A fentanyl citrate@PLGA-PEG nanosphere and its preparation method

Abstract

The present invention relates to the field of pharmaceutical technologies, and particularly relates to a fentanyl citrate@PLGA-PEG nanosphere and a preparation method thereof. The preparation method includes: Step S1, preparing a fentanyl citrate solution and a PLGA-PEG solution; Step S2, dropping the fentanyl citrate solution into the PLGA-PEG solution, and performing ultrasonic emulsification after mixing to obtain a primary emulsion; Step S3, dropping the primary emulsion into a PVA solution, and performing ultrasonic emulsification after mixing to obtain a multiple emulsion; Step S4, dropping the multiple emulsion into water, stirring to volatilize the organic solvent, and then performing centrifugal filtration on it to obtain the fentanyl citrate@PLGA-PEG nanosphere. In the present invention, a PLGA-PEG nanocarrier is used as a drug carrier to carry fentanyl citrate, and compared with the existing fentanyl citrate preparations for oral administration, sustained release of fentanyl citrate can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a fentanyl citrate@PLGA-PEG nano-microsphere and a preparation method thereof. Background Art

[0002] Fentanyl citrate is an analgesic, applicable to severe pain without shock and respiratory depression, used in pre-hospital first aid for trauma, surgical anesthesia, and also for breakthrough cancer pain, etc.

[0003] Currently, the orally administered fentanyl citrate preparations (such as transmucosal buccal tablets buccal effervescent tablets buccal patches sublingual tablets ) are generally released relatively fast, but the duration of drug effect is insufficient, with a maximum of only 2.5 - 5h. In the case of insufficient medical evacuation force at the disaster accident site, the short duration of drug effect will lead to repeated use of analgesics, increasing the pain of patients and the risk of side effects. Therefore, it cannot meet the need for analgesia in disaster rescue under insufficient evacuation conditions. Summary of the Invention

[0004] In view of the above analysis, the present invention aims to provide a fentanyl citrate@PLGA-PEG nano-microsphere and a preparation method thereof to solve the problem that the orally administered fentanyl citrate preparations in the prior art cannot achieve drug sustained release.

[0005] On the one hand, an embodiment of the present invention provides a preparation method of a fentanyl citrate@PLGA-PEG nano-microsphere, including: Step S1, preparing a fentanyl citrate solution and a PLGA-PEG solution; Step S2, dropping the fentanyl citrate solution into the PLGA-PEG solution, mixing and then performing ultrasonic emulsification to obtain a primary emulsion; Step S3, dropping the primary emulsion into a PVA solution, mixing and then performing ultrasonic emulsification to obtain a double emulsion; Step S4, dropping the double emulsion into water, stirring to volatilize the organic solvent, and then performing centrifugal filtration to obtain the fentanyl citrate@PLGA-PEG nano-microsphere.

[0006] Further, in Step S1, preparing the PLGA-PEG solution includes: preparing a mixed solution of dichloromethane and acetone, and dissolving PLGA-PEG in the mixed solution of dichloromethane and acetone to obtain the PLGA-PEG solution.

[0007] Further, the mass percentage of PEG in the used PLGA-PEG is less than or equal to 33%.

[0008] Further, in Step S3, when performing ultrasonic emulsification on the primary emulsion, the ultrasonic power is 15 - 30W.

[0009] Further, in step S1, preparing a fentanyl citrate solution includes: dissolving fentanyl citrate in a 1% PVA solution to obtain a fentanyl citrate solution.

[0010] Further, the volume ratio of the 1% PVA solution to the mixed solution of dichloromethane and acetone is 1:10 to 1:5.

[0011] Further, in step S1, the mass percentage of fentanyl citrate and PLGA-PEG is 5% to 25%.

[0012] Further, in step S1, the mass percentage of PEG in the used PLGA-PEG is 33%; in step S3, the ultrasonic power during ultrasonic emulsification of the primary emulsion is 20 W; in step S1, the volume ratio of the 1% PVA solution to the mixed solution of dichloromethane and acetone is 1:7; in step S1, the mass percentage of fentanyl citrate and PLGA-PEG is 25%.

