Ropivacaine hydrochloride sustained-release microgel as well as preparation method and application thereof

Abstract

The invention provides a ropivacaine hydrochloride (RVC) sustained-release microgel as well as a preparation method and application thereof, and belongs to the technical field of local anesthetics. The RVC sustained-release microgel provided by the invention is prepared from the following raw materials in parts by weight: 21 to 42 parts of RVC, 2.4 parts of collagen (COL) and 71.4 parts of laponite (LAP). According to the invention, two materials of COL and LAP are adopted to jointly deliver RVC to prepare the RVC sustained-release microgel (RVC-LAP / COL), and the purpose of long-acting analgesia of local anesthetics is achieved by slowly releasing RVC. Compared with the traditional dosage form, the RVC-LAP / COL disclosed by the invention has the advantages that the administration frequency can be reduced, the compliance of a patient is improved, the toxic and side effects are reduced, the safety and timeliness are enhanced, the medicine absorption is better controlled, the safety is high, the clinical application space of local anesthetics is greatly expanded, and a new choice is provided for analgesia management of patients with acute and chronic pains.

Description

Technical Field

[0001] The invention belongs to the technical field of local anesthetics, and in particular relates to a ropivacaine hydrochloride sustained-release microgel and a preparation method and application thereof. Background Art

[0002] In recent years, with the fast-paced social life, the demand for pain management has increased. Among them, chronic pain lasts for a long time and is difficult to control, which is one of the thorny problems in pain management. The long-term effects of current clinical drug therapy and interventional therapy are not ideal. Regional nerve block based on local anesthetics still has an indispensable position in the management of chronic pain. Ropivacaine hydrochloride (RVC) is a new type of long-acting amide local anesthetic with a wide clinical safety dose, rapid onset, and ability to penetrate the mucosa. It is currently widely used in clinical practice. However, its single administration can only maintain analgesia for 2-6 hours. Therefore, it is currently used in combination with other drugs and multiple administrations of catheters to maintain its long-term analgesic effect. However, for patients with chronic pain, whether it is catheter indwelling or ultrasound-guided nerve block, each operation is accompanied by many complications, such as catheter displacement, obstruction, local hematoma, nerve damage, infection, local anesthetic poisoning, paralysis and other problems. Therefore, if the duration of action of local anesthetics can be prolonged and the efficacy of local anesthetics can be improved, it will bring great convenience to the treatment of both postoperative acute pain and chronic pain. Summary of the invention

[0003] In view of this, one of the purposes of the present invention is to provide a ropivacaine hydrochloride sustained-release microgel with long-term analgesic effect. Another purpose of the present invention is to provide a method for preparing the ropivacaine hydrochloride sustained-release microgel with long-term analgesic effect.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a ropivacaine hydrochloride sustained-release microgel. The ropivacaine hydrochloride sustained-release microgel comprises the following raw materials in parts by weight: 21 to 42 parts of ropivacaine hydrochloride, 2.4 parts of collagen and 71.4 parts of lithiasis earth.

[0006] The present invention also provides a method for preparing the above-mentioned ropivacaine hydrochloride sustained-release microgel, comprising the following steps: mixing the ropivacaine hydrochloride with water to obtain a ropivacaine hydrochloride aqueous solution; mixing the ropivacaine hydrochloride aqueous solution with the lithium ion, and freeze-drying to obtain a powder; dissolving the collagen to obtain a collagen solution; mixing the collagen solution with the powder, and standing at 37° C. to obtain the ropivacaine hydrochloride sustained-release microgel.

[0007] Preferably, the weight-to-volume ratio of ropivacaine hydrochloride to collagen solution is 21-42 mg:600 μL.

[0008] Preferably, the preparation method of the collagen solution is: mixing collagen with glacial acetic acid solution, adjusting the pH to 7.0, and then adding water to obtain the collagen solution.

[0009] Preferably, the concentration of the glacial acetic acid solution is 0.25 mol / L.

[0010] Preferably, in the collagen solution, the volume ratio of glacial acetic acid solution to water is 7:3.

[0011] Preferably, the freeze-drying is performed at -50°C and 30 mtorr for 48 hours.

[0012] Preferably, the standing time is 30 min.

[0013] The present invention also provides application of the ropivacaine hydrochloride sustained-release microgel or the preparation method in preparing analgesic products.

[0014] Beneficial effects of the present invention:

[0015] The present invention uses collagen and lithium diatomite to deliver ropivacaine hydrochloride to prepare ropivacaine hydrochloride sustained-release microgel (RVC-LAP / COL), and achieves the long-acting analgesic purpose of local anesthetics by slowly releasing RVC. Compared with traditional dosage forms, the RVC-LAP / COL provided by the present invention can reduce the frequency of administration, improve patient compliance, reduce toxic and side effects, enhance safety and timeliness, better control drug absorption, and has high safety. It greatly improves the clinical application space of local anesthetics, has broad application prospects, and provides a new choice for analgesic management of patients with acute and chronic pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figures are the gel morphology and principle diagram, wherein (a) is a blank hydrogel without drug; (b) is a ropivacaine hydrochloride microgel; (c) is a schematic diagram of the structure of lithium terephthalate; (d) is a schematic diagram of the preparation principle of the present invention;

[0017] Figure 2 The results of NTA particle size detection, where (a) is the particle size distribution diagram of 3.5% RVC-LAP / COL; (b) is the particle size distribution diagram of 7.0% RVC-LAP / COL; the black area in the figure is the average of three times, and the red area is the standard deviation area;

[0018] Figure 3 are the results of microscopic morphological observation, where a is the microgel morphology under an optical microscope; b is the microgel morphology under an electron microscope;

[0019] Figure 4 is the standard curve of ropivacaine hydrochloride;

[0020] Figure 5 is the in vitro drug release curve;

[0021] Figure 6 The results of water content and swelling rate determination, where a is the water content of ropivacaine hydrochloride microgel, and b is the swelling performance determination result of blank hydrogel without drug;

[0022] Figure 7 Cell morphology observation after 24 h of treatment in each group;

[0023] Figure 8 The results of in vitro cytotoxicity experiments at different time points of each group, where a is the cell activity of each group after 24h co-incubation of PC12 cells, b is the cell activity of each group after 48h co-incubation of PC12 cells, c is the cell activity of each group after 24h co-incubation of BV2 cells, d is the cell activity of each group after 48h co-incubation of BV2 cells, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001 are comparisons between RVC group and RVC-LAP / COL group;

[0024] Fig. 9 The live and dead cell staining results, where a is the live and dead staining fluorescence image of PC12 cells, b is the live and dead percentage statistical graph of PC12 cells, c is the live and dead staining fluorescence image of BV2 cells, and d is the live and dead percentage statistical graph of BV2 cells;

[0025] Fig.10 The results of NO levels in the supernatant of BV2 cells in each group, where a is the NO level of the control group and the blank material group, b is the NO level of the high-concentration group, c is the NO level of the medium-concentration group, and d is the NO level of the low-concentration group. ****P<0.0001 is the comparison between the control group and the RVC and RVC-LAP / COL groups, and ns indicates no significant difference.

[0026] Fig.11 For the technical route;

[0027] Fig.12 The modeling process and schematic diagram of the selective sciatic nerve branch injury (SNI) in rats;

[0028] Fig.13 The mechanical pain threshold of rats in each group, (b) is the mechanical pain threshold of rats in each group before administration, (c) is the mechanical pain threshold of rats in each group. **P<0.005, ***P<0.001, ****P<0.0001 are compared between 0.5% RVC, 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL groups and normal saline (NS) group, and the red arrows are the administration time points;

[0029] Fig.14 Figure 2 is the thermal pain threshold of rats in each group, where (b) is the thermal pain threshold of rats in each group on the day before administration; Figure (c) is the thermal pain threshold of rats in each group; *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001 are the comparisons between 0.5% RVC, 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL groups and NS group, and the red arrows are the administration time points;

[0030] Fig.15 The cold pain threshold of rats in each group, (b) is the cold pain threshold of rats in each group on the day before administration; (c) is the cold pain threshold of rats in each group. **P<0.005, ***P<0.001, ****P<0.0001 are compared between 0.5% RVC, 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL groups and NS group, and the red arrows are the administration time points;

[0031] Fig.16 This is the trajectory diagram of the rat open field experiment;

[0032] Fig.17 Total movement distance of each group at different time points, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001 are compared with NS group; #P<0.05, ##P<0.005, ###P<0.0005, ####P<0.0001 are compared with 0.5% RVC group;

[0033] Fig.18 Images of each group were taken 7 and 14 days after injection around the sciatic nerve;

[0034] Fig.19 Hematoxylin-eosin staining of the heart, liver, spleen, lung, brain and kidney on the 7th day after drug injection in each group;

