A method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry

Abstract

This invention discloses a method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry, belonging to the field of pharmaceutical analysis technology. The method includes: S1, obtaining a standard curve equation; S2, performing low-temperature high-speed centrifugation on a docetaxel nanoliposome suspension to separate a supernatant containing free drug and a precipitate containing liposomes; S3, obtaining the absorbance of the test solution; S4, substituting the absorbance of the test solution into the standard curve equation to calculate the free drug content or encapsulated drug content; S5, calculating the encapsulation efficiency. This invention uses low-temperature high-speed centrifugation to separate free drug and liposomes. The low-temperature condition of 4℃ reduces drug leakage. (13800 rpm·min) ‑1 High-speed centrifugation ensures complete liposome precipitation and reliable separation. Compared to HPLC, this invention uses ultraviolet spectrophotometry, which eliminates the need for complex chromatographic separation conditions and large amounts of mobile phase, resulting in faster detection speeds; a single sample determination can be completed within one hour.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically to a method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry. Background Technology

[0002] Docetaxel is a taxane-based antitumor drug widely used to treat various malignant tumors, including breast cancer, non-small cell lung cancer, and prostate cancer. However, due to its poor water solubility and high toxicity, it is often formulated into nanoliposomes in clinical practice to improve drug targeting, prolong its circulation time in vivo, and reduce toxic side effects.

[0003] Encapsulation efficiency is a key indicator for evaluating liposome quality, directly affecting the in vivo behavior and efficacy of the formulation. Currently, common methods for determining liposome encapsulation efficiency include high-performance liquid chromatography (HPLC) and dextran gel column chromatography. While HPLC offers high accuracy, it is expensive, complex to operate, and has high detection costs, requiring large amounts of organic solvents as the mobile phase. Gel column chromatography is time-consuming and prone to inaccurate results due to drug adsorption or leakage.

[0004] Ultraviolet (UV) spectrophotometry offers advantages such as ease of operation, widespread instrument availability, and low detection costs. However, its direct application in encapsulation efficiency determination is limited by the light scattering effect of liposomes themselves and the challenge of effectively separating free drug from liposomes. Therefore, developing a method that can effectively separate free drug from liposomes and rapidly and accurately determine the encapsulation efficiency of docetaxel nanoliposomes using UV spectrophotometry is of significant practical value.

[0005] To address this issue, this application proposes a method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry, in order to solve the problems in the prior art that limit the direct application of liposome formulations in encapsulation efficiency determination due to the light scattering effect of the liposomes themselves and the effective separation of free drug from the liposomes.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry includes:

[0009] S1. Prepare a series of standard working solutions of docetaxel and perform ultraviolet scanning. After measuring the absorbance, perform linear regression of absorbance against concentration to obtain the standard curve equation.

[0010] S2. Take the docetaxel nanoliposome suspension and centrifuge it at low temperature and high speed to separate the supernatant containing free drug and the precipitate containing liposomes.

[0011] S3. The supernatant is treated by the supernatant method or the precipitate is treated by the precipitation method to obtain the test solution and the absorbance is measured to obtain the absorbance of the test solution.

[0012] S4. Substitute the absorbance of the test solution into the standard curve equation to calculate the free drug content or the encapsulated drug content;

[0013] S5. Based on the calculated free drug content or encapsulated drug content, and combined with the total dosage of docetaxel, calculate the encapsulation rate using the encapsulation rate formula.

[0014] Furthermore, in step S1, the process of obtaining the standard curve equation is as follows:

[0015] Accurately weigh the docetaxel standard, dissolve it in anhydrous ethanol, and dilute it stepwise to prepare a series of standard working solutions with concentrations of 25, 20, 15, 10, and 5 μg / mL. Perform ultraviolet scanning in the wavelength range of 190–500 nm to determine the maximum absorption wavelength as 225 nm. At this wavelength, measure the absorbance of the series of standard working solutions with ethanol as a blank control. Perform linear regression of absorbance against concentration to obtain the standard curve equation.

[0016] Furthermore, in step S2, the conditions for the low-temperature high-speed centrifugation separation are as follows:

[0017] The docetaxel nanoliposome suspension was placed in a pre-cooled centrifuge tube. The formulation ratio of docetaxel nanoliposomes (docetaxel: soy lecithin: cholesterol = 1:20:4) was used to centrifuge the sample at 13800 r·min under low temperature conditions of 4℃. -1 Centrifuge at a speed of 30 min to ensure that the free drug is completely retained in the supernatant and that the liposomes are completely precipitated at the bottom of the tube.

[0018] Furthermore, the supernatant processing procedure is as follows:

[0019] Accurately transfer 0.5 mL of the supernatant obtained after centrifugation, add 9.5 mL of anhydrous ethanol to dilute it 20 times and shake well. Using anhydrous ethanol as a blank control, measure the absorbance of the diluted solution at a wavelength of 225 nm. This absorbance is used to calculate the content of free docetaxel.

