolysis, and the degradation is accelerated compared with that of untreated microspheres; FIG. 3B, C is a diagram showing the result of quantitative analysis of the degradation of microspheres, and it can be seen that the degradation of modified microspheres is accelerated.
Example 5 compatibility and anti-inflammatory Properties of antagonist functionalized L-polylactic acid porous microspheres
1. Nucleus pulposus cell extraction
The nucleus pulposus cells used in this experiment were extracted from Sprague-Dawley rats (SD rats). The caudal vertebra of the rat is taken out under aseptic condition, the paraspinal muscles are stripped, the fibrous ring is cut by a sharp blade, and the nucleus pulposus tissues of all the segments are sequentially taken out by using forceps of the ophthalmology department. The nucleus pulposus tissue was digested with 0.25% collagenase type II for 2h at 37 ℃. After filtration through a 200 mesh sieve, the supernatant was centrifuged and discarded. After three washes with sterile PBS, DMEM medium containing 10% PBS was added. Placing nucleus pulposus cells at 37 deg.C and containing 5% CO2The culture was carried out in a volumetric incubator, with the medium changed every 3 days.
2. Cell culture
The microspheres were sterilized by first placing in 75% ethanol for 30min, then washed three times with sterile PBS, and then added to DMEM medium overnight. Cells were washed three more times with sterile PBS prior to seeding. Rat nucleus pulposus cells were digested with 0.25% pancreatin and counted three times. Containing 4x10540ml of the cell culture medium and 200 microspheres were added to a spinner flask and stirred at 50rpm for 24 h. Thereafter, the microspheres were aspirated, washed three times with PBS, and non-adherent cells were removed. The microspheres were seeded in 96-well plates, two microspheres per well. As a control, 5000 cell adherent cultures were added per well in 96-well plates. The medium was DMEM, 10% FBS, 1% double antibody. The cells were incubated at 37 ℃ with 5% CO2The culture box is used for culturing, and the liquid is changed every other day. In immunofluorescence, PCR, ELISA experiments, in order to simulate in vivo regressionCells for microsphere culture were previously cultured with a medium containing TNF-. alpha.at a concentration of 25ng/ml for three days, with varying inflammatory environment.
3. Cell viability assay
To assess the viability of nucleus pulposus cells after inoculation, we performed live/dead staining experiments. On days 1, 3, 5 after cell culture, live and dead staining reagents were added to the well plates and incubated at room temperature for 30 min. The stained cells were observed with a fluorescence microscope. Meanwhile, to further demonstrate cell viability, we resuspended the cell-loaded microspheres in 5ml of culture medium and injected into 6-well plates with a syringe containing a 24G needle to continue culturing. Observing the adherent and proliferation conditions of the cells migrating from the microspheres to the pore plate by using an optical microscope on time, and calculating the cell amount in unit area at different times by using Image J software.
FIG. 4A is a graph showing the results of live-dead staining with fewer dead cells as the number of cells increases with time; FIG. 4B is an electron micrograph of cells cultured in microspheres, and FIG. 4C is a graph showing the cell mass per unit area at different times, and it can be seen from the graph that the cell mass per unit area gradually increases with time.
4. Cell proliferation assay
In order to detect cell proliferation among different groups, cell adherent culture was used as a control and evaluated by using CCK-8 kit. At appropriate time points (1, 3, 5, 7d) medium containing 10% by volume of CCK-8 reagent was added. After 4 hours of incubation, 100ul of medium per well was pipetted into a new 96 well plate and absorbance at 450nm was measured using a microplate reader.
From the results in FIG. 4D, it can be seen that the cells were more active in the microspheres and that the cells in CCK-8 experiments proliferated faster in the microspheres compared to adherent cultures.
5. Expression of inflammatory and extracellular matrix-associated genes
And detecting the expression of the genes related to the inflammation and the extracellular matrix by using a qRT-PCR method. The cells were cultured for 3 and 7 days, respectively, and total RNA was extracted. Reverse transcription into complementary DNA (cDNA) followed by gene expression analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference.
FIG. 5A is a graph showing the in vitro anti-inflammatory effect of the antagonist functionalized microspheres evaluated by PCR, and compared with other groups, the MS-BNP group significantly inhibited the expression of inflammatory genes by releasing the antagonist.
6. Detection of concentration of inflammatory factor and extracellular matrix-related protein in cell culture medium
The concentrations of inflammatory factors and extracellular matrix-related proteins in the nucleus pulposus cell culture medium were determined by enzyme-linked immunosorbent assay (ELISA). Culturing nucleus pulposus cells of different groups for 3 and 7 days respectively, sucking supernatant, measuring the concentration of the supernatant by using an ELISA kit, and repeating each index for three times.
FIG. 5B is a graph showing the results of ELISA assay of the cell supernatant inflammatory factor concentration, from which it can be seen that the cell supernatant inflammatory factor concentration in the MS-BNP group is significantly lower.
Example 6 in vivo experiment of antagonist functionalized L-polylactic acid porous microspheres
1. Establishment of animal model
Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate in an amount of 3 ml/kg. After complete anesthesia, rat tails were disinfected with 75% alcohol and were sequentially punctured with a 21G needle into Co7-10 intervertebral discs to induce degeneration. In order to ensure the puncturing effect, the needle is rotated for 5s after puncturing the fibrous ring and then is maintained for 30 s. Each disc was injected with 20ul of microsphere solution and the negative control group was injected with PBS as a control. After the surgery was completed, the rats were placed in a warm and ventilated place.