[0035] Fig. 20 HE staining and ELISA expression of inflammatory factors in the tissues around the sciatic nerve after 7 days of drug injection in each group, where a is HE staining of the tissues around the sciatic nerve on the 7th day after drug injection, and the black arrow indicates the residual gel structure; b is the expression of IL-1β in the tissues around the sciatic nerve detected by ELISA; c is the expression of IL-6 in the tissues around the sciatic nerve detected by ELISA; d is the expression of TNF-α in the tissues around the sciatic nerve detected by ELISA; *Compared with the NS group, P<0.05; **Compared with the NS group, P<0.005; ***Compared with the NS group, P<0.0005; ****Compared with the NS group, P<0.0001. #Compared with the RVC group, P<0.05; ##Compared with the RVC group, P<0.005; ####Compared with the RVC group, P<0.0001;

[0036] Fig.21 HE staining of tissues around the sciatic nerve in each group 14 days after injection and the inflammatory scores at 7 and 14 days after injection, where a is HE staining of tissues at 14 days after injection of drugs around the sciatic nerve, and the black arrow indicates the residual gel structure; b is the inflammatory score of tissues at 7 days after injection of drugs around the sciatic nerve; c is the inflammatory score of tissues at 14 days after injection of drugs around the sciatic nerve; d is the myotoxicity score at 7 days after injection of drugs around the sciatic nerve; e is the myotoxicity score at 14 days after injection of drugs around the sciatic nerve;

[0037] Fig. 22 TB staining of sciatic nerve in each group 7 days after drug injection;

[0038] Fig.23 The liver and kidney function of the rats in each group were measured 7 days after injection, where a was the alanine aminotransferase (ALT) content in the blood of the rats in each group; b was the aspartate aminotransferase (AST) content in the blood of the rats in each group; c was the γ-glutamyl transferase (γ-GT) content in the blood of the rats in each group; d was the urea nitrogen (BUN) content in the blood of the rats in each group; e was the creatinine (CREA) content in the blood of the rats in each group; f was the uric acid (UA) content in the blood of the rats in each group. DETAILED DESCRIPTION

[0039] The invention provides a ropivacaine hydrochloride sustained-release microgel. The ropivacaine hydrochloride sustained-release microgel comprises the following raw materials in parts by weight: 21 to 42 parts of ropivacaine hydrochloride, 2.4 parts of collagen and 71.4 parts of lithiasis earth.

[0040] The present invention has no special limitation on the specific sources of ropivacaine hydrochloride (RVC), collagen (COL) and laetrile (LAP), and any conventional commercially available products in the art may be used. The dosage form of the ropivacaine hydrochloride is preferably a powder.

[0041] The present invention also provides a method for preparing the above-mentioned ropivacaine hydrochloride sustained-release microgel, comprising the following steps: mixing the ropivacaine hydrochloride with water to obtain a ropivacaine hydrochloride aqueous solution; mixing the ropivacaine hydrochloride aqueous solution with the lithium ion, and freeze-drying to obtain a powder; dissolving the collagen to obtain a collagen solution; mixing the collagen solution with the powder, and standing at 37° C. to obtain the ropivacaine hydrochloride sustained-release microgel.

[0042] The present invention uses collagen and lithium diatomite to cross-link into a mesh microgel as a delivery carrier for ropivacaine hydrochloride. It can quickly gel at 37°C, and is a green gel preparation method. Figure 1As shown in (a) of FIG. 3 , the blank hydrogel without drug is an injectable liquid at low temperature and can be quickly cross-linked into a non-flowable integral hydrogel at 37°C. Figure 1 As shown in (b), after encapsulating the drug ropivacaine hydrochloride, it becomes micron-sized hydrogel particles, which are liquid at low temperatures and still have a certain fluidity at 37°C. Lithium phosphate (LAP) is a white synthetic montmorillonite clay, a layered hydrated magnesium silicate. When LAP powder is dispersed in an aqueous medium, water will reach the middle layer of LAP and cause it to swell. This induces Na + dissociation of Na and causing them to diffuse into the medium due to the osmotic gradient. + The release of α-lactamase leads to a permanent negative charge on the surface of LAP, which can be used to load cationic drugs, such as Figure 1 As shown in (c) in FIG. 1 . Ropivacaine hydrochloride exists in the form of hydrochloride, with a positive charge on the surface, which combines with the negative charge on the surface of LAP by electrostatic action to achieve drug delivery. The outer layer is wrapped by collagen to form a ropivacaine hydrochloride sustained-release microgel, as shown in FIG. Figure 1 As shown in (d) in the figure, the ropivacaine hydrochloride sustained-release microgel prepared by the present invention is in the form of a white gel with certain fluidity and a porous network structure.

[0043] In the present invention, when the ropivacaine hydrochloride is mixed with water, the amount of water is not particularly limited, as long as it can dissolve the ropivacaine hydrochloride powder. In the present invention, the freeze-drying is preferably performed at -50°C and 30 mtorr for 48 hours; the freeze-drying is preferably performed using a vacuum freeze dryer (manufacturer: GOLD-SIM, specification FD5-3B).

[0044] In the present invention, the preparation method of the collagen solution is preferably: mixing collagen with glacial acetic acid solution, adjusting the pH to 7.0, and then adding water to obtain the collagen solution; the concentration of the glacial acetic acid solution is preferably 0.25 mol / L, and the volume ratio of the glacial acetic acid solution to water is preferably 7:3. In the present invention, the solution used to adjust the pH is 0.1% sodium hydroxide.

[0045] In the process of preparing ropivacaine hydrochloride sustained-release microgel of the present invention, the weight volume ratio of ropivacaine hydrochloride to collagen solution is 21-42 mg: 600 μL. When the collagen solution and powder are mixed and allowed to stand, the standing time is preferably 30 minutes.

[0046] The present invention also provides the use of the above ropivacaine hydrochloride sustained-release microgel or the above preparation method in preparing analgesic products. In the present invention, the product preferably includes a drug.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] The ropivacaine hydrochloride powder used in the following examples was purchased from Selleck with the product number S405801; collagen was purchased from Dubuwuqi Biomedical Technology.

[0051] Example 1

[0052] A ropivacaine hydrochloride sustained-release microgel is prepared by the following method:

[0053] (1) Weigh 21 mg of ropivacaine hydrochloride powder (RVC), add ddH2O until it is completely dissolved, and obtain a ropivacaine hydrochloride aqueous solution. Then weigh 71.4 mg of lithium pyrochloride (LAP) and mix with the above ropivacaine hydrochloride aqueous solution, put it in a -80°C refrigerator and freeze it overnight, and then use a vacuum freeze dryer (manufacturer: GOLD-SIM, specification FD5-3B) at -50°C and 30 mtorr for lyophilization for 2 days to obtain a powder.

[0054] (2) Weigh 2.4 mg of collagen (COL) and add 420 μL of 0.25 mol / L glacial acetic acid to dissolve it, and place it at 4°C overnight for use. Adjust the pH of the previously dissolved collagen to 7.0 with 0.1% sodium hydroxide on ice, and add ddH2O to a final volume of 600 μL to prepare a collagen solution with a concentration of 4 mg / mL.

[0055] (3) The collagen solution obtained in step (2) was fully mixed with the powder obtained in step (1), and allowed to stand at 37° C. for 30 min to obtain a ropivacaine hydrochloride sustained-release microgel with a mass volume percentage of 3.5% (referred to as 3.5% RVC-LAP / COL).

[0056] Example 2

[0057] A ropivacaine hydrochloride sustained-release microgel is prepared by the following method:

[0058] (1) Weigh 42 mg of ropivacaine hydrochloride powder (RVC), add ddH2O until it is completely dissolved, and obtain a ropivacaine hydrochloride aqueous solution. Then weigh 71.4 mg of lithium phosphate (LAP) and mix with the above ropivacaine hydrochloride aqueous solution, freeze in a -80°C refrigerator overnight, and then freeze-dry for 2 days to obtain a powder.

[0059] (2) Weigh 2.4 mg of collagen (COL) and add 420 μL of 0.25 mol / L glacial acetic acid to dissolve it, and place it at 4°C overnight for use. Adjust the pH of the previously dissolved collagen to 7.0 with 0.1% sodium hydroxide on ice, and add ddH2O to a final volume of 600 μL to prepare a collagen solution with a concentration of 4 mg / mL.

[0060] (3) The collagen solution obtained in step (2) was fully mixed with the powder obtained in step (1), and allowed to stand at 37° C. for 30 min to obtain a ropivacaine hydrochloride sustained-release microgel with a mass volume percentage of 7.0% (referred to as 7.0% RVC-LAP / COL).

[0061] Example 3

[0062] The ropivacaine hydrochloride sustained-release microgels prepared in Example 1 and Example 2 were subjected to the following tests. The following test data were statistically analyzed and plotted using GraphPadPrism 9 software and IBM SPSS Statistics 22.0 software. Quantitative data were expressed as mean ± standard deviation. All experimental data were tested for normal distribution before analysis. Data that met the normal distribution were tested using t-tests; data that did not meet the normal distribution were tested using non-parametric tests. P < 0.05 was considered statistically significant:

[0063] 1. NTA particle size detection

[0064] The successfully prepared 3.5% RVC-LAP / COL and 7% RVC-LAP / COL were measured using a ZetaView PMX 110 nanoparticle tracking analyzer (Particle Metrix, Germany). The sample pool was rinsed with a deionized water solution, then calibrated with polystyrene microspheres (100nm), and after calibration, the sample pool was rinsed with a 1×PBS solution. The sample was diluted one thousand times with a 1×PBS solution and then injected for inspection. The measurement was repeated three times. The particle size and particle size distribution coefficient Span were obtained, and the calculation formula is shown below.