[0013] On the other hand, an embodiment of the present invention also provides a fentanyl citrate@PLGA-PEG nanomicroparticle, which is obtained by the preparation method of the above embodiment and includes fentanyl citrate and a PLGA-PEG nanocarrier carrying fentanyl citrate.

[0014] Further, the sustained release time of the fentanyl citrate@PLGA-PEG nanomicroparticle exceeds 5 days, the drug release rate in the first 15 minutes before in vitro release is not less than 5.36%, the drug loading rate is not lower than 10.68%, and the encapsulation rate is not lower than 99%.

[0015] Compared with the prior art, the present invention can at least achieve one of the following beneficial effects:

[0016] 1. The present invention uses a PLGA-PEG nanocarrier as a drug carrier to carry fentanyl citrate. Compared with the existing fentanyl citrate preparations used orally it can achieve the sustained release of fentanyl citrate and provide a reference basis for the drug research and development of oral preparations for disaster relief and dysmenorrhea relief.

[0017] 2. The present invention adjusts the dosage of fentanyl citrate, the mass percentage of PEG in PLGA-PEG, the ultrasonic power during the formation of the multiple emulsion, the volume ratio of the inner aqueous phase and the oil phase, etc., so that the drug loading rate, encapsulation rate, sustained release time and rapid drug release percentage of fentanyl citrate@PLGA-PEG can simultaneously achieve good effects.

[0018] 3. The fentanyl citrate @PLGA-PEG of the present invention can achieve the following performance: the sustained release time exceeds 5 days, the drug release rate in the first 15 minutes before in vitro release is not less than 5.36%, the drug loading rate is not less than 10.68%, and the encapsulation rate is not less than 99%.

[0019] In the present invention, the above technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent specification, and some advantages can be made obvious from the specification, or understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the examples in the specification and the content specifically pointed out in the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings are only for the purpose of showing specific embodiments and are not considered as a limitation to the present invention. Among them:

[0021] Figure 1 is a schematic diagram of the fentanyl citrate @PLGA-PEG microspheres of Example 1 of the present invention;

[0022] Figure 2 in (a)-(i) are the particle size distribution diagrams of the samples of Examples 1-9 respectively;

[0023] Figure 3 is the standard curve of fentanyl citrate for detecting the encapsulation rate by high performance liquid chromatography;

[0024] Figure 4 is the comparison diagram of the percentage of released drugs of fentanyl citrate at different time points in Examples 1-9; among them, a is the curve diagram of the percentage of released drugs from 15 min to 12 h, and b is the bar chart of the percentage of released drugs at the 15th minute;

[0025] Figure 5 is the standard curve of detecting the concentration of fentanyl citrate in the supernatant of the sustained release solution by high performance liquid chromatography;

[0026] Figure 6 is the comparison diagram of the percentage of released drugs of fentanyl citrate from 1 to 6 days in Examples 1-9; among them, a is the curve diagram of the percentage of released drugs from 1 to 6 days, and b is the bar chart of the percentage of released drugs at the 5th day;

[0027] Figure 7 is the schematic diagram of the influence relationship of four parameters A, B, C, and D on the drug performance of fentanyl citrate in Examples 1-9. Among them, a is the influence of the four parameters on the particle size; b is the influence of the four parameters on the dispersity index; c is the influence of the four parameters on the drug loading rate; d is the influence of the four parameters on the drug release rate at 15 min; e is the influence of the four parameters on the drug release rate at 5 days. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The preferred embodiments of the present invention will be specifically described below in conjunction with the accompanying drawings. The accompanying drawings form a part of the present invention and are used together with the embodiments of the present invention to explain the principles of the present invention, rather than to limit the scope of the present invention.

[0029] To accurately explain the implementation scheme of the present invention, further explanations are made on relevant professional terms:

[0030] PLGA-PEG: poly(lactic-co-glycolic acid)-polyethylene glycol; wherein, PLGA is poly(lactic-co-glycolic acid) and PEG is polyethylene glycol.

[0031] PLGA-w%PEG: w% represents the mass percentage of PEG in the composite.

[0032] PVA: polyvinyl alcohol.

