Diosmetin temperature-sensitive liposome and preparation method thereof

Abstract

The invention discloses diosmetin temperature-sensitive liposome and a preparation method thereof, the diosmetin temperature-sensitive liposome is prepared by adopting a film hydration method, and dipalmitoyl phosphatidylcholine and 1-myristoyl-2-stearoyl phosphatidylcholine are combined through hydrogen bonds and hydrophobic force to form a stable liposome structure. The dipalmitoyl phosphatidylcholine provides a basic phospholipid double-layer structure, the 1-myristoyl-2-stearoyl phosphatidylcholine enables the liposome to have a structural change at a specific temperature, so that the fluidity and the structure of a liposome membrane are changed, the thermo-sensitive liposome is formed, the diosmetin is encapsulated in a hydrophobic cavity of the thermo-sensitive liposome, and the stability of the diosmetin is improved. According to the diosmetin temperature-sensitive liposome, active ingredients are protected, the bioavailability of the diosmetin temperature-sensitive liposome is improved, remarkable drug release can be achieved at the target temperature, accurate control over the drug release rate is achieved, the treatment effect is enhanced, and side effects are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to diosmetin thermosensitive liposomes and a preparation method thereof. Background Art

[0002] Dioscorea is a flavonoid compound widely found in citrus fruits, with multiple pharmacological effects such as antibacterial, antioxidant, and anti-inflammatory. Recent studies have shown that dioscorea has an inhibitory effect on various tumor cells such as breast cancer, liver cancer, lung cancer, and prostate cancer. However, flavonoids have the disadvantages of poor water solubility, high first-pass effect, and low oral bioavailability, which seriously limit the clinical application of this type of compound.

[0003] Thermosensitive liposomes are a new type of drug controlled release carrier that is temperature sensitive and can undergo phase change at a specific temperature. The phospholipid state changes from a gel state to a liquid crystal state, thereby releasing the encapsulated drug. In clinical applications, the diseased area needs to be heated up using thermal therapy to cause the liposome to undergo phase change. Thermosensitive liposomes can not only improve drug targeting, but also reduce drug leakage and reduce the toxic side effects of chemotherapy drugs on normal cells and tissues. The unique thermosensitive targeted drug release of thermosensitive liposomes has shown broad development prospects and application value in the field of drug carriers.

[0004] Although the research on thermosensitive liposomes has made significant progress, there is no research report on the combination of diosmetin and thermosensitive liposomes, which limits the wider clinical application of diosmetin. Therefore, the development of a thermosensitive liposome that can effectively encapsulate diosmetin and has good thermosensitive drug release properties has become an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a diosmin thermosensitive liposome and a preparation method thereof.

[0006] One of the technical solutions adopted by the present invention to solve the technical problem is:

[0007] Provided is a method for preparing diosmetin thermosensitive liposomes, the method comprising: placing diosmetin, dipalmitoylphosphatidylcholine, and 1-myristoyl-2-stearoylphosphatidylcholine in an eggplant-shaped bottle at a mass ratio of 2-2.6:15-18:2, adding methanol to mix with the diosmetin, dissolving the solid under ultrasonic conditions, and performing reduced pressure rotary evaporation until the methanol reacts completely, wherein the dipalmitoylphosphatidylcholine and 1-myristoyl-2-stearoylphosphatidylcholine are bonded to the inner wall of the eggplant-shaped bottle through hydrogen bonds and hydrophobic forces. The liposomes are formed into lipid films, ultrapure water is added into the bottle, and the films are de-membraned and hydrated for 10 minutes, so that the lipid films swell to form lipid bilayer structures to form liposomes. Meanwhile, the 1-myristoyl-2-stearoylphosphatidylcholine causes the liposomes to undergo phase change to form thermosensitive liposomes, and the dioscorea lignin is encapsulated in the hydrophobic cavity of the thermosensitive liposomes. The hydrated liposomes are transferred to a beaker, and are completely dispersed under ultrasound. The liposomes are repeatedly passed through the film at 50°C using a liposome extruder to make the particle size more uniform, and the dioscorea lignin thermosensitive liposomes are obtained by storing at low temperature.