[0065]

[0066] Among them, X10, X50 and X90 are the particle sizes when the sample accumulates to 10%, 50% and 90%. The smaller the Span value, the narrower the particle size distribution and the more uniform the size.

[0067] The results are as follows Figure 2As shown. The average particle size of 3.5% RVC-LAP / COL was 271.47±4.68nm, Span0.9. ​​The average particle size of 7.0% RVC-LAP / COL was 255.7±6.94nm, Span 0.9. The particle sizes of these two concentrations are within the typical size range of microgels, and the Span values ​​are less than 1, indicating that the particle size is relatively uniform.

[0068] 2. Scanning electron microscope (SEM) observation

[0069] The prepared 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL were completely freeze-dried for 48 hours and fixed on the sample stage of a scanning electron microscope. Then they were placed in a gold spraying pool for gold spraying, and the internal structure of the microgel was observed using a scanning electron microscope (SEM).

[0070] The results are as follows Figure 3 As shown. Under an optical microscope, multiple irregularly shaped translucent tiny gel particles ( Figure 3 a). Under electron microscopy, 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL both showed a sponge-like porous structure with irregular pores of different sizes. The pores are interconnected, providing a structural basis for drug loading. The surface is rough and uneven, which is conducive to delaying the release of drugs.

[0071] 3. Determination of in vitro release behavior

[0072] (1) Determination of ropivacaine hydrochloride standard curve

[0073] Weigh 10 mg of ropivacaine hydrochloride powder and dissolve it in 10 mL of ddH2O to prepare a 1000 μg / mL ropivacaine hydrochloride standard solution, then dilute it with ddH2O to prepare 750 μg / mL, 500 μg / mL, 250 μg / mL, 100 μg / mL, and 20 μg / mL ropivacaine hydrochloride standard solutions with concentration gradients. Then use an ELISA instrument to detect the absorbance (OD value) of the standard solution at 262 nm, take the average value of multiple measurements, and draw a standard curve of ropivacaine hydrochloride concentration and absorbance. The results are as follows Figure 4 As shown, the linear regression equation is: Y = 4 × 10 -5 X+3.3524,R 2 =0.9995. This indicates that the standard curve of ropivacaine hydrochloride has a good linear relationship within the concentration range.

[0074] (2) Release experiment

[0075] Ropivacaine hydrochloride powder was diluted to 30 mg / mL with ddH2O, and then 1 mL of the solution was injected into a dialysis bag (8-14 kDa). The sealed dialysis bag was placed on a shaker containing 300 mL of ddH2O, and 1 mL of equal amounts of ddH2O were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 120, and 168 h. Each time, 1 mL of fresh ddH2O was replaced. The release medium was analyzed using an ELISA reader. The data are expressed as the cumulative ropivacaine release rate for each sample (n=5).

[0076] The results are as follows Figure 5 As shown in the figure, the in vitro release rate of ropivacaine hydrochloride aqueous solution (0.5% RVC) is fast and the release degree is high. The drug release can reach more than 85% in 6 hours, and the cumulative release rate basically reaches equilibrium in 8 hours. Both 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL show sustained release behavior in vitro, and the cumulative release time in vitro can reach about 7 days.

[0077] 4. Determination of water content

[0078] The prepared 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL were centrifuged at 5000rpm and 25°C for 10 minutes. The supernatant was removed and weighed, and the initial weight was recorded as W1. Then, the mixture was dried to a constant weight and weighed again as W2. The measurement was repeated 5 times and the average value was taken. The moisture content (g / g) was calculated according to the moisture content formula: moisture content (g / g) = [(W2-W1) / W1] × 100%.

[0079] The results are as follows Figure 6 As shown in a, the water content of 3.5% RVC-LAP / COL was 64.91±1.02%, and the water content of 7.0% RVC-LAP / COL was 65.30±0.43%, and the difference was not statistically significant (P>0.05).

[0080] 5. Swelling test

[0081] Place the sample (blank hydrogel without drug, that is, the difference from Example 1 is that ropivacaine hydrochloride is not added, and the rest is the same as Example 1, recorded as LAP / COL) in an electric constant temperature incubator at 50-60°C and dry in a drying oven for 72-84 hours to constant weight, weigh it and record it as the initial dry mass M1, place it in deionized water, and then place it in a 37°C constant temperature water bath. Remove moisture from the surface of the sample at different time points, weigh the mass after swelling with an electronic balance, and record it as M2, until the swelling equilibrium is reached and the mass no longer changes. Repeat the measurement 5 times and take the average value. Calculate the swelling rate (%) using the formula: Swelling rate (%) = [(M2-M1) / M1] × 100%, where M1 is the initial dry mass of the sample, and M2 is the wet mass of the sample after absorbing water at different time points, so as to draw a swelling curve of the swelling rate relative to time. The results are as follows Figure 6 As shown in b, it shows that the LAP and COL cross-linked product reaches swelling equilibrium in about 6 hours, the equilibrium swelling rate is small, and the water absorption capacity is relatively weak. These two characteristics make the ropivacaine hydrochloride microgel have abundant water content to nourish the nerves when applied around the nerves, but will not cause local nerve compression due to its strong water absorption performance and excessive swelling.

[0082] Example 4

[0083] In vitro cytotoxicity analysis of ropivacaine hydrochloride sustained-release microgels: Adrenal medullary pheochromoma cells (PC12) and microglial cells (BV2), which are commonly used to evaluate neurotoxicity, were selected to detect the in vitro cytotoxicity and inflammatory response of ropivacaine hydrochloride sustained-release microgels. PC12 cell line (highly differentiated) was purchased from Hechuang Biotechnology Co., Ltd.; BV2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. The following data were statistically analyzed and plotted using GraphPadPrism 9 software and IBM SPSS Statistics 22.0 software. Quantitative data are expressed as mean ± standard deviation. All experimental data were first tested for normal distribution before analysis. Data that met the normal distribution were tested using t-test; data that did not meet the normal distribution were tested using nonparametric tests, and P < 0.05 was considered statistically significant.

[0084] 1. Experimental methods

[0085] 1.1 Culture and grouping of PC12 cells and BV2 cells

[0086] (1) PC12 cell culture and passaging: PC12 cells were cultured in a 37°C, 5% CO2 incubator with a medium containing 10% FBS1640. When the PC12 cells grew to 80% to 90% fusion rate, the medium was discarded, and the cells were washed 2-3 times with PBS. The PBS was discarded and 1 mL of 0.25% trypsin (containing 0.02% EDTA) was added. After digestion at 37°C for 1 min, the cells were gently shaken under a microscope to observe the cell detachment. After the cells were completely detached, complete medium was added to terminate the digestion, and the cells were collected into a centrifuge tube. The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. After adding 5 mL of the prepared complete medium and mixing by pipetting, 1-2 mL was inoculated into a new T25 culture flask, and then the complete medium was added to make up to 5 mL to complete the passaging.

[0087] (2) BV2 cell culture and passaging: BV2 cells were cultured in a 37°C, 5% CO2 incubator with a 10% FBSDMEM high-glucose medium. When the BV2 cells grew to 80% to 90% fusion rate, the medium was discarded, and the cells were washed 2-3 times with PBS. The PBS was discarded and 1 mL of 0.25% trypsin (containing 0.02% EDTA) was added. After digestion at 37°C for 1 min, the cells were gently shaken under a microscope to observe the cell detachment. After the cells were completely detached, complete medium was added to terminate the digestion, and the cells were collected in a centrifuge tube. The tube was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. After adding 5 mL of the prepared complete medium and mixing by pipetting, 1-2 mL was inoculated into a new T25 culture flask, and then the complete medium was added to make up to 5 mL to complete the passaging.

[0088] (3) Cell cryopreservation: When the cell density of the T25 culture flask reaches about 80% to 90% under a microscope, the cells can be cryopreserved. Discard the old culture medium in the culture flask, wash 2-3 times with PBS solution, and discard the PBS. Add 1mL of 0.25% trypsin to digest the cells for 1 minute, then gently shake under a microscope to observe the cell detachment. After the cells are completely detached, add complete culture medium to terminate digestion, transfer to a centrifuge tube with a Pasteur tube, centrifuge at 1000rpm for 5 minutes, and discard the supernatant. Add 1mL of serum-free cell freezing solution and gently blow to resuspend. Transfer to a cryotube, seal, mark, and freeze in a -80℃ refrigerator.

[0089] (4) Grouping and processing of in vitro cell tests:

[0090] Blank control group (Control group): No treatment was performed and the cells were directly cultured with complete medium.

[0091] Ropivacaine hydrochloride group (RVC group): The half inhibitory concentration (IC 50: 402.7 μM), according to mass (mg) = concentration (mM) × volume (mL) × molecular weight (g / mol), the concentration of ropivacaine hydrochloride is 125 μg / mL, and the cells are cultured after being prepared with complete culture medium.