[0020] Furthermore, the specific processing procedure of the precipitation method is as follows:

[0021] After centrifugation, the supernatant was discarded, and 1 mL of anhydrous ethanol was added to the liposome precipitate. The liposomes were sonicated for 5 min to completely demulsify and release the encapsulated docetaxel. The mixture was then filtered through a 0.45 μm microporous membrane. The filtrate was diluted 40 times with anhydrous ethanol and shaken well. The absorbance of the diluted solution was measured at a wavelength of 225 nm. This absorbance was used to calculate the content of encapsulated docetaxel.

[0022] Furthermore, the formula for calculating the encapsulation efficiency EE is as follows:

[0023] When using the supernatant method, the encapsulation efficiency EEup = (total dosage - free drug content) / total dosage × 100%;

[0024] When using the precipitation method, the encapsulation efficiency EEsediment = (encapsulated drug content / total drug dosage) × 100%.

[0025] Furthermore, the method also includes the determination of drug loading:

[0026] Based on the calculated free drug content or encapsulated drug content, and combined with the amount of soybean lecithin, cholesterol, and docetaxel added in the prescription, the drug loading amount is calculated according to the drug loading formula.

[0027] The formula for the drug loading (DL%) in the supernatant method is as follows:

[0028] DL% = (Total drug dosage - Free drug content) / (Phospholipid + Cholesterol + Total drug dosage) × 100%;

[0029] The formula for the drug loading (DL%) of the precipitation method is:

[0030] DL sedimentation% = Encapsulated drug content / (phospholipid + cholesterol + total drug input) × 100%.

[0031] Furthermore, the method also includes a methodological verification step:

[0032] The relative standard deviation of multiple measurements was calculated through precision tests, the recovery rates at low, medium, and high concentration levels were calculated through spiking recovery tests, and the absorbance changes of the test solution at different time points were examined through stability tests to verify the precision, accuracy, and stability of the measurement results.

[0033] Compared with existing technologies, this invention provides a method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry. The method effectively separates free drug from liposomes using low-temperature high-speed centrifugation, and the 4°C low-temperature condition reduces drug leakage. (13800 rpm·min) -1High-speed centrifugation ensures complete liposome precipitation and reliable separation. Compared to HPLC, this invention employs ultraviolet spectrophotometry, eliminating the need for complex chromatographic separation conditions and large mobile phases. It offers rapid detection, with single-sample determinations completed within one hour. Ultraviolet spectrophotometers are standard laboratory equipment with high availability; the detection process eliminates the need for expensive chromatographic columns and organic solvents, significantly reducing detection costs. Cross-validation using two methods (supernatant method and precipitation method) further enhances the reliability of the results. Methodological validation demonstrates good precision and meets the required recovery rate. This method is suitable for quality control and encapsulation efficiency determination in the R&D stage of docetaxel nanoliposomes, and also provides a reference method for encapsulation efficiency determination of other liposomal drugs. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0035] Figure 1 This is a schematic diagram of the method flow provided in an embodiment of the present invention;

[0036] Figure 2 The ultraviolet absorption scanning spectrum of docetaxel provided in the embodiments of the present invention shows that the maximum absorption wavelength is 225 nm;

[0037] Figure 3 The standard curve of docetaxel provided for embodiments of the present invention. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0039] As attached Figure 1 To be continued Figure 3 As shown:

[0040] Example 1:

[0041] like Figure 1 As shown, the standard curve for docetaxel was established:

[0042] Accurately weigh 10 mg of docetaxel standard and place it in a 50 mL volumetric flask. Dissolve and dilute to the mark with anhydrous ethanol, and shake well to prepare a stock solution with a concentration of 200 μg / mL. Accurately measure an appropriate amount of the stock solution and dilute it stepwise with anhydrous ethanol to prepare a series of standard working solutions with concentrations of 25, 20, 15, 10, and 5 μg / mL.

[0043] like Figure 2 As shown, the above solution was taken and subjected to ultraviolet scanning in the wavelength range of 190–500 nm, and the maximum absorption wavelength of docetaxel was determined to be 225 nm. The absorbance of a series of standard solutions was measured at 225 nm, and a linear regression was performed on the absorbance (A) against the concentration (C, μg / mL) to obtain the standard curve equation. Figure 3 As shown, the results indicate that docetaxel exhibits good linearity in the concentration range of 5–25 μg / mL, with a typical regression equation of A = 0.01368x + 0.009110 and a correlation coefficient r = 0.9990.