[0033] The embodiments of the present invention provide a method for preparing fentanyl citrate@PLGA-PEG nanospheres, comprising the following steps:

[0034] Step S1, preparing a fentanyl citrate solution and a PLGA-PEG solution;

[0035] Step S2, dropping the fentanyl citrate solution into the PLGA-PEG solution, mixing and then performing ultrasonic emulsification to obtain a primary emulsion;

[0036] Step S3, dropping the primary emulsion into the PVA solution, mixing and then performing ultrasonic emulsification to obtain a double emulsion;

[0037] Step S4, dropping the double emulsion into water, stirring to volatilize the organic solvent, and then performing centrifugal filtration to obtain fentanyl citrate@PLGA-PEG nanospheres.

[0038] PLGA is a biodegradable drug-controlled release material. In the present invention, PLGA-PEG nanocarriers are used as drug carriers to carry fentanyl citrate. Compared with the existing fentanyl citrate preparations for oral use ( etc.) using glucose powder, magnesium stearate, etc. as excipients, it can achieve the sustained release of fentanyl citrate, providing a reference basis and technical support for the research of disaster relief analgesic drugs. In addition, the present invention uses the emulsion solvent evaporation method to prepare fentanyl citrate@PLGA-PEG nanospheres, and the method is classic, stable and reliable, and easy to operate.

[0039] In some embodiments of the present invention, in step S1, preparing the PLGA-PEG solution includes: preparing a mixed solution of dichloromethane and acetone, and dissolving PLGA-PEG in the mixed solution of dichloromethane and acetone to obtain the PLGA-PEG solution. Wherein, the volume ratio of dichloromethane to acetone is: V 二氯甲烷 :V 丙酮 = 3:2. When dissolving PLGA-PEG, 3.5 mL of the mixed solution of dichloromethane and acetone can be used to dissolve every 100 mg of PLGA-PEG.

[0040] In some embodiments of the present invention, the mass percentage of PEG in the used PLGA-PEG is less than or equal to 33%. By adjusting the mass percentage of PEG in PLGA-PEG to a suitable range, the fentanyl citrate@PLGA-PEG of the present invention has excellent sustained-release effect and rapid-release effect, thereby increasing the sustained-release time of fentanyl citrate and achieving the rapid release of fentanyl citrate.

[0041] In some embodiments, preparing the fentanyl citrate solution includes: dissolving fentanyl citrate in a 1% PVA solution to obtain the fentanyl citrate solution.

[0042] In some embodiments, when preparing the fentanyl citrate solution and the PLGA-PEG solution in step S1, the mass percentage of fentanyl citrate to PLGA-PEG is 5% - 25%. The present invention controls the dosage of fentanyl citrate within 5% - 25% of PLGA-PEG, so that the finally obtained fentanyl citrate@PLGA-PEG has a large drug loading amount, the drug loading amount is above 2%, and can reach up to more than 10% at most; at the same time, it can also improve the sustained-release effect and rapid-release effect of fentanyl citrate@PLGA-PEG to a certain extent. Among them, the sustained release in the present invention means that fentanyl citrate can be slowly released for a long time after use to extend its action time; the rapid release means that fentanyl citrate starts to be released within a short time after use.

[0043] In some embodiments, when preparing the fentanyl citrate solution and the PLGA-PEG solution in step S1, the volume ratio of the 1% PVA solution to the mixed solution of dichloromethane and acetone is 1:10 - 1:5, so that in the primary emulsion formed after the two are mixed, the volume ratio of the internal aqueous phase to the oil phase (i.e., the 1% PVA solution and the mixed solution of dichloromethane and acetone) is 1:10 - 1:5. By adjusting the volume ratio of the internal aqueous phase to the oil phase within a suitable range, the present invention can further improve the sustained-release effect and rapid-release effect of fentanyl citrate@PLGA-PEG.

[0044] In step S2, the fentanyl citrate solution is added dropwise to the PLGA-PEG solution to obtain an O / W type solution; the solution is ultrasonically emulsified so that the fentanyl citrate solution is evenly dispersed in the PLGA-PEG solution to obtain colostrum. The ultrasonic emulsification can be achieved by using a cell disruptor. The ultrasonic power during ultrasonic emulsification can be 15 to 25 W, for example, 15 W, 18 W, 20 W, 22 W, 25 W.