[0092] Ropivacaine hydrochloride microgel group (RVC-LAP / COL group): Preparation of ropivacaine hydrochloride microgels with the same content as the above RVC group: The ropivacaine hydrochloride microgels were prepared by the method of Example 1, except that the final concentration of ropivacaine hydrochloride in the microgels was 125 μg / mL, and the amounts of other raw materials such as lithium talc and collagen were converted in geometric proportions according to the proportional relationship in Example 1, and the rest were the same as Example 1. The cells were cultured in the supernatant after being placed in complete culture medium for 24 hours using an extract.

[0093] Blank material group (LAP / COL group): Preparation of drug-free microgels: The difference from the above RVC-LAP / COL group is that ropivacaine hydrochloride was not added, and the rest was the same as the above RVC-LAP / COL group. The cells were cultured by extracting the supernatant after being placed in complete culture medium for 24 hours.

[0094] 1.2 CCK8 method to measure the cytotoxicity of ropivacaine hydrochloride sustained-release microgels

[0095] The cells were digested with trypsin, and PC12 and BV2 cell suspensions were prepared and counted. 4 The density of cells / well was inoculated into a 96-well plate, and after incubation in a 37°C, 5% CO2 incubator for 24 hours, the culture medium was discarded, and 1, 5, 10, 20, 50, 100, 500 μM continuous concentrations of ropivacaine hydrochloride aqueous solution and ropivacaine hydrochloride microgel soaking extract were prepared with complete culture medium, and then added to the cells for 24 hours and 48 hours. After the corresponding culture time, 10 μL CCK8 reagent was added to each well and continued to incubate in the incubator for 1 hour. After incubation, tin foil was used to protect from light, and the OD value was recorded at 450nm, and the cell viability was calculated.

[0096] 1.3 Live and dead cell staining

[0097] (1) Inoculation and culture: Prepare PC12 and BV2 cell suspensions and count them. 5 The cells were seeded into 6-well plates at a density of 1.54 μg / well and cultured for 24 h. Then, the cells were treated with cell intervention for 24 h according to the above grouping method.

[0098] (2) Washing: Discard the supernatant and wash once with PBS. Be careful to avoid excessive washing that may cause loss of dead cells.

[0099] (3) Staining: CalceinAM (1000×) and PI (1000×) were diluted 1000 times with complete culture medium to CalceinAM (1×) and PI (1×), and then 1 mL of CalceinAM (1×) and PI (1×) were added to each well. The wells were wrapped in tin foil and incubated in a 37°C, 5% CO2 incubator for 30 min.

[0100] (4) Observation: After staining is completed, observe the staining effect under a fluorescence microscope (live cells are stained with green fluorescence by Calcein AM, and dead cells are stained with red fluorescence by PI). Note that the entire process must be kept away from light.

[0101] 1.4 Griess method to measure the expression of nitric oxide (NO) in cell supernatant induced by LPS

[0102] (1) Preparation of LPS: Weigh 10 mg of LPS using an electronic balance and add 10 mL of sterile PBS to dissolve, filter using a 0.22 μM filter to prepare a 1 mg / mL solution, and then dilute to 250 ng / mL using complete culture medium. Dispense into EP tubes and place in a -80°C refrigerator for later use.

[0103] (2) Cellular inflammation model: BV2 cells with good growth status were prepared into suspension and counted. Then, 1×10 4 The cells were inoculated at a density of 100 cells / well, and after the cells adhered to the wall, each group of cells was treated with complete medium containing 250 ng / mL LPS for 24 h.

[0104] (3) According to the above grouping method, three groups of low, medium and high concentrations were set up, with ropivacaine hydrochloride IC 50 Medium concentration group, 10 times IC 50 High concentration group, 1 / 10 times IC 50 For the low concentration group, after 24 hours of cell intervention treatment, the cell supernatant of each group was collected. According to the instructions, the standard was diluted to a concentration range of 1-100μM, and the standard and sample were added to the 96-well plate at 50μL / well, with 3 replicates for each group. Then, room temperature Griess Reagent I and room temperature Griess Reagent Ⅱ were added to each well at 50μL / well. The sample absorbance at 540nm was measured. A standard curve was drawn, and the sample NO concentration was calculated based on the standard curve.

[0105] 2. Experimental results

[0106] 2.1 Observation of PC12 and BV2 cell growth morphology

[0107] The changes in each group after 24 hours of treatment are as follows Figure 7As shown in the figure, under normal circumstances, these PC12 cells are in a highly differentiated state, with spindle-shaped cells and clear outlines. Multiple slender synapses of varying numbers can be seen, similar to neuronal axons. The cell morphology of the LAP / COL group and the RVC-LAP / COL group showed no increase in synaptic cell bodies, and other changes were not obvious. The cell morphology of the RVC group changed significantly, cell differentiation was inhibited, cell bodies increased, and synapses were significantly reduced and shortened. BV2 cells exist in two types: round and spindle, with rich cytoplasm and nuclei, in a semi-suspended and semi-adherent state. Suspended cells are round, and adherent cells are both round and spindle-shaped. Round cells have a high refractive index and transparent edges. Spindle cells have slender black protrusions. There was no obvious change in cell morphology in the LAP / COL group and the RVC-LAP / COL group. The cells in the RVC group were clumps with blurred boundaries, poor refractive index, and some cell death.

[0108] 2.2 Effect of CCK8 assay on the activity of BV2 and PC12 cells

[0109] The results are as follows Figure 8 As shown, after culturing PC12 cells for 24 hours, the RVC group and RVC-LAP / COL group showed statistical differences in cell growth inhibition at 100μM (P<0.05), with cell proliferation rates of 77.99±5.03% and 90.58±1.89%, respectively; the cell proliferation rates at 500μM were 69.99±5.00% and 86.71±3.18%, respectively. After culturing for 48 hours, the RVC group and RVC-LAP / COL group showed statistical differences in cell growth inhibition at 50μM (P<0.05), with cell proliferation rates of 80.88±3.29% and 91.19±1.69%, respectively; the cell proliferation rates at 100μM were 74.22±2.26% and 89.07±1.59%, respectively; the cell proliferation rates at 500μM were 60.23±1.69% and 81.40±1.44%, respectively. After culturing BV2 cells for 24 hours, the growth inhibition of RVC group and RVC-LAP / COL group also showed statistical difference at 100μM (P<0.05), and the cell proliferation rates were 82.16±2.03% and 93.90±3.62%, respectively; the cell proliferation rates at 500μM were 65.67±8.02% and 85.29±1.73%, respectively. After culturing for 48 hours, the growth inhibition of RVC group and RVC-LAP / COL group showed statistical difference at 100μM (P<0.05), and the cell proliferation rates were 76.01±4.37% and 90.96±3.08%, respectively; the cell proliferation rates at 500μM were 57.24±4.44% and 85.29±1.73%, respectively.

[0110] The above experimental results show that ropivacaine hydrochloride and ropivacaine hydrochloride microgels show time- and concentration-dependent toxicity to cells as the concentration and time change. The cell growth inhibition is not obvious at low concentrations, and the difference in cell viability is not statistically significant (P>0.05). However, as the concentration increases, the RVC group has a more obvious inhibition on cell growth, and the cell viability shows a statistical difference. This shows that RVC-LAP / COL, as a sustained-release agent, can reduce the impact on cell proliferation, reduce cytotoxicity, and improve safety through smooth release.

[0111] 2.3 Live and dead cell staining

[0112] The results are as follows Fig. 9 As shown in the results, most of the PC12 cells in the LAP / COL and RVC-LAP / COL groups were stained with green fluorescence after culture, and the cell survival rates were high, 95.86% and 92.17, respectively. The red area in the RVC group increased significantly, and the cell survival rate was 75.20%. The same phenomenon was observed in BV2 cells, with the survival rates of the LAP / COL and RVC-LAP / COL groups being 97.88% and 93.98%, respectively, and the cell survival rate in the RVC group being only 76.43%. This experiment shows that LAP / COL has low cytotoxicity and can be used as an ideal biomaterial; RVC-LAP / COL sustained-release microgels can slow down the drug release rate, continuously and stably release drugs, thereby reducing the toxic side effects of drugs and improving safety.

[0113] 2.4 Determination of NO expression in BV2 cell supernatant by Griess method

[0114] The results are as follows Fig.10 As shown in the results, NO is produced by nitric oxide synthase (iOS) and plays an important role in promoting inflammation. By measuring the expression of NO, the results showed that there was no statistical difference in NO expression between the LAP / COL group and the blank control group (P>0.05), indicating that the blank carrier did not cause increased NO expression. At high concentrations, there was no significant difference in NO expression between the RVC-LAP / COL group and the control group (P>0.05); the NO expression in the RVC group was increased compared with the control group and the RVC-LAP / COL group, and the difference was statistically significant (P<0.0001). At low and medium concentrations, there was no significant difference in NO expression among the RVC group, the RVC-LAP / COL group, and the control group. This shows that at lower concentrations, RVC-LAP / COL sustained-release microgels have no obvious advantages over RVC. With the increase in concentration, the advantages of RVC-LAP / COL slow and stable release are more prominent, avoiding the peak and valley phenomenon and reducing toxic side effects and inflammatory reactions.