[0008] Furthermore, the temperature of the rotary evaporation is 40°C to 50°C.

[0009] Furthermore, the demoulding hydration temperature is 40°C to 50°C.

[0010] Furthermore, the liposome was repeatedly passed through a 400 nm membrane 15 times and then through a 200 nm membrane 10 times using a liposome extruder.

[0011] The second technical solution adopted by the present invention to solve its technical problem is:

[0012] Provided is a diosmetin thermosensitive liposome, which is prepared by the above-mentioned diosmetin thermosensitive liposome preparation method, and is transported to a lesion area in a patient's body.

[0013] Furthermore, the phase transition temperature of the dioscorea thermosensitive liposome is 53.04°C.

[0014] Compared with the background technology, the technical scheme has the following advantages: the present invention adopts a thin film hydration method to prepare diosmetin thermosensitive liposomes, and utilizes dipalmitoylphosphatidylcholine and 1-myristoyl-2-stearoylphosphatidylcholine to combine through hydrogen bonds and hydrophobic forces to form a stable liposome structure, dipalmitoylphosphatidylcholine provides a basic phospholipid bilayer structure, 1-myristoyl-2-stearoylphosphatidylcholine causes the liposome to undergo structural changes at a specific temperature, thereby changing the fluidity and structure of the liposome membrane to form thermosensitive liposomes, diosmetin is encapsulated in the hydrophobic cavity of the thermosensitive liposome to form diosmetin thermosensitive liposomes, protect the active ingredients and improve their bioavailability, and the results show that the encapsulation rate of diosmetin thermosensitive liposomes is 89.89±0.0016%, the drug loading is 6.64%, the particle size is 88.31nm, the potential is -10.30mV, and the phase transition temperature is 53.04℃. At 37°C, the lignin thermosensitive liposome released only 44.5% in 12 hours, and the cumulative release remained at 55.2% after 36 hours. At 42°C, the cumulative release was 67.4% in 12 hours, and the cumulative release remained at 82.9% after 24 hours. At 53°C, equilibrium was reached after 8 hours, with a cumulative release rate of 83.8%, indicating that it can achieve significant drug release at the target temperature, achieve precise control of the drug release rate, enhance the therapeutic effect and reduce side effects, and has potential application value in drug delivery and treatment, especially in cases where it is necessary to control drug release and improve drug stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0016] Figure 1 is the standard curve of dioscorea lignin;

[0017] Figure 2 The figure is the effect of the ratio of DPPC to MSPC on the thermosensitive liposomes of dioscorea lignin;

[0018] Figure 3 This is the effect of the ratio of DPPC to diosmetin on diosmetin thermosensitive liposomes;

[0019] Figure 4 This is the effect of rotary evaporation temperature on dioscorea thermosensitive liposomes;

[0020] Figure 5 This is the effect of hydration temperature on dioscorea thermosensitive liposomes;

[0021] Figure 6 The contour map and three-dimensional response surface diagram of the factors affecting the encapsulation efficiency of dioscorea thermosensitive liposomes;

[0022] Figure 7The contour map and three-dimensional response surface map of the particle size of the influencing factors of dioscorea thermosensitive liposomes;

[0023] Figure 8 The contour map and three-dimensional response surface diagram of the absolute value of the potential of the influencing factors of the thermosensitive liposomes of dioscorea lignin;

[0024] Fig. 9 Optimal prescription prediction plot for the model built for response surface methodology;

[0025] Fig.10 is the particle size distribution diagram of dioscorea thermosensitive liposomes;

[0026] Fig.11 This is the Zeta potential diagram of diosgenin thermosensitive liposomes;

[0027] Fig.12 This is the temperature responsiveness diagram of dioscorea thermosensitive liposomes;

[0028] Fig.13 The in vitro release of dioscorea thermosensitive liposomes at 37℃, 42℃ and 53℃ in the same period of time;