[0045] In step S3, colostrum is uniformly added to the PVA solution, mixed evenly, and then ultrasonically emulsified to obtain a double emulsion. The PVA solution is a PVA solution with a mass fraction of 5%. In addition, during the dropping process, the dropping can be stirred, for example, by stirring and mixing by magnetic stirring, and the speed of the magnetic stirring can be 300 to 500 rpm to avoid stirring too fast and splashing the solution.

[0046] In this embodiment, the PVA solution is used as a dispersant or emulsifier to disperse the raw materials into the continuous phase to prevent the microsphere droplets from sticking and merging; at the same time, the microsphere droplets are evenly distributed by uniform dripping and stirring.

[0047] In some embodiments, when the colostrum is ultrasonically emulsified in step S3, the ultrasonic power is 15 to 30 W. The present invention controls the ultrasonic power when preparing the emulsion, so that fentanyl citrate @PLGA-PEG has excellent sustained release effect, rapid release effect and high drug loading, thereby increasing the sustained release time of fentanyl citrate and achieving rapid release of fentanyl citrate.

[0048] In step S4, after the ultrasonic emulsification in step S3 is completed, the emulsion is immediately dripped into pure water at a uniform speed, and volatilized for a certain period of time, such as 4 hours, under magnetic stirring, so that the organic solvent in the emulsion is volatilized, and the spherical material is precipitated in the water, and finally solidified by filtering and drying to form fentanyl citrate@PLGA-PEG microspheres, such as Figure 1 The speed of magnetic stirring can be 300-500 rpm, for example, 300 rpm, 400 rpm, 500 rpm, to ensure uniform dispersion of the drug in the emulsion during the volatilization process, avoid too slow stirring speed, resulting in uneven distribution of the particle size of the obtained nano-microspheres, and avoid too fast stirring, resulting in splashing of the solution.

[0049] Specifically, vacuum freeze drying can be used to dry the drug to avoid drug ineffectiveness.

[0050] In some embodiments, by simultaneously controlling the above parameters, namely, the mass percentage of PEG in PLGA-PEG, the mass percentage of fentanyl citrate and PLGA-PEG, the volume ratio of 1% PVA solution to the mixture of dichloromethane and acetone, and the ultrasonic power during the preparation of double emulsion, and enabling their synergistic effect, the drug loading amount, encapsulation efficiency, sustained release time, and rapid drug release percentage of fentanyl citrate@PLGA-PEG can be improved.

[0051] In some embodiments, when the preparation process of fentanyl citrate@PLGA-PEG meets the following conditions, that is, the mass percentage of PEG in PLGA-PEG is 33%, the volume ratio of 1% PVA solution to the mixture of dichloromethane and acetone in step S1 is 1:7, the dosage percentage of fentanyl citrate and PLGA-PEG is 25%, and the ultrasonic power during the ultrasonic emulsification of the primary emulsion in step S3 is 20 W, the drug loading amount, encapsulation efficiency, sustained release time, and rapid drug release percentage of the finally obtained fentanyl citrate@PLGA-PEG can simultaneously achieve good effects. Its sustained release time exceeds 5 days, the drug release rate in the first 15 minutes before in vitro release is not less than 5.36%, the drug loading rate is not less than 10.68%, and the encapsulation efficiency is not less than 99%.

[0052] In addition, an embodiment of the present invention also provides a fentanyl citrate@PLGA-PEG nanosphere, which can be obtained by using the preparation method of the above embodiment, and it includes fentanyl citrate and a PLGA-PEG nanocarrier carrying fentanyl citrate.

[0053] The present invention uses a PLGA-PEG nanocarrier as a drug carrier to carry fentanyl citrate, and compared with the existing orally used fentanyl citrate preparation, it can achieve the sustained release of fentanyl citrate, enabling it to meet the needs of disaster relief analgesics under the condition of insufficient medical evacuation conditions.