[0115] The above results show that the in vitro safety evaluation of the ropivacaine hydrochloride sustained-release microgel of the present invention shows good biocompatibility and no obvious cytotoxicity. Compared with ropivacaine hydrochloride, it has no obvious effect on cell proliferation, differentiation and inflammatory response, and its advantages become more obvious as the concentration increases.

[0116] Example 5

[0117] Application of the ropivacaine hydrochloride sustained-release microgels prepared in Example 1 and Example 2 in the rat SNI model

[0118] The chronic pain model was established by constructing a selective injury (Spared Nerve Injury, SNI) model of sciatic nerve branches, and the effect and duration of ropivacaine hydrochloride sustained-release microgel were evaluated by sciatic nerve block, and the safety of various tissues and organs of the body was evaluated. The data of this embodiment were statistically analyzed and plotted using GraphPad Prism 9 software and IBM SPSS Statistics 22.0 software. Quantitative data were expressed as mean ± standard deviation. All experimental data were first tested for normal distribution before analysis, behavioral analysis used repeated measures analysis of variance, and post hoc test used Bonferroni, and P < 0.05 was considered statistically significant.

[0119] This study used SPF male SD rats (200-250g), which were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. and kept in an SPF environment with free access to food and water. Six rats were fed in a cage, with free access to food and water, and the room temperature was maintained at 22-25°C to maintain a normal circadian rhythm. This study was approved by the Animal Ethics Committee of Central South University (CSU-2023-0353).

[0120] 1. Experimental methods

[0121] 1.1 Experimental animal grouping and technical route

[0122] (1) Experimental grouping: 30 male SD rats were randomly divided into 5 groups (n=6): ropivacaine hydrochloride group (0.5% RVC, the mass volume percentage of RVC in the ropivacaine hydrochloride aqueous solution was 0.5%), normal saline group (NS), blank material group (LAP / COL), 3.5% RVC-LAP / COL group (3.5% RVC-LAP / COL) and 7.0% RVC-LAP / COL group (7.0% RVC-LAP / COL). The left SNI model was established in rats under general anesthesia. Behavioral measurements were performed after modeling. After the hyperalgesia was stabilized on the 5th day after surgery, the left sciatic nerve was blocked for the 5 groups of rats, and 0.6 mL of the corresponding drugs were injected respectively. Behavioral tests, movement assessments, open field tests, etc. were performed on the 5 groups of rats 2h, 4h, 6h, 1d, 3d, 5d, and 7d after injection, including mechanical paw withdrawal threshold (PWT), cold withdrawal latency (ColdWithdrawal Latency, CWL), thermal withdrawal latency (ThermalWithdrawal Latency, TWL), kinematic scores, and open field tests.

[0123] (2) Technical route: Fig.11 shown.

[0124] 1.2 Establishment of the rat sciatic nerve branch selective injury (SNI) model

[0125] One day before surgery, the rats were subjected to behavioral baseline tests and their body weights were recorded. The rats were fasted from food and water the night before modeling. A 1% concentration of sodium pentobarbital was prepared in advance and injected intraperitoneally into the rats at 50 mg / kg for anesthesia. After the rats were anesthetized, the hair on the left hind limb was removed and disinfected with iodine 3 times. A longitudinal incision of approximately 1-2 cm was made on the back of the left thigh of the rat. After blunt separation of the biceps femoris, the sciatic nerve and its three main branches were exposed: the common peroneal nerve, the tibial nerve, and the sural nerve. Fig.12 As shown in the figure, the sural nerve is located on the far right side of the left leg and is the thinnest of the nerves. Referring to the Woolf method, the tibial nerve and the common peroneal nerve were ligated with 4-0 silk thread. The lower limb flexion can be observed and the nerve was cut off about 2 mm distal to the ligature. During the operation, pay attention to protecting the integrity of the sural nerve, otherwise it will lead to modeling failure. Finally, the wound muscle and skin were intermittently sutured and disinfected again after suturing.

[0126] 1.3 Paw withdrawal threshold (PWT) in rats

[0127] Rats were selected for mechanical pain threshold measurement 1 day before modeling, 3 days and 5 days after surgery, and 2 hours, 4 hours, 6 hours, 1 day, 3 days, 5 days and 7 days after drug administration. Rats in each group were placed individually on an elevated wire mesh, separated by a 10 cm × 10 cm glass grid, and covered with a perforated acrylic plate lid. After the rats were adapted for 30 minutes and were quiet, VonFrey needles (1.4, 2, 4, 6, 8, 10, 15, 26 g) were used to vertically stimulate the 1 / 3 skin of the outer side of the plantar lateral lower limb of the rat on the surgical side. The fiber was slightly bent into an "S" shape for 3-5 seconds. The rat's rapid licking or retraction of the foot was considered a positive sign. The interval between each test was at least 5 minutes. The measurement was repeated 5 times and the average value was taken. The curvature of the cilia of the VonFrey needle was kept the same as much as possible for each measurement. If no positive reaction occurred after 26 g acupuncture, 26 g was taken as the pain threshold for this measurement. The minimum force was 1.4 g. If a positive reaction occurred less than this value, it was recorded as 1.4 g.

[0128] 1.4 Thermal withdrawal latency (TWL) in rats

[0129] Rats were selected for heat pain threshold measurement 1d before modeling, 3d, 5d after surgery, and 2h, 4h, 6h, 1d, 3d, 5d, and 7d after drug administration. The heat withdrawal latency in response to radiant heat stimulation was measured using a hot and cold plate analgesia instrument. The rats were allowed to adapt to the pain threshold plate for 30min before the measurement began. The temperature of the hot plate was then set to 52.5±0.5℃, and the measurement began after the temperature of the hot plate became constant. The rat body was held upright with one hand so that the sole of the left foot of the rat touched the hot plate, and the timing switch was pressed at the same time. When the rat withdrew its foot due to heat, the timing switch was pressed again to stop the timing. The screen will display the time of contact with the hot plate, which is the heat withdrawal latency. The maximum duration of each test is 30s. The measurement was repeated 5 times, the maximum and minimum values ​​were removed, and the remaining 3 values ​​were averaged as the final withdrawal latency. The interval time for each test was at least 5min.

[0130] 1.5 Cold withdrawal latency (CWL) of rats

[0131] Rats were selected for cold pain threshold measurement 1d before modeling, 3d, 5d after surgery, and 2h, 4h, 6h, 1d, 3d, 5d, and 7d after drug administration. The cold foot withdrawal latency in response to radiant cold stimulation was measured using a hot and cold plate analgesia instrument. The rats were allowed to adapt to the pain threshold plate for 30min before the measurement began. The temperature of the cold plate was then set to 4±0.5℃, and the measurement was started after the temperature of the cold plate was constant. The rat body was held upright with one hand so that the sole of the left foot of the rat touched the cold plate, and the timing switch was pressed at the same time. When the rat withdrew its foot due to the cold, the timing switch was pressed again to stop the timing. The screen will display the time of contact with the cold plate, which is the cold foot withdrawal latency, and the cutoff time is set to 120s. Each rat was measured 5 times, the maximum and minimum values ​​were removed, and the remaining 3 values ​​were averaged as the final foot withdrawal latency, with an interval of at least 5min each time.

[0132] 1.6 Motor function scores of rats

[0133] After the pain threshold test, the rats were tested for motor function at the same time point and the basic conditions of each group of rats, such as walking ability, body weight, drinking water, and eating habits, were observed and recorded. The motor function score of rats was performed according to the following method:

[0134] (1) Placement reflex: The rat's forelimbs are placed slightly higher than its hind limbs. The operator observes whether the rat's hind limbs will subconsciously be lifted onto the table when the dorsal surface of the hind limb touches the edge of the table.

[0135] (2) Grasping reflex: The operator places the rat on a wire mesh and records whether the rat's hind paws grasp the wire mesh.

[0136] (3) Righting reflex: The operator places the rat on its back on a flat surface and records whether the rat immediately corrects to an upright position.

[0137] 1.7 Open field test (OFT) in rats

[0138] Rats were selected for open field experiments 1 day before modeling, 3 days and 5 days after surgery, and 2 hours, 4 hours, 6 hours, 1 day, 3 days, 5 days and 7 days after drug administration. The rats were placed in a 40cm×100cm×100cm open field box to adapt to the environment for 10 minutes, and then the rats were taken out. The rats were placed in the center of the open field box in turn, and a digital camera was set up about 2 meters above it to start recording and observing the data of each rat within 10 minutes. After observing each rat, the feces and urine of the rats were cleaned with 75% alcohol to prevent the residual feces and odor of the rats in this experiment from affecting the results of the next test. After waiting for drying, the rats were replaced and the above experiment was repeated. The movement trajectory of the rats was analyzed using Smartv3.0 software.