[0029] Fig.14 This is the DSC graph of dioscorea lignin thermosensitive liposomes. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1: Preparation method of dioscorea thermosensitive liposomes

[0032] 4 mg of dianthus, an appropriate amount of dipalmitoylphosphatidylcholine (hereinafter referred to as DPPC) and an appropriate amount of 1-myristoyl-2-stearoylphosphatidylcholine (hereinafter referred to as MSPC) were weighed in a mass ratio of 2-2.6:15-18:2, and they were placed in 100 mL eggplant-shaped bottles respectively, 20 mL of methanol was added and mixed therewith, the solid was dissolved under ultrasonic conditions, and the methanol was evaporated under reduced pressure and rotary evaporated at 40° C. to complete the reaction of methanol, the dipalmitoylphosphatidylcholine and 1-myristoyl-2-stearoylphosphatidylcholine were combined on the inner wall of the eggplant-shaped bottle through hydrogen bonds and hydrophobic forces to form a lipid membrane, 20 mL of ultrapure water was added to the bottle, and the membrane was further dehydrated at 40° C. to 50° C. for 10 minutes, so that the lipid membrane expanded to form a lipid bilayer structure to constitute a liposome, and at the same time, the The 1-myristoyl-2-stearoylphosphatidylcholine causes the liposome to undergo a phase change to form a thermosensitive liposome, and the diosmetin is encapsulated in the hydrophobic cavity of the thermosensitive liposome. The hydrated liposome is transferred to a beaker, and is completely dispersed under 30KHz frequency ultrasound. At 50°C, the liposome is repeatedly passed through a 400nm membrane 15 times and then through a 200nm membrane 10 times using a liposome extruder to make the particle size relatively more uniform. The liposome aqueous solution is stored in a refrigerator at 4°C to obtain the diosmetin thermosensitive liposome. The fatty acid chain in the 1-myristoyl-2-stearoylphosphatidylcholine causes the phase change temperature of the liposome to change, the myristic acid increases the rigidity or fluidity of the liposome membrane and affects the thermosensitivity of the liposome, and the stearic acid increases the melting point of the membrane, so that the liposome remains stable at a specific temperature.

[0033] Test Example 1: Drawing of the standard curve of dioscorea lignin

[0034] Accurately weigh 5 mg of lignin standard and place it in a beaker, add an appropriate amount of methanol and stir to dissolve. After complete dissolution, drain it into a 10 mL volumetric flask with a glass rod, titrate it to the scale with methanol, and store it at 4 ° C for later use. Prepare lignin solutions according to 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 and 40.0 μg / mL, and measure the absorbance A at each concentration with a UV spectrophotometer. A = 0.01705 + 0.01538C (R 2 =0.99932). The linear relationship between the absorbance and concentration of dioscorea lignin was good in the concentration range of 0.5-40.0 μg / mL, indicating that the method was feasible.

[0035] Experimental Example 2: Optimization of the preparation process of dioscorea thermosensitive liposomes

[0036] 1. Single factor investigation

[0037] (1) Determination of the ratio of DPPC to MSPC

[0038] Dioscorea thermosensitive liposomes were prepared according to the ratio of DPPC to MSPC of 9:1, 10:1 and 11:1. The ratio of these two substances was determined by encapsulation efficiency, particle size and Zeta potential. The experimental results are shown in Figure 2 and Table 1, where Figure 2 Where A is the encapsulation efficiency, B is the particle size, and C is the potential.

[0039] Table 1 Analysis of variance table of the effect of DPPC to MSPC ratio on encapsulation efficiency, particle size and potential

[0040]

[0041] Depend on Figure 2 From the results in Table 1, it can be seen that the ratio of DPPC to MSPC has no significant effect on encapsulation efficiency, particle size and potential. The encapsulation efficiency and particle size are more dispersed at 9:1 and 11:1, and the potential conditions at the three levels are somewhat dispersed, so 10:1 was selected for subsequent experiments.