[0054] In some embodiments, after detection, the sustained release time of fentanyl citrate@PLGA-PEG exceeds 5 days, the drug release rate in the first 15 minutes before in vitro release is not less than 5.36%, the drug loading rate is not less than 10.68%, and the encapsulation efficiency is not less than 99%, indicating that the PLGA-PEG nanocarrier is suitable for loading fentanyl citrate.

[0055] The following further illustrates the fentanyl citrate@PLGA-PEG nanosphere and its preparation method of the present invention with specific examples.

[0056] Example 1

[0057] The preparation process of fentanyl citrate@PLGA-PEG is specifically as follows.

[0058] Step S1: Prepare fentanyl citrate solution and PLGA-PEG solution.

[0059] Prepare 5% PVA solution: Weigh 2 g of PVA and place it in a beaker. Add 40 mL of pure water to it, seal it with tin foil and a rubber band, place it in a water bath at 100 °C and heat it in a water bath for 2 h to obtain 5% PVA solution.

[0060] Pour 10 mL of the swollen 5% PVA into 3 centrifuge tubes, add magnetic stirrers, place them on a magnetic stirrer, and set the rotation speed to 600 rpm for standby.

[0061] Take 50 mL of pure water and place it in a beaker for standby.

[0062] Prepare PLGA-PEG solution: Weigh 100 mg of PLGA-33% PEG for standby. Prepare a 15 mL mixed solution of dichloromethane and acetone, V 二氯甲烷 :V 丙酮 =3:2. Add 3.5 mL of the V 二氯甲烷 :V 丙酮 =3:2 mixed solution to PLGA-33% PEG to dissolve it.

[0063] Prepare fentanyl citrate solution: Weigh 25 mg of fentanyl citrate and add 0.35 mL of 1% PVA solution (diluted from the 5% PVA solution prepared above).

[0064] Step S2: Prepare the primary emulsion.

[0065] Drop the fentanyl citrate solution into the PLGA-33% PEG solution, and place the obtained O / W solution in a cell disruptor and ultrasonicate it 5 times with an ultrasonic power of 20 W to obtain the primary emulsion.

[0066] Step S3: Prepare the multiple emulsion.

[0067] Drop the primary emulsion into the 5% PVA solution on a magnetic stirrer at a constant speed. After mixing evenly, remove the centrifuge tube and take out the magnetic stirrer, and use a cell crusher to ultrasonicate the primary emulsion for 2 min with an ultrasonic power of 30 W.

[0068] Place the magnetic stirrer into a beaker containing 50 mL of pure water, put the beaker on a magnetic stirrer and adjust the rotation speed to 400 rpm. Immediately after the ultrasonication, drop the multiple emulsion into the beaker containing 50 mL of pure water at a constant speed. After the multiple emulsion is dropped, volatilize it at 400 rpm on the magnetic stirrer for 4 h.

[0069] Step S4: Collect the microspheres.

[0070] After 4 hours of volatilization, remove the beaker from the magnetic stirrer. Filter each sample through a 0.45 μm filter into a centrifuge tube, centrifuge for 1 hour at a speed of 18,000 G, and retain the supernatant for later use. Add 10 mL of pure water to the centrifuge tube, dissolve the precipitate with the help of an ultrasonic cleaner, and then centrifuge for 1 hour again (speed 18,000 G), and retain the supernatant for later use.

[0071] Quick-freeze the precipitate in the centrifuge tube with liquid nitrogen. Immediately after quick-freezing, place it in a vacuum freeze dryer for drying (temperature -51 °C, pressure 0.035 mbar). Take it out after 24 hours to obtain fentanyl citrate@PLGA-PEG, as Figure 1 shown, and store it in a -20 °C refrigerator for later use.

[0072] Examples 2 - 9

[0073] The preparation processes of Examples 2 - 9 are basically similar to that of Example 1, with the differences lying in the following 4 parameters: A. Dosage of fentanyl citrate (mg); B. Mass percentage of PEG in PLGA-PEG (X%); C. Ultrasonic power (W) when forming the double emulsion; D. Volume ratio of the inner aqueous phase to the oil phase (mL:mL).

[0074] The settings of each parameter in Examples 1 - 9 are shown in Table 1. Examples 2 - 9 are respectively used to prepare fentanyl citrate@PLGA-PEG microspheres according to the set parameters shown in Table 1.