[0139] 1.8 Specimen Collection

[0140] (1) Perfusion: First, 1% sodium pentobarbital was injected intraperitoneally to deeply anesthetize the rat. After reaching the appropriate depth of anesthesia, the rat's chest and abdominal hair were removed, the rat's limbs were fixed on the operating table, the rat's abdominal cavity and diaphragm were cut open, the ribs were cut open, the xiphoid process was pulled up with tissue forceps to expose the rat's heart and liver, the scalp needle was inserted and fixed from the rat's apex, and a small incision was quickly made in the rat's right atrial appendage. The needle tail was connected to a syringe, and about 100 mL of 0.9% NaCl was slowly perfused to make the rat's liver and lungs change from red to white, and the right atrium outflow fluid became clear. Then, 50 mL of 4% paraformaldehyde (PFA) at 4°C was slowly perfused. The rat's limbs and tail twitched, which was a sign of successful perfusion.

[0141] (2) Sampling and processing: After the perfusion, the rats were quickly placed on an ice box, and the heart, liver, brain, lung, kidney, spleen, sciatic nerve, and muscle tissue around the injection were removed. They were placed in a centrifuge tube and fixed in 4% PFA for 48 hours. They were then stored in a 4°C refrigerator.

[0142] 1.9 Tissue paraffin embedding and sectioning

[0143] (1) Fixation: After sampling, place the tissue block in PFA at 4°C for 48 hours. (2) Washing: After fixation, rinse with running water three times, 20 minutes each time, to wash off the PFA. (3) Dehydration: Place the tissue in 75% ethanol solution, 85% ethanol solution, and 95% ethanol solution for 2 hours each, and dehydrate with 100% ethanol for 30 minutes. Adjust the dehydration time according to the size and type of the tissue. (4) Transparent: Immerse the dehydrated tissue block in environmentally friendly dewaxing and transparent liquid for 1 hour, and transparent twice. (5) Wax immersion: After the paraffin is melted, place the tissue block in paraffin for a total of three times. The first time is 15 minutes, the second time is 30 minutes, and the third time is 1 hour. (6) Embedding: Pour the melted paraffin into the mold, place the tissue block in the mold, wait for it to cool and solidify naturally, then remove the wax block, number it and save it. (7) Slicing and baking: Use a paraffin slicer to cut the wax block into 4μm slices and place it in a warm water bath to flatten it naturally. Then insert the slide into the water surface at an angle so that the slice is attached to the center of the slide. Then bake the slide in a 60℃ oven overnight, take it out and store it at room temperature for later use.

[0144] 1.10 Hematoxylin-eosin (HE) staining

[0145] (1) Dewaxing of paraffin sections: Place the paraffin sections of the heart, liver, spleen, lung, kidney, brain and muscle tissues in an environmentally friendly dewaxing transparent solution, dewax for 30 minutes at room temperature, and dewax twice. (2) Nuclear staining: Place the sections in a hematoxylin staining solution for 5 minutes, and wash off the excess staining solution with tap water. (3) Differentiation: The sections are differentiated with 1% hydrochloric acid ethanol for 5 seconds, and then rinsed with tap water. (4) Blueing: The sections are blued with a hematoxylin blueing solution for 5 seconds, and then rinsed with tap water. (5) Staining: The sections are stained with eosin staining solution for 2 to 5 minutes, and then rinsed with tap water. (6) Dehydration: After wiping off the moisture on the slide, immerse in 80% alcohol for dehydration for about 1 to 2 minutes; transfer to 90% alcohol for dehydration for about 1 to 2 minutes; and finally transfer to anhydrous ethanol for dehydration for 1 to 2 minutes. (7) Transparency: The sections are transparentized with an environmentally friendly dewaxing transparent solution for 5 minutes, and then replaced with a fresh environmentally friendly dewaxing transparent solution for another 5 minutes. (8) Sealing: Add neutral gum to seal the slides and place them flat to dry. (9) Microscopic examination: Use an optical microscope with different magnifications to observe the morphological changes of each tissue and organ and take photos.

[0146] 1.11 Toluidine blue (TB) staining

[0147] (1) Dewaxing of paraffin sections: Immerse the paraffin sections of the sciatic nerve in an environmentally friendly dewaxing transparent liquid, dewax at room temperature for 30 minutes according to the thickness of the sections, and dewax twice. (2) Toluidine blue staining: Stain the tissue sections in toluidine blue dye solution for 5 minutes, and then rinse them thoroughly with tap water. (3) Differentiation: After washing the sections with water, differentiate them with 1% hydrochloric acid ethanol. Observe the differentiation under a microscope until the nerves are purple-blue and clear. Wash with tap water to terminate the differentiation. (4) Transparency: The sections are then dehydrated with fresh anhydrous ethanol for 5 minutes, and then transparentized with environmentally friendly dewaxing transparent liquid for 5 minutes. Replace with fresh environmentally friendly dewaxing transparent liquid and transparentize for another 5 minutes. (5) Sealing: Add neutral gum for sealing and lay flat to dry. (6) Microscopic examination: Use an optical microscope of different magnifications to observe the morphological changes of the sciatic nerve and take pictures.

[0148] 1.12 Inflammation score

[0149] The samples were scored for inflammation and myotoxicity according to Table 1 below. The inflammation and myotoxicity scores are subjective assessments. The myotoxicity score reflects two characteristics of local anesthetic myotoxicity: nuclear internalization and regeneration. Nuclear internalization is characterized by muscle cells that are normal in size and color, but the nucleus is away from the normal position of the cell periphery. Regeneration is characterized by atrophy of muscle cells, cells with a small amount of eosinophilic cytoplasm and darkly stained nuclei.

[0150] Table 1 Inflammation and myotoxicity scores

[0151]

[0152] 1.13 Rat liver and kidney function determination

[0153] On the 7th day after administration, before specimen collection, about 5 mL of blood was collected by cardiac puncture and placed in a heparin anticoagulant tube. Then centrifuged at 3000 rpm and 4°C for 10 min, and the upper serum was taken into a clean centrifuge tube. Biochemical indicators reflecting liver function and kidney function, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (γ-GT), urea nitrogen (BUN), creatinine (CREA), and urea (UA), were tested by a biochemical detector.

[0154] 1.14ELISA

[0155] 1.14.1. Sample collection and storage

[0156] Take 40 mg of muscle tissue around the sciatic nerve at the injection site and wash with PBS to remove blood stains. Use scissors to disassemble large pieces of tissue and place them in a centrifuge tube. Add an appropriate amount of PBS and sterilized zirconium oxide grinding balls, and place the centrifuge tube on a grinder to grind into a homogenate. Then centrifuge the muscle tissue homogenate at 4°C and 5000 rpm for 10 minutes to take the supernatant.

[0157] 1.14.2. BCA protein quantification

[0158] (1) Dilute the standard: dilute the standard to 2μg / μL, 1μg / μL, 0.5μg / μL, 0.25μg / μL, 0.12μg / μL, 0.0625μg / μL, 0μg / μL with sample diluent according to the instructions. (2) Preparation of BCA working solution: thoroughly mix BCA-A and BCA-B at a volume ratio of 50:1. (3) Quantitative detection: add 50μL / well of the standard and the sample to be tested to a 96-well plate, add 200μL BCA working solution to each well, mix thoroughly and react at room temperature for 30 minutes. Then use an enzyme reader to measure the absorbance (OD value) at a wavelength of 562nm and record it. (4) Draw the standard protein curve, fit the regression equation and calculate the protein concentration in the sample to be tested.

[0159] 1.14.3. Reagent preparation

[0160] (1) Preparation of standard: After centrifugation, dissolve the standard in 1 mL of sample diluent, pipette repeatedly to mix thoroughly, and set aside for later use. Take 7 1.5 mL clean centrifuge tubes and label them A0-A6, and add 250 μL of sample diluent to each. Pipette 250 μL of standard into A6 and gently pipette to mix. Then pipette 250 μL from A6 into A5 and gently pipette to mix. Continue to dilute the standard in equal proportions. A0 is the sample diluent. (2) Preparation of washing solution: Dilute the concentrated washing solution with deionized water at a ratio of 1:25. (3) Preparation of biotin-labeled antibody working solution: Dilute the biotin-labeled antibody solution with biotin-labeled antibody diluent at a ratio of 1:100. (4) Preparation of horseradish peroxidase-labeled avidin working solution: Dilute the horseradish peroxidase-labeled avidin with horseradish peroxidase-labeled avidin diluent at a ratio of 1:100.

[0161] 1.14.4. Operation steps (1) Sample addition: Add 100 μL / well of the standard and the sample to be tested to a 96-well plate, shake gently to mix, and incubate at 37°C for 2 hours. Discard the liquid in the plate and spin dry. (2) Add 100 μL / well of the biotin-labeled antibody working solution, cover with a sealing film, and then incubate at 37°C for 1 hour. Discard the liquid in the plate, add the washing solution to wash the plate 3 times according to the instructions, and spin dry. (3) Add 100 μL / well of the horseradish peroxidase-labeled avidin working solution, cover with a sealing film, and incubate at 37°C for 1 hour. Discard the liquid in the plate, add the washing solution to wash the plate 3 times according to the instructions, and spin dry. (4) Add 90 μL / well of the substrate solution, and then color develop at 37°C for 30 minutes in the dark. (5) Stop the reaction: Add 50 μL / well of the stop solution. (6) Immediately after the reaction is terminated, measure the OD value of each well at a wavelength of 450 nm using an ELISA reader.