[0044] Example 2:

[0045] Determination of encapsulation efficiency and drug loading using the supernatant method:

[0046] Take 1 mL of docetaxel nanoliposome suspension and place it in a pre-cooled 2 mL centrifuge tube. Incubate at 4℃ for 13800 rpm·min. -1 Centrifuge for 30 minutes.

[0047] Accurately transfer 0.5 mL of the supernatant and dilute 20-fold with 9.5 mL of anhydrous ethanol, then shake well. Using anhydrous ethanol as a blank control, measure the absorbance at 225 nm. Substitute the absorbance into the standard curve to calculate the content of free docetaxel (W_free). Calculate the drug loading (DL) and encapsulation efficiency (EE) using the following formulas:

[0048]

[0049]

[0050] In the formula, Wtotal is the dosage of the drug (mg), Wphospholipid and Wcholesterol are the dosages of soybean lecithin and cholesterol in the prescription (mg), respectively, and Wdrug is the total dosage of the drug (mg).

[0051] Each batch of samples was measured in triplicate, and the average value was taken. The results showed that the average encapsulation efficiency of the three batches of samples was 91.00%, with an RSD of 1.26%, and the drug loading was 3.79%, with an RSD of 1.32%.

[0052] The measurement results are shown in Table 1 below;

[0053] Table 1. Results of encapsulation efficiency and drug loading of three batches of samples (x ± s, n=3)

[0054]

[0055] Example 3:

[0056] Determination of encapsulation efficiency and drug loading by precipitation method:

[0057] Take 1 mL of docetaxel nanoliposome suspension and place it in a pre-cooled 2 mL centrifuge tube. Centrifuge at 4°C for 30 min.

[0058] After centrifugation, carefully discard the supernatant. Add 1 mL of anhydrous ethanol to the liposome precipitate and sonicate for 5 min to demulsify the liposomes, releasing the encapsulated docetaxel. Filter through a 0.45 μm microporous membrane. Dilute the filtrate 40 times with anhydrous ethanol, mix well, and measure the absorbance at 225 nm. Substitute the absorbance into the standard curve to calculate the drug content (W_encapsulation). Calculate DL_precipitation% and EE_precipitation% using the following formulas:

[0059]

[0060]

[0061] In the formula, Wtotal is the dosage of the drug (mg), Wphospholipid and Wcholesterol are the dosages of soybean lecithin and cholesterol in the prescription (mg), respectively, and Wdrug is the total dosage of the drug (mg).

[0062] The measurement results are shown in Table 2 below;

[0063] Table 2. Results of encapsulation efficiency and drug loading of three batches of samples (x ± s, n=3)

[0064]

[0065] Each batch of samples was measured in triplicate, and the average value was taken. The results showed that the average encapsulation efficiency of the three batches of samples was 90.47%, with an RSD of 1.75%, and the drug loading was 3.77%, with an RSD of 1.73%, which was basically consistent with the results of the supernatant method.

[0066] As can be seen from the above, the present invention has the following advantages:

[0067] (1) Good separation effect: Low temperature high speed centrifugation method effectively separates free drug and liposomes. The low temperature condition of 4℃ reduces drug leakage. 13800 rpm·min -1 High-speed centrifugation ensures complete liposome precipitation and reliable separation.

[0068] (2) Simple and fast operation: Compared with HPLC, the present invention uses ultraviolet spectrophotometry, which does not require complex chromatographic separation conditions and a large amount of mobile phase. The detection speed is fast, and the determination of a single sample can be completed within 1 hour.

[0069] (3) Low cost: UV spectrophotometers are routine laboratory equipment with high penetration rate; the detection process does not require expensive chromatographic columns and organic solvents, which significantly reduces the detection cost.

[0070] (4) Good accuracy: The results are more reliable by cross-validation of two determination methods (supernatant method and precipitation method); the method validation shows that the method has good precision and the recovery rate meets the requirements.

[0071] (5) Strong applicability: It is applicable to the quality control and encapsulation rate determination of docetaxel nanoliposomes in the R&D stage, and also provides a reference method for the encapsulation rate determination of other liposomal drugs.

[0072] Example 4:

[0073] Methodological validation:

[0074] (1) Precision test:

[0075] Docetaxel nanoliposome samples from the same batch were used, and the encapsulation efficiency was determined using both the supernatant method and the precipitation method. Each method was performed in parallel for six times, and the relative standard deviation (RSD) was calculated. The results showed that the RSD of the supernatant method was 0.98%, and the RSD of the precipitation method was 1.17%, both less than 2.0%, indicating that the method has good precision.

[0076] (2) Recovery test:

[0077] Docetaxel standard was accurately weighed and added to blank liposome solution to prepare recovery test solutions at low, medium, and high concentrations (5, 10, and 15 μg / mL). The solutions were then analyzed according to the method of this invention, with each concentration measured in triplicate, and the recovery rate was calculated. The results showed that the average recoveries for the low, medium, and high concentrations were 93.77%, 96.76%, and 95.27%, respectively, with an overall average recovery rate of 95.27% and an RSD of 1.50%, indicating good accuracy of the method.