[0042] (2) Determination of the ratio of DPPC to diosmin

[0043] Thermosensitive liposomes of dioscorea lignin were prepared according to the ratio of DPPC to dioscorea lignin of 16:2, 17:2 and 18:2. The ratio of the two substances was determined by encapsulation efficiency, particle size and Zeta potential. The experimental results are shown in Figure 3 and Table 2, where Figure 3 Where A is the encapsulation efficiency, B is the particle size, and C is the potential.

[0044] Table 2 Analysis of variance table of the effect of DPPC to lignin ratio on encapsulation efficiency, particle size and potential

[0045]

[0046] Depend on Figure 3 It can be seen that there are extreme values ​​when the ratio of DPPC to dioscorea is 8.5:1. With the increase of the ratio of DPPC to dioscorea, the encapsulation efficiency and the absolute value of the potential show a trend of first increasing and then decreasing. With the increase of the ratio of DPPC to dioscorea, the particle size shows a trend of first decreasing and then increasing.

[0047] From the variance analysis table 2, it can be seen that when the significance level α = 0.05, the significance p of the ratio of DPPC to diosmetin on the encapsulation efficiency, particle size and potential of diosmetin thermosensitive liposomes is p < 0.05, indicating that the ratio of DPPC to diosmetin has a significant effect on these three indicators. The ratio of DPPC to diosmetin was determined to be 8.5:1.

[0048] (3) Determination of rotary evaporation temperature

[0049] The diosmetin thermosensitive liposomes were prepared at 40°C, 45°C and 50°C, and the temperature of the rotary evaporation was determined by the encapsulation efficiency, particle size and Zeta potential. Figure 4 and Table 3, where Figure 4 Where A is the encapsulation efficiency, B is the particle size, and C is the potential.

[0050] Table 3 Variance analysis table of the effect of rotary evaporation temperature on encapsulation efficiency, particle size and potential

[0051]

[0052]

[0053] Depend on Figure 4 It can be seen that there are extreme values ​​when the rotary evaporation temperature is 45°C. With the increase of the rotary evaporation temperature, the encapsulation efficiency and the absolute value of the potential show a trend of first increasing and then decreasing. With the increase of the rotary evaporation temperature, the particle size shows a trend of first decreasing and then increasing.

[0054] From the variance analysis table 3, it can be seen that when the significance level α=0.05, the significance p of the rotary evaporation temperature on the encapsulation efficiency, particle size and potential of the thermosensitive liposomes of dioscorea lignin is p<0.05, indicating that the rotary evaporation temperature has a significant effect on these three indicators. The rotary evaporation temperature is determined to be 45°C.

[0055] (4) Determination of hydration temperature

[0056] The diosmetin thermosensitive liposomes were prepared at hydration temperatures of 40°C, 45°C and 50°C, and the hydration temperature was determined by encapsulation efficiency, particle size and Zeta potential. Figure 5 and Table 4, where Figure 5 Where A is the encapsulation efficiency, B is the particle size, and C is the potential.

[0057] Table 4 Analysis of variance table of the effect of hydration temperature on encapsulation efficiency, particle size and potential

[0058]

[0059] Depend on Figure 5 It can be seen that extreme values ​​appear when the hydration temperature is 45°C. With the increase of hydration temperature, the encapsulation efficiency and the absolute value of potential show a trend of first increasing and then decreasing, and with the increase of hydration temperature, the particle size shows a trend of first decreasing and then increasing.

[0060] From the variance analysis table 4, it can be seen that when the significance level α = 0.05, the significance p of hydration temperature on the encapsulation efficiency, particle size and potential of the thermosensitive liposomes of dioscorea lignin is p < 0.05, indicating that the hydration temperature has a significant effect on these three indicators. The hydration temperature is determined to be 45°C.

[0061] 2. Box-Behnken design-response surface method optimization

[0062] (1) Optimize coding and level

[0063] On the basis of single factor experiment, in order to screen out the best formula for the preparation of thermosensitive liposomes of diosmetin, the ratio of DPPC to diosmetin (X1), rotary evaporation temperature (X2) and hydration temperature (X3) were used as conditions, and the encapsulation efficiency (Y1), particle size (Y2) and potential absolute value (Y3) were used as references to optimize the formula of thermosensitive liposomes of diosmetin by response surface methodology. The factor levels are shown in Tables 5 and 6.