[0162] 2. Experimental results

[0163] 2.1 General Observations

[0164] The rats in each group were in good mental state, drank and ate normally, reacted quickly, had smooth and shiny hair, and had no obvious infection in the postoperative wound of the left leg, which healed well. It was observed that the gait of the rats in each group was basically normal, and there was no obvious deformity or paralysis of the lower limbs on the operated side.

[0165] 2.2 Mechanical pain threshold of rats in each group

[0166] After the rat SNI model was established, the mechanical pain threshold began to decrease significantly on the third day, and the mechanical pain threshold dropped to a stable level around the fifth day, and the drug was injected around the sciatic nerve. Fig.13 As shown in (b), there was no significant difference in mechanical pain thresholds among the groups before drug injection. Fig.13As shown in (c), the pain threshold of rats in the NS group and LAP / COL group did not change significantly after injection, indicating that saline and blank materials themselves had no analgesic effect; 2 hours after injection, the mechanical pain threshold of the 0.5% RVC group was higher than that of the NS group (P<0.0001), the mechanical pain threshold of the 3.5% RVC-LAP / COL group was higher than that of the NS group (P<0.001), and the mechanical pain threshold of the 7.0% RVC-LAP / COL group was higher than that of the NS group (P<0.0001), and the differences were statistically significant. 4 hours after injection, the mechanical pain threshold of the 0.5% RVC group was higher than that of the NS group (P<0.0001), the mechanical pain threshold of the 3.5% RVC-LAP / COL group was higher than that of the NS group (P<0.005), and the mechanical pain threshold of the 7.0% RVC-LAP / COL group was higher than that of the NS group (P<0.0001), and the differences were statistically significant. Six hours after injection, the mechanical pain threshold of the 0.5% RVC group was not significantly different from that of the NS group. The mechanical pain threshold of the 3.5% RVC-LAP / COL group was still higher than that of the NS group 1 day after injection (P<0.001), and the difference was statistically significant. Three days after injection, there was no significant difference between the NS group and the 7.0% RVC-LAP / COL group. The mechanical pain threshold of the 7.0% RVC-LAP / COL group was still higher than that of the NS group 5 days after injection (P<0.005), and the difference was statistically significant. There was no significant difference between the 7.0% RVC-LAP / COL group and the NS group. The mechanical pain threshold of the 0.5% RVC group was higher than that of the 3.5% RVC-LAP / COL group and the 7.0% RVC-LAP / COL group only in the first 2 hours after administration, and there was no obvious advantage in analgesia at other times.

[0167] 2.3 Thermal pain threshold of rats in each group

[0168] After the rat SNI model was established, the thermal pain threshold of the rats did not change significantly. Some literature reports show that the SNI model is not sensitive to the thermal pain threshold. Fig.14 As shown in (b), there was no significant difference in the heat pain thresholds of the groups before drug injection. Fig.14As shown in (c), the pain thresholds of rats in the NS group and LAP / COL group did not change significantly after injection. Two hours after injection, the thermal pain threshold of the 0.5% RVC group was higher than that of the NS group (P<0.0001), the thermal pain threshold of the 3.5% RVC-LAP / COL group was higher than that of the NS group (P<0.0005), and the thermal pain threshold of the 7.0% RVC-LAP / COL group was higher than that of the NS group (P<0.0001). The differences were statistically significant. Four hours after injection, the thermal pain threshold of the 0.5% RVC group was higher than that of the NS group (P<0.05), the thermal threshold of the 3.5% RVC-LAP / COL group was higher than that of the NS group (P<0.005), and the thermal pain threshold of the 7.0% RVC-LAP / COL group was higher than that of the NS group (P<0.0001). There were statistically significant differences. After 6 hours after injection, there was no significant difference in the thermal pain threshold between the 0.5% RVC group and the NS group. The heat pain threshold of the 3.5% RVC-LAP / COL group was still higher than that of the NS group 1 day after injection (P<0.001), and the difference was statistically significant. There was no significant difference between the NS group and the NS group 3 days after injection. The heat pain threshold of the 7.0% RVC-LAP / COL group was still higher than that of the NS group 5 days after injection (P<0.05), and the difference was statistically significant. There was no significant difference between the NS group and the NS group starting from 7 days after injection. The heat pain threshold of the 0.5% RVC group was also higher than that of the 3.5% RVC-LAP / COL group and the 7.0% RVC-LAP / COL group only in the first 2 hours after administration.

[0169] 2.4 Cold pain threshold of rats in each group

[0170] After the rat SNI model was established, the cold pain threshold began to decrease significantly on the third day and dropped to a stable level around the fifth day. Fig.15 As shown in (b), there was no significant difference in the cold pain thresholds of the groups before drug injection. Fig.15As shown in (c), 2 hours after injection, the cold pain threshold of the 0.5% RVC group was higher than that of the NS group (P<0.0001), the cold pain threshold of the 3.5% RVC-LAP / COL group was higher than that of the NS group (P<0.001), and the cold pain threshold of the 7.0% RVC-LAP / COL group was higher than that of the NS group (P<0.0001). The differences were statistically significant. 4 hours after injection, the cold pain threshold of the 0.5% RVC group was higher than that of the NS group (P<0.001), the mechanical cold threshold of the 3.5% RVC-LAP / COL group was higher than that of the NS group (P<0.0001), and the cold pain threshold of the 7.0% RVC-LAP / COL group was higher than that of the NS group (P<0.0001). The differences were statistically significant. Starting 6 hours after injection, there was no significant difference in the cold pain threshold between the 0.5% RVC group and the NS group. The cold pain threshold of the 3.5% RVC-LAP / COL group was still higher than that of the NS group 1 day after injection (P<0.005), and the difference was statistically significant. There was no significant difference between the NS group and the NS group from 3 days after injection. The cold pain threshold of the 7.0% RVC-LAP / COL group was still higher than that of the NS group 5 days after injection (P<0.001), and the difference was statistically significant. There was no significant difference between the NS group and the NS group from 7 days after injection. The cold pain threshold of the 0.5% RVC group was also higher than that of the 3.5% RVC-LAP / COL group and the 7.0% RVC-LAP / COL group only in the first 2 hours after administration.

[0171] 2.5 Kinematic evaluation of rats in each group

[0172] The weight of rats in each group increased normally, their appearance was basically normal, there was no hemiplegia, and there was no significant change in drinking and eating. After testing the mechanical withdrawal threshold and the cold and hot withdrawal latency, the rats were tested for placement reflex, grasping reflex, and corrective reflex to determine the motor function of the rats. The results are shown in Table 2. Compared with the saline group, the placement reflex, grasping reflex, and corrective reflex of the RVC group were inhibited 2 hours after sciatic nerve block (P < 0.0001), and the placement reflex (P < 0.005) and grasping reflex (P < 0.05) of the 7.0% RVC-LAP / COL group were inhibited. At the other time points, there was no obvious motor dysfunction in the rats.

[0173] Table 2 Kinematic evaluation of rats in each group (n=6)

[0174]

[0175] N=6 / group; 5trials; Mean (SEM)

[0176] Note: *P<0.05, **P<0.005, ****P<0.0001 are compared between 0.5% RVC, 7.0% RVC-LAP / COL group and NS group

[0177] 2.6 Rat open field test

[0178] The open field test was used to evaluate the changes in spontaneous motor activity of rats in each group and indirectly reflect the pain conditions of rats. Fig.16 and Fig.17 As shown in the figure, there was no significant difference in the movement distance of rats in each group before modeling, and the rats explored the entire area extensively. Five days after surgery, as the pain threshold of rats gradually decreased to a stable level, the total movement distance of rats in each group decreased significantly, the range of movement of rats was limited to the periphery of the open field, and the stay in the central area was significantly reduced. After administration, the drug took effect, the range of movement of rats gradually became wider, and the exploration of the central area increased. Two hours after administration, the movement distance of rats in the 0.5% RVC group, 3.5% RVC-LAP / COL group, and 7.0% RVC-LAP / COL group was significantly increased compared with the NS group and LAP / COL group, and the difference was statistically significant (P < 0.005). At the same time, the movement distance of rats in the 0.5% RVC group was longer than that in the 3.5% RVC-LAP / COL group and the 7.0% RVC-LAP / COL group, and the difference was statistically significant (P < 0.05). This indicates that saline and the material itself have no obvious analgesic effect. 0.5% RVC, 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL can relieve pain and restore the rats' ability to move and explore. In addition, according to the aforementioned pain threshold measurement results and kinematic evaluation, 0.5% RVC has a better analgesic effect than 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL 2 hours after administration. Although the movement of the limbs on the surgical side was slightly inhibited 2 hours after administration, its overall movement distance was still longer than that of the 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL groups. However, after 2 hours, the movement distance of rats in the 3.5% RVC-LAP / COL and 7.0% RVC-LAP / COL groups was longer than that of the 0.5% RVC group, which is consistent with the results of the pain threshold measurement. The total movement distance of the rats in the 0.5% RVC group was still higher than that of the NS group until 6 hours after administration, and the difference was statistically significant (P < 0.0001). The total movement distance of the rats in the 3.5% RVC-LAP / COL group and the 7.0% RVC-LAP / COL group was still higher than that of the NS group until 3 days after administration, and the difference was statistically significant (P < 0.05). There was no significant difference in the movement distance of the rats in each group 5 days after administration.