[0078] (3) Stability test:

[0079] The absorbance of the same test solution was measured at 0, 2, 4, 8, 12, and 24 h. The results showed that the RSD of the absorbance value was 0.103%, indicating that the test solution had good stability within 24 h.

[0080] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for determining the encapsulation efficiency of docetaxel nano-liposomes based on low temperature high speed centrifugation-ultraviolet spectrophotometry, characterized in that, include: S1. Prepare a series of standard working solutions of docetaxel and perform ultraviolet scanning. After measuring the absorbance, perform linear regression of absorbance against concentration to obtain the standard curve equation. S2. Take the docetaxel nanoliposome suspension and centrifuge it at low temperature and high speed to separate the supernatant containing free drug and the precipitate containing liposomes. S3. The supernatant is treated by the supernatant method or the precipitate is treated by the precipitation method to obtain the test solution and the absorbance is measured to obtain the absorbance of the test solution. S4. Substitute the absorbance of the test solution into the standard curve equation to calculate the free drug content or the encapsulated drug content; S5. Based on the calculated free drug content or encapsulated drug content, and combined with the total dosage of docetaxel, calculate the encapsulation rate using the encapsulation rate formula.

2. The method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry according to claim 1, characterized in that, In step S1, the process of obtaining the standard curve equation is as follows: Accurately weigh the docetaxel standard, dissolve it in anhydrous ethanol, and dilute it stepwise to prepare a series of standard working solutions with concentrations of 25, 20, 15, 10, and 5 μg / mL. Perform ultraviolet scanning in the wavelength range of 190–500 nm to determine the maximum absorption wavelength as 225 nm. Measure the absorbance of the series of standard working solutions at this wavelength, and perform linear regression of absorbance against concentration to obtain the standard curve equation.

3. The method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry according to claim 1, characterized in that, In step S2, the conditions for the low-temperature high-speed centrifugation separation are: The sample of the docetaxel nanoliposome suspension was placed in a pre-cooled centrifuge tube, and centrifuged at 13800 r·min -1 at 4°C for 30 min, so that the free drug was completely retained in the supernatant and the liposomes were completely precipitated at the bottom of the tube.

4. The method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry according to claim 1, characterized in that, The supernatant processing procedure is as follows: Accurately transfer 0.5 mL of the supernatant obtained after centrifugation, add 9.5 mL of anhydrous ethanol to dilute it 20 times and shake well. Using anhydrous ethanol as a blank control, measure the absorbance of the diluted solution at a wavelength of 225 nm. This absorbance is used to calculate the content of free docetaxel.

5. The method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry according to claim 1, characterized in that, The specific processing procedure of the precipitation method is as follows: After centrifugation, the supernatant was discarded, and 1 mL of anhydrous ethanol was added to the liposome precipitate. The liposomes were sonicated for 5 min to completely demulsify and release the encapsulated docetaxel. The mixture was then filtered through a 0.45 μm microporous membrane. The filtrate was diluted 40 times with anhydrous ethanol and shaken well. The absorbance of the diluted solution was measured at a wavelength of 225 nm. This absorbance was used to calculate the content of encapsulated docetaxel.

6. The method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry according to claim 1, characterized in that, The formula for calculating the encapsulation ratio EE is: When using the supernatant method, the encapsulation efficiency EEup = (total dosage - free drug content) / total dosage × 100%; When using the precipitation method, the encapsulation efficiency EEsediment = (encapsulated drug content / total drug dosage) × 100%.

7. The method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry according to claim 1, characterized in that, The method also includes the determination of drug loading: Based on the calculated free drug content or encapsulated drug content, and combined with the amount of soybean lecithin, cholesterol, and docetaxel added in the prescription, the drug loading amount is calculated according to the drug loading formula. The formula for the drug loading (DL%) in the supernatant method is as follows: DL% = (Total drug dosage - Free drug content) / (Phospholipid + Cholesterol + Total drug dosage) × 100%; The formula for the drug loading (DL%) of the precipitation method is: DL sedimentation% = Encapsulated drug content / (phospholipid + cholesterol + total drug input) × 100%.

8. The method for determining the encapsulation efficiency of docetaxel nanoliposomes based on low-temperature high-speed centrifugation-ultraviolet spectrophotometry according to claim 1, characterized in that, The method also includes a methodological verification step: The relative standard deviation of multiple measurements was calculated through precision tests, the recovery rates at low, medium, and high concentration levels were calculated through spiking recovery tests, and the absorbance changes of the test solution at different time points were examined through stability tests to verify the precision, accuracy, and stability of the measurement results.

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