[0064] Table 5 Factors and levels of Box-Behnken design

[0065]

[0066] Table 6 Independent variables and dependent variables

[0067]

[0068]

[0069] (2) Establishment of multivariate quadratic regression equation

[0070] With encapsulation rate as the dependent variable, the model significance test p<0.05 was obtained through Design-Expert 12 software analysis, indicating that the model is statistically significant. 2 , B 2 , C 2 The results were significantly different (p<0.05), and variance analysis and multivariate quadratic regression equation fitting were performed to obtain equation 1. In equation 1, X1(P=0.0010), X2(P=0.0327), X3(P=0.0005), X2X3(P=0.0137), X1 2 (P<0.0001), X2 2 (P<0.0001) and X3 2 (P<0.0001) had a very significant effect, and the other items were not significant.

[0071] Y1=89.77+1.50X1+0.7363X2+1.68X3+1.28X2X3-8.35X1 2 -6.17X2 2 -3.16X3 2 (1)

[0072] The model P value is <0.0001, and the determination coefficient R 2=0.9929, correction factor R adj 2 =0.9837, and the lack-of-fit F=0.2062>0.05, indicating that the measured encapsulation efficiency has no significant lack-of-fit, indicating that the regression model has a high degree of fitting, and the unknown factors have little interference on the model. The established mathematical model has a high credibility, and this model can be used for the formulation analysis and prediction of lignin thermosensitive liposomes.

[0073] Design-Expert 12 software was used to draw contour maps and three-dimensional response surface diagrams of the factors affecting the encapsulation efficiency of dioscorea thermosensitive liposomes, such as Figure 6 As shown, A1 and B1: the influence of independent variables X1 and X2, C1 and D1: the influence of independent variables X1 and X3, E1 and F1: the influence of independent variables X2 and X3, Figure 6 It can be seen that when one of the single factors takes the median value, the other two single factors continue to increase, and the encapsulation efficiency of dioscorea thermosensitive liposomes maintains a trend of first increasing and then decreasing.

[0074] Taking particle size as the dependent variable, the results of the analysis by Design-Expert 12 software showed that the model significance test p<0.05, indicating that the model is statistically significant. 2 , C 2 The results were subjected to variance analysis and multivariate quadratic regression equation fitting to obtain equation 2. In equation 2, X1X3 (P = 0.0156), X2X3 (P = 0.0013), X2 2 (P=0.0035) and X3 2 (P<0.0001) had a very significant effect, and the other items were not significant.

[0075] Y2=83.39+27.98X1X3+45.63X2X3+37.02X2 2 +77.47X3 2 (2)

[0076] Model P value < 0.0001, determination coefficient R 2 =0.9557, correction factor R adj 2 =0.8987, and the lack-of-fit F=35.51>0.05, indicating that the measured particle size has no significant lack-of-fit, indicating that the regression model has a high degree of fitting and the unknown factors have little interference on the model. Therefore, the established mathematical model has a high credibility and can be used to analyze and predict the formulation of lignin thermosensitive liposomes.

[0077] Design-Expert 12 software was used to draw the contour map and three-dimensional response surface diagram of the influencing factors of the particle size of the thermosensitive liposomes of dioscorea lignin, such as Figure 7 As shown, A2 and B2: the influence of independent variables X1 and X2, C2 and D2: the influence of independent variables X1 and X3, E2 and F2: the influence of independent variables X2 and X3, Figure 7 It can be seen that when one of the single factors takes the median value, the other two single factors continue to decrease, and the particle size of the dioscorea thermosensitive liposomes maintains a trend of first decreasing and then increasing.