[0075] Table 1 Settings of each parameter in Examples 1 - 9

[0076]

[0077] Perform performance tests on the fentanyl citrate@PLGA-PEG microspheres in Examples 1 - 9:

[0078] (1) Detect the drug loading rate

[0079] Use the high-performance liquid chromatography method to detect the drug loading rate of the sample. The calculation formula for the drug loading rate is as follows.

[0080]

[0081] The detection results of the drug loading rate of Examples 1 - 9 are shown in Table 3.

[0082] (2) Detect the particle size

[0083] Take the precipitate obtained by centrifugation before drying in step S4 of each example as the sample, and record them as PLGA E1 - 9 respectively. Add 2 mL of pure water to the sample to fully dissolve it to obtain a sample solution; filter the sample solution with a 0.45 μm filter, drop it into a cuvette, and place the cuvette to be measured in a particle size analyzer to measure the particle size.

[0084] The particle size detection results of Examples 1 to 9 are shown in Table 3 and Figure 2 as shown. The particle size of the samples is between 144 and 231 nm; and the particle size distributions of the samples in each example are relatively concentrated, and the particle sizes are relatively uniform. In addition, the dispersity index PDI of the samples in each example was also detected. As shown in Table 3, it also indicates that the particle size distributions of the samples in each example are relatively uniform.

[0085] (3) Detection of encapsulation efficiency

[0086] Take 10 mg of fentanyl citrate@PLGA-PEG microspheres from each example and place them in a centrifuge tube, and add 10 mL of pure water to dissolve them. After observation, the freeze-dried materials, especially the samples of Examples 3, 6, and 9, are flaky and insoluble in water. Filter the sample aqueous solution with a 0.45 μm filter, take 9 mL of each sample and place it in a retention tube, and centrifuge for 15 min (3000 rpm). Take the supernatant at the lower layer of the retention tube for standby.

[0087] Prepare V 甲醇 :V 水 = 7:3 mixed solution of 60 mL. Weigh 5 mg of fentanyl citrate, add 5 mL of the above-prepared mixed solution to fentanyl citrate to obtain a 1 mg / mL fentanyl citrate standard solution. Dilute the 1 mg / mL fentanyl citrate standard solution to concentrations of 100 μg / mL, 80 μg / mL, 60 μg / mL, 40 μg / mL, and 20 μg / mL for standby. Prepare 500 mL of buffer salt solution using 500 mg of anhydrous sodium sulfate, 1000 mg of ammonium acetate, and 500 mL of water. Use a pH meter to detect its pH value and adjust it to 6.3 ± 0.1 with glacial acetic acid for standby.

[0088] Turn on the high-performance liquid chromatograph. The mobile phases are placed in the order of A methanol, B acetonitrile, C pure water, and D buffer salt solution. After discharging the air bubbles, rinse the chromatographic column with methanol and water in a proportional gradient. Continue to rinse the chromatographic column with a ratio of methanol:acetonitrile:buffer salt = 1:4:5. After the column pressure is stable, start injecting samples. Transfer the fentanyl citrate standard solution and the supernatant at the lower layer of the retention tube to the injection vials respectively. Inject samples into each injection vial 3 times to obtain the peak areas. Use the standard solution concentration as the X-axis and the peak area percentage as the Y-axis to draw a calibration curve, as Figure 3 shown. Measure the mass of fentanyl citrate in the supernatant at the lower layer of the retention tube in each example in this way and calculate the encapsulation efficiency. Among them, the encapsulation efficiency calculation formula is:

[0089]

[0090] Since the release peak areas measured for the samples in each example are all less than the detection limit of the standard curve, and the calculated drug concentrations are all negative values, as shown in Table 2, the encapsulation efficiencies of each example measured by this method are all 100%.