2. X-ray and MRI examination
At 4W, 8W post-surgery, 5 rats per group were randomly picked for X-ray and MRI examination before sacrifice. The rat was kept in a supine position with the tail in line on the molybdenum target radiographic imaging unit. The X-ray number parameters were set to 66cm collimator to film distance, 63mAs exposure time, and 35kV voltage. MRI was performed using a 1.5T system (GE), with T2 weighted images obtained in the coronal plane with the parameters: the repetition time is 3000 ms; echo time 80 ms; the visual field is 200 multiplied by 200 mm; the slice thickness was 1.4 mm. The DHI of each group of discs was obtained by measurement and analysis in a medical imaging software system by experienced radiologists blinded to the study. The ratio of the change in DHI of the discs in the experimental group to the normal discs was expressed as DHI%. MRI images were classified into classes I to IV by evaluating the signal intensity of T2 weighted images according to the modified Thomson classification method.
FIG. 6A is a graph showing the results of evaluating the heights of the intervertebral spaces of different treatment groups by X-ray, and the intervertebral spaces of the MS-BNP group are significantly better than those of the other experimental groups; FIG. 6B is a graph showing the results of using MRI to evaluate the intervertebral disc signal intensity of different treatment groups, and it can be seen that the intervertebral disc signal of MS-BNP group is better; FIG. 6C is a diagram showing the quantitative analysis of the X-ray and MRI results; figure 6D is a schematic in vivo experiment showing that microspheres loaded with antagonist are injected into disc to inhibit disc degeneration through inhibition of inflammatory factor TNF- α. Reference:
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Claims (6)

1. a preparation method of an antagonist functionalized L-polylactic acid porous microsphere is characterized by comprising the following steps:
(1) preparation of antagonist-loaded BSA nanoparticles
Preparing a BSA aqueous solution, and adding an antagonist into the BSA aqueous solution; pumping ethanol into BSA solution, and stirring at room temperature overnight; adding a chitosan solution dissolved in acetic acid into the mixed solution after stirring overnight, and mixing; then pumping the ethanol solution, and stirring and mixing at room temperature; centrifuging the mixed solution, and washing the mixed solution for multiple times by using a 50% ethanol solution to obtain BSA (bovine serum albumin) nanoparticles loaded with antagonists;
(2) preparation of porous levorotatory polylactic acid microspheres
Preparing microspheres by using a microfluidic device by using levorotatory polylactic acid, gelatin and polyvinyl alcohol as raw materials; then, placing the microspheres in alkaline water for treatment, and then cleaning with deionized water to obtain the levorotatory polylactic acid porous microspheres;
(3) preparation of antagonist functionalized L-polylactic acid porous microspheres
And (2) coupling the BSA nanoparticles loaded with the antagonist and the L-polylactic acid porous microspheres by adopting an EDC/NHS method to prepare the antagonist functionalized L-polylactic acid porous microspheres.
2. The method for preparing the antagonist functionalized L-polylactic acid porous microspheres according to claim 1, wherein the flow rate of ethanol pumped into the BSA solution for the first time in the step (1) is 2 mL/min; the flow rate of the BSA solution pumped in by the ethanol for the second time is 0.5mL/min, and the time for stirring and mixing the BSA solution pumped in by the ethanol for the second time at room temperature is 8 h.
3. The method for preparing the levorotatory polylactic acid porous microsphere with functionalized antagonist according to claim 1, wherein the specific preparation steps of the step (2) are as follows: dissolving levorotatory polylactic acid in dichloromethane, dissolving gelatin and polyvinyl alcohol in deionized water, and then ultrasonically and uniformly mixing the three raw materials to obtain emulsion; taking the emulsion as a discontinuous phase and the polyvinyl alcohol solution as a continuous phase, preparing microspheres by using a micro-fluidic device with two channels and a coaxial needle, collecting the microspheres in ice water at 4 ℃, and slowly stirring overnight; then placing the mixture into warm water at 45 ℃ for stirring, washing the mixture with deionized water for three times, and storing the mixture after freeze-drying.
4. The method for preparing levorotatory polylactic acid porous microspheres with functionalized antagonist according to claim 1, wherein the weight fraction of levorotatory polylactic acid dissolved in dichloromethane in step (2) is 2wt%, and the weight fractions of gelatin and polyvinyl alcohol dissolved in deionized water are 7.5 wt% and 1wt%, respectively.
5. The method for preparing the levorotatory polylactic acid porous microsphere with functionalized antagonist according to claim 1, wherein the specific preparation steps of the step (3) are as follows: ultrasonically dispersing the microspheres prepared in the step (2) in MES buffer solution with the pH value of 6.0, respectively adding EDC and NHS with the mass ratio of 2:3, reacting for 15min at 37 ℃ after ultrasonic dispersion, centrifuging and removing supernatant; dispersing the BSA nanoparticles loaded with the antagonist prepared in the step (1) in deionized water, adding the centrifuged microspheres into the deionized water, ultrasonically mixing, and reacting at 37 ℃ overnight; finally, centrifuging again, and repeatedly cleaning with deionized water to obtain the antagonist functionalized L-polylactic acid porous microsphere.
6. An antagonist functionalized L-polylactic acid porous microsphere prepared by the preparation method of any one of claims 1 to 5.
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