[0078] The absolute value of potential was used as the dependent variable, and the model significance test p<0.05 was obtained through the Design-Expert 12 software analysis, indicating that the model is statistically significant. 2 , B 2 The results were analyzed by variance analysis and multivariate quadratic regression equation fitting. The fitting equation 3 was obtained. In equation 3, X1X3 (P = 0.0156), X2X3 (P = 0.0013), X2 2 (P=0.0035) and X3 2 (P<0.0001) had a very significant effect, and the other items were not significant.

[0079] Y3=9.84+0.3088X2+0.4050X3+0.4525X1X2-2.46X1 2 -1.04X2 2 (3)

[0080] Model P value <0.0001, determination coefficient R 2 =0.9807, correction factor R adj 2 =0.9560, and the lack of fit F=19.57>0.05, indicating that the absolute value of the measured potential has no significant lack of fit, indicating that the regression model has a high degree of fit, and the unknown factors have little interference on the model. Therefore, the established mathematical model has a high credibility and can be used for the formulation and process research of diosmetin thermosensitive liposomes. This model can be used to analyze and predict the formulation of diosmetin thermosensitive liposomes.

[0081] Design-Expert 12 software was used to draw contour maps and three-dimensional response surface maps of the absolute value of the potential of the influencing factors of dioscorea thermosensitive liposomes, such as Figure 8 As shown, A3 and B3: the influence of independent variables X1 and X2, C3 and D3: the influence of independent variables X1 and X3, E3 and F3: the influence of independent variables X2 and X3, Figure 8It can be seen that when one of the single factors takes the median value, the other two single factors continue to increase, and the absolute value of the potential of the dioscorea thermosensitive liposomes maintains a trend of first increasing and then decreasing.

[0082] (3) Response surface optimization, prediction and verification

[0083] In order to achieve the maximum encapsulation efficiency, the minimum particle size and the maximum absolute potential, the optimized prescription obtained by analysis using Design-Expert software is as follows Fig. 9 The solution is X1=8.510, X2=45.198°C, X3=45.363°C, at which time Y1=89.924%, Y2=85.483nm, and Y3=9.874mV.

[0084] Weigh 4 mg of dianthus lignin, 34 mg of dipalmitoylphosphatidylcholine and 3.4 mg of 1-myristoyl-2-stearoylphosphatidylcholine, respectively, put them into 100 mL eggplant-shaped bottles, add 20 mL of methanol and mix them, dissolve the solid under ultrasonic conditions, and evaporate under reduced pressure and rotary evaporation at 45°C until the methanol reacts completely. When a thin film appears on the inner wall of the eggplant-shaped bottle, add 20 mL of ultrapure water into the bottle, and continue to demembrane and hydrate at 45°C for 10 minutes to make the lipid membrane swell to form a lipid bilayer structure to form liposomes. The hydrated liposomes are transferred to a beaker and dispersed completely under 30KHz frequency ultrasound. At 50°C, a liposome extruder is used to repeatedly pass through a 400nm membrane 15 times and a 200nm membrane 10 times to make the particle size relatively more uniform. Finally, the liposome aqueous solution is stored in a 4°C refrigerator to obtain dianthus lignin thermosensitive liposomes for subsequent testing.

[0085] Experimental Example 3: Particle size distribution and Zeta potential measurement of dioscorea thermosensitive liposomes

[0086] The particle size was measured using a Malvern quartz cuvette. Fig.10 As shown, the average particle size was measured to be 88.13 nm and the PID (polydispersity index) was 0.22, indicating that the particle size data was stable.

[0087] The potential of the liposome stock solution was measured using a Malvern Zeta potential sample cell. Fig.11 As shown, the average potential was measured to be -10.4 mV, indicating that the lignin thermosensitive liposomes have a certain stability.

[0088] Test Example 4: Determination of encapsulation efficiency and drug loading of dioscorea thermosensitive liposomes

[0089] Three batches of diosmetin thermosensitive liposomes were prepared. Under the same conditions, the drug loading was 6.64% and the encapsulation efficiency was 89.89%, indicating that the encapsulation effect of diosmetin in liposomes was good.