[0179] 2.7 In vivo safety evaluation

[0180] 2.7.1 General Observations

[0181] The results are as follows Fig.18As shown in the figure, 7 days after the injection of drugs around the sciatic nerve, the sciatic nerve and surrounding muscles were collected for histological analysis. After the sciatic nerve was exposed, it was found that the structure of the NS group and the 0.5% RVC group was intact, and there was no obvious abnormality around the sciatic nerve. There was light yellow drug residue around the sciatic nerve in the LAP / COL group, the 3.5% RVC-LAP / COL group, and the 7.0% RVC-LAP / COL group. Fig.18 As indicated by the arrows. One was found in the LAP / COL group, one in the 3.5% RVC-LAP / COL group, and two in the 7.0% RVC-LAP / COL group (n=6). Therefore, in order to further observe the drug residue, the sciatic nerve and surrounding muscles were collected again on the 14th day after administration for histological analysis, and no obvious residue was found. There was no edema, discoloration, adhesion or other signs of tissue damage in each group.

[0182] 2.7.2 Tissue and organ toxicity

[0183] In order to evaluate the tissue and organ toxicity of the drug, HE staining was performed on important tissues and organs of rats. Fig.19 As shown, on the 7th day after drug injection, the structures of important tissues and organs in each group were normal, and there were no obvious pathological changes.

[0184] On the 7th day after drug injection, the muscles around the sciatic nerve were harvested for histological analysis. Fig. 20 As shown in a, muscle cells in the normal saline (NS) group were neatly arranged, with red cytoplasm and blue nuclei located around the muscle cells, and no inflammatory cell infiltration. In the 0.5% RVC group, connective tissue hyperplasia and dark-stained inflammatory cell infiltration were observed in the muscle gaps. Scattered light red or gray homogeneous sustained-release microgel residues were observed in the LAP / COL group, the 3.5% RVC-LAP / COL group, and the 7.0% RVC-LAP / COL group. Fig. 20 As shown by the black arrow in a. Surrounding it are inflammatory cell infiltration and a small number of dark-stained megakaryocytes, as well as a small number of muscle cells undergoing nuclear internalization and regeneration, with the nucleus being away from the usual position of the cell periphery and eosinophilic cytoplasm and dark-stained nucleus.

[0185] The expression of proinflammatory factors IL-1β, IL-6, and TNF-α in the tissue surrounding the sciatic nerve was detected by ELISA. Fig. 20As shown in b to d, the expression levels of IL-1β and IL-6 in the 0.5% RVC group were higher than those in the NS group, and the difference was statistically significant (P < 0.005). The expression levels of IL-1β, IL-6, and TNF-α in the 3.5% RVC-LAP / COL group and the 7.0% RVC-LAP / COL group were higher than those in the NS group, and the difference was statistically significant (P < 0.005). The expression level of TNF-α in the 3.5% RVC-LAP / COL group was higher than that in the 0.5% RVC group, and the difference was statistically significant (P < 0.05). The expression levels of IL-1β, IL-6, and TNF-α in the 7.0% RVC-LAP / COL group were higher than those in the 0.5% RVC group, and the difference was statistically significant (P < 0.05).

[0186] The results showed that the 0.5% RVC group, 3.5% RVC-LAP / COL group, and 7.0% RVC-LAP / COL group all caused different degrees of inflammatory response in the body, and the inflammatory response in the sustained-release microgel group was more obvious. This may be because it takes a certain amount of time for the microgel to decompose, resulting in microgel residues causing inflammatory response.

[0187] To further explore the residual status of microgels in vivo, histological analysis of the muscles surrounding the sciatic nerve was performed again on the 14th day after administration. Fig.21 As shown in a, the inflammation disappeared in the 0.5% RVC group, the muscle cells were arranged neatly, and no inflammatory cell infiltration was observed. The muscle cells in the LAP / COL group, the 3.5% RVC-LAP / COL group, and the 7.0% RVC-LAP / COL group were arranged regularly, the cytoplasm was red, and there was no inflammatory cell infiltration in the muscle gap. The microgel residues were also significantly reduced, with only a small amount of scattered microgel residues and inflammatory cells in the connective tissue around the muscles.

[0188] Combined with the myotoxicity and inflammation scores on the 7th and 14th days after administration, Fig.21 b~ Fig.21 As shown in Figure e, mild inflammation and myotoxicity were observed in all treatment groups. Inflammation mainly occurred in the soft tissues where the residual microgels adhered, and myotoxicity was manifested by the degeneration and regeneration of myocytes in the perifascicular area of ​​the injection site. However, at 14 days after administration, the inflammatory response was significantly reduced compared with that at 7 days after administration, indicating that local inflammation was a reversible reaction with self-limitation.

[0189] 2.7.3 TB staining of sciatic nerve

[0190] In the safety evaluation of sciatic nerve block in rats, whether RVC-LAP / COL causes nerve damage is a question of close concern. The sciatic nerve was obtained on the 7th day after peri-injection of the sciatic nerve for toluidine blue (TB) staining. Fig. 22Light microscopy showed that the myelinated nerve fibers in each group had normal morphology, dense fibers, intact outer membrane structure, regular arrangement, and uniform distribution. No obvious morphological changes were found, including axon density, myelin sheath structure, etc. This shows that RVC-LAP / COL sustained-release microgel is a safe local anesthetic sustained-release agent, which does not cause nerve damage when used for sciatic nerve block in rats.

[0191] 2.7.4 Rat liver and kidney function determination

[0192] After 7 days of administration, the toxicity of the rats in each group was evaluated by measuring the physiological indicators of liver and kidney function. The alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, urea nitrogen, creatinine, and uric acid in the rats were measured. Fig.23 As shown, the range between the two red dotted lines is the normal value range. The liver and kidney functions of rats in each group are generally normal. The 3.5% RVC-LAP / COL group and the 7.0% RVC-LAP / COL group have large encapsulated drug amounts and high concentrations, but they do not obviously cause abnormal liver and kidney functions in rats. This indicates that RVC-LAP / COL sustained-release microgels can maintain the stability of blood drug concentrations and reduce liver and kidney toxicity caused by sudden increases in blood drug concentrations through continuous and slow release.

[0193] In summary, RVC-LAP / COL can prolong the duration of drug action in vivo by slowly and steadily releasing RVC, achieving long-term analgesia. The analgesic duration of 3.5% RVC-LAP / COL is 1-3 days, and the analgesic duration of 7.0% RVC-LAP / COL can reach 5-7 days, while the effect on motor function is slight. As the drug degrades, a self-limiting inflammatory response is produced, with no obvious organ toxicity or neurotoxicity.

[0194] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ropivacaine hydrochloride sustained-release microgel, characterized in that: The ropivacaine hydrochloride sustained-release microgel comprises the following raw materials in parts by weight: 21 to 42 parts of ropivacaine hydrochloride, 2.4 parts of collagen and 71.4 parts of lithium terephthalate.

2. The method for preparing the ropivacaine hydrochloride sustained-release microgel according to claim 1, characterized in that: The method comprises the following steps: mixing the ropivacaine hydrochloride with water to obtain a ropivacaine hydrochloride aqueous solution; mixing the ropivacaine hydrochloride aqueous solution with the lithium ion, and freeze-drying to obtain powder; and dissolving the collagen to obtain a collagen solution. The collagen solution and the powder were mixed and allowed to stand at 37° C. to obtain ropivacaine hydrochloride sustained-release microgel.

3. The preparation method according to claim 2, characterized in that: The weight-to-volume ratio of ropivacaine hydrochloride to collagen solution is 21-42 mg:600 μL.

4. The preparation method according to claim 2, characterized in that: The preparation method of the collagen solution is: mixing collagen with glacial acetic acid solution, adjusting the pH to 7.0, and then adding water to obtain the collagen solution.

5. The preparation method according to claim 4, characterized in that: The concentration of the glacial acetic acid solution is 0.25 mol / L.

6. The preparation method according to claim 4, characterized in that: In the collagen solution, the volume ratio of glacial acetic acid solution to water is 7:

3.

7. The preparation method according to claim 2, characterized in that: The freeze drying is performed at -50°C and 30 mtorr for 48 hours.

8. The preparation method according to claim 2, characterized in that: The standing time is 30 min.

9. Use of the ropivacaine hydrochloride sustained-release microgel according to claim 1 or the preparation method according to any one of claims 2 to 8 in the preparation of analgesic products.

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