[0091] Table 2 Concentrations of Fentanyl Citrate in the Lower Clear Liquid in Examples 1 - 9

[0092]

[0093] (4) Detection of the Immediate Release Performance of the Drug

[0094] Take the fentanyl citrate@PLGA-PEG microspheres of Examples 1 - 9, denoted as PLGA E1 - 9 respectively, add artificial gastric juice to dilute them into a 12 mg / mL solution. After fully dissolving, aliquot them into (E1 - E9)×8 centrifuge tubes with 1 mL of solution in each tube for standby. Take the time of aliquoting as the 0 time point, and set 8 time points of 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h for centrifugation. Take out 1 portion of the sample solution (E1 - E9) at T0, centrifuge for 15 min (14000 rpm), and place it in a -20°C refrigerator for standby. Place the other 7 portions of the samples in a 37°C constant temperature environment, centrifuge them in sequence according to the set time points. After centrifugation, store all the supernatant liquids in a -20°C refrigerator for retention. Use high performance liquid chromatography to detect the peak area in the supernatant liquid, and calculate the drug release percentage at each time point by the difference method.

[0095] The percentages of the released drug of fentanyl citrate at each time point calculated are as Figure 4 shown. According to Figure 4 it can be seen that the fentanyl citrate in each example can be rapidly released, and the release rate in the first 15 minutes can reach more than 3.6%, and most can reach more than 5%.

[0096] (5) Detection of the Sustained Release Performance of the Drug

[0097] Take the weighed fentanyl citrate@PLGA-PEG microspheres in Examples 1 - 9, dilute them into a 6 mg / mL solution with physiological saline according to the mass, and aliquot the samples into (E1 - E9)×6 centrifuge tubes with 1 mL of solution in each tube for standby.

[0098] Take the time of aliquoting as the 0 time point, and set 6 time points of 1 d, 2 d, 3 d, 4 d, 5 d, and 6 h for centrifugation. Take out 1 portion of the sample solution (E1 - E9) at T0, centrifuge for 15 min (14000 rpm), and place it in a -20°C refrigerator for standby. Place the other 5 portions of the samples in a 37°C constant temperature environment, centrifuge them in sequence at the same time point every day. After centrifugation, store all the supernatant liquids in a -20°C refrigerator for retention and waiting for measurement.

[0099] Weigh 5 mg of fentanyl citrate, add 5 mL of normal saline to the fentanyl citrate to obtain a 1 mg / mL fentanyl citrate standard solution. Dilute the 1 mg / mL fentanyl citrate standard solution to concentrations of 100 μg / mL, 80 μg / mL, 60 μg / mL, 40 μg / mL, and 20 μg / mL for standby. Prepare 500 mL of buffer salt solution using 500 mg of anhydrous sodium sulfate, 1000 mg of ammonium acetate, and 500 mL of water. Use a pH meter to detect its pH value and adjust it to 6.3 ± 0.1 with glacial acetic acid for standby.

[0100] Turn on the high-performance liquid chromatograph. The mobile phases are arranged in the order of A methanol, B acetonitrile, C pure water, and D buffer salt solution. After discharging the air bubbles, wash the chromatographic column with methanol and water in a proportional gradient, and continue to wash the chromatographic column with the ratio of methanol:acetonitrile:buffer salt = 1:4:5. Start injecting samples after the column pressure is stable. Transfer the fentanyl citrate standard solution and the supernatant obtained from the sustained-release experiment to the injection vial. Inject each injection vial 3 times to obtain the peak area. Use the standard solution concentration as the X-axis and the peak area percentage as the Y-axis to plot the calibration curve, as Figure 5 shown.

[0101] According to the standard curve, calculate the drug concentration in the supernatant of the sustained-release solution, and use the difference method to calculate the drug release percentage at the same time point every day during the experiment. The calculation results are shown in Table 3 and Figure 6 shown. According to Figure 6 it can be seen that the sustained-release effects of fentanyl citrate in each example are all excellent. The drug release rate has not reached 100% on the 6th day, indicating that the drug sustained-release time is not less than 6 days (the release time has exceeded 5 days).

[0102] Table 3 Performance comparison table of fentanyl citrate nanospheres in each example

[0103]

[0104] According to the above test results, analyze the influence of different parameters on each performance, Figure 7 showing the influence relationship diagrams of different parameters with drug particle size, dispersity index, drug loading rate, 15-minute release rate, and 5-day release rate.