[0090] Table 7 Results of encapsulation efficiency and drug loading

[0091]

[0092] Test Example 5: Determination of the temperature-responsive drug release of dioscorea thermosensitive liposomes

[0093] The dialysis bag method was used for the study. 1 mL of the prepared lignin thermosensitive liposomes was added to the treated dialysis bag (molecular weight cutoff of 8000), and both ends were sealed to ensure that the solution in the dialysis bag did not leak. The dialysis bag was placed in a centrifuge tube containing 35 ml of 1% Tween-80 aqueous solution; the centrifuge tube was placed in a constant temperature shock box at 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C and 44°C, and released at a speed of 150r / min for 4 hours, and then immediately taken out of the ice bath, the lignin concentration was measured, and the release rate of the lignin thermosensitive liposomes at different temperatures was calculated.

[0094] like Fig.12 As shown, with the increase of temperature, the release amount of dioscorea lignin from liposomes gradually increases. At 37-41°C, the release rate increases slowly. At 42°C, it suddenly rises by 9.2%. At 43°C, it only increases by 0.69%, indicating that the optimal release temperature of dioscorea lignin thermosensitive liposomes is 42°C. At this time, the dioscorea lignin thermosensitive liposomes change from the original orderly arranged gel state to the loosely arranged liquid crystal state, and the drug is released from it.

[0095] Experimental Example 6: Drawing of in vitro drug release curve of dioscorea thermosensitive liposomes

[0096] 1 mL of dioscorea thermosensitive liposomes was added to a treated dialysis bag (molecular weight cutoff of 8000), and both ends were sealed to ensure that the solution in the dialysis bag did not leak. The dialysis bag was placed in a centrifuge tube containing 35 ml of 1% Tween-80 aqueous solution; in a constant temperature shock box at 37°C and 42°C, at a speed of 150 r / min, 0.5 ml of samples were taken at 1h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h (while supplemented with an equal amount of 1% Teween-80 aqueous solution at the same temperature), the dioscorea content was determined, the cumulative release rate was calculated according to the following formula, and the release curve was drawn, as shown in FIG. Fig.13 shown.

[0097]

[0098] Where C n : sample concentration at the nth time point; V: total volume of release medium; C i: The sample concentration at the i-th time point (C0=0); Vi: The sampling volume at the i-th time point (V0=0); M0 represents the initial amount of dioscorea in the liposome.

[0099] from Fig.13 It can be seen that the thermosensitive liposomes of dioscorea lignin only released 44.5% in 12 hours at 37°C, and then the arc of continuous rise was small. After 36 hours, the cumulative release rate remained at 55.2% with the increase of time. Under 42°C, 67.4% was released in 12 hours, and 82.9% was released in 24 hours. As time increased, the cumulative release rate no longer increased significantly. Under 53°C, equilibrium was reached in 8 hours, and the cumulative release rate was 83.8%. This shows that at 53°C, the liposome structure was greatly disordered, indicating that temperature changes can control the release of dioscorea lignin from liposomes, and the liposomes are temperature-sensitive liposomes.

[0100] Test Example 7: DSC Analysis of Dioscorea Thermosensitive Liposomes

[0101] The results were obtained by differential scanning calorimetry. Fig.14 As shown in the figure, the thermosensitive liposomes of dioscorea lignin have a peak with obvious changes near 41.68℃, which is consistent with the phase transition temperature of the main material DPPC. At this time, the phospholipid bilayer of the liposome will undergo a phase transition from gel state to liquid state, causing the structure of the liposome to change from the initial tight arrangement to loose arrangement, and finally allowing the encapsulated drug to be rapidly released from the liposome. In addition, there is a very sharp peak at 53.04℃, indicating that its phase transition reaches the maximum degree of disorder at this time, and at the same time, it shows that: with the addition of MSPC, the hydrophobic drug dioscorea lignin is encapsulated in the hydrophobic cavity of the thermosensitive liposome, causing its phase transition temperature to increase from the original 41.68℃ to 53.04℃.

[0102] The invention adopts a thin film hydration method to prepare diosmetin thermosensitive liposomes, and utilizes dipalmitoylphosphatidylcholine and 1-myristoyl