[0105] As Figure 7 shown in a of

[0106] Figure 7 Figure 7 shown in b of

[0107] As shown Figure 7 in c, under the conditions that the dosage of fentanyl citrate is 25 mg, PLGA-PEG contains 25% PEG, the ultrasonic power is 20 W, and the ratio of internal aqueous phase to external oil phase is 0.5 / 3.5 mL, the drug loading capacity of the formed nanospheres is larger and more suitable for drug loading.

[0108] As shown Figure 7 in d, the PEG ratio in PLGA-PEG and the ultrasonic power have the greatest influence on the drug release rate. Among them, under the conditions of 25% PEG and an ultrasonic power of 20 W, the release rate of fentanyl citrate in the first 15 minutes is the largest, indicating that these conditions are more suitable for rapid release.

[0109] As shown Figure 7 in e, the PEG ratio in PLGA-PEG and the ultrasonic power have the greatest influence on the drug release rate. Among them, under the conditions of 33% PEG and an ultrasonic power of 20 W, the release rate of fentanyl citrate on the 5th day is the largest, indicating that these conditions are more suitable for sustained release.

[0110] In order to enable the drug of the embodiment of the present invention to simultaneously meet the performance indicators of drug loading rate, encapsulation rate, rapid release, and sustained release, a dosage of 25 mg of fentanyl citrate, PLGA-20% PEG, an ultrasonic power of 20 W, and an internal aqueous phase to external oil phase volume ratio of 0.5 / 3.5 mL can be adopted, so that the sustained release time of fentanyl citrate@PLGA-PEG is not less than 6 days (the release time has exceeded 5 days), not less than 5.36% in the first 15 minutes of in vitro release, the drug loading rate is not less than 10.68%, and the encapsulation rate is not less than 99%.

[0111] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for preparing fentanyl citrate@PLGA-PEG nanoparticles, characterized in that: include: Step S1, preparing a fentanyl citrate solution and a PLGA-PEG solution; wherein the mass percentage of PEG in the PLGA-PEG used is 25% to 33%, and the mass percentage of the fentanyl citrate and the PLGA-PEG is 5% to 25%; Step S2, adding the fentanyl citrate solution dropwise to the PLGA-PEG solution, mixing and then performing ultrasonic emulsification to obtain colostrum; Step S3, adding the colostrum dropwise to the PVA solution, mixing and then performing ultrasonic emulsification to obtain a double emulsion; wherein the ultrasonic power when performing ultrasonic emulsification on the colostrum is 15-30W; Step S4, adding the double emulsion dropwise into water, stirring to volatilize the organic solvent, and then centrifuging and filtering the water to obtain the fentanyl citrate@PLGA-PEG nanospheres; wherein the stirring speed is 300-500 rpm; In step S1, preparing the PLGA-PEG solution comprises: preparing a mixed solution of dichloromethane and acetone, and dissolving PLGA-PEG in the mixed solution of dichloromethane and acetone to obtain the PLGA-PEG solution; In step S1, preparing a fentanyl citrate solution comprises: dissolving fentanyl citrate in 1% PVA solution to obtain the fentanyl citrate solution; The volume ratio of the 1% PVA solution to the mixed solution of dichloromethane and acetone is 1:10-1:

5.

2. The preparation method according to claim 1, characterized in that: In step S1, the mass percentage of PEG in the PLGA-PEG used is 33%; In step S3, the ultrasonic power is 20 W when the colostrum is ultrasonically emulsified; In step S1, the volume ratio of 1% PVA solution to the mixture of dichloromethane and acetone is 1:7; In step S1, the mass percentage of fentanyl citrate and PLGA-PEG is 25%.

3. A fentanyl citrate@PLGA-PEG nanoparticle, characterized in that: The preparation method according to any one of claims 1 to 2 is used to obtain the fentanyl citrate and a PLGA-PEG nanocarrier carrying the fentanyl citrate.

4. The fentanyl citrate@PLGA-PEG nanoparticles according to claim 3, characterized in that: The sustained release time of the fentanyl citrate@PLGA-PEG nano-microspheres is more than 5 days, the drug release rate in the first 15 minutes of in vitro release is not less than 5.36%, the drug loading rate is not less than 10.68%, and the encapsulation rate is not less than 99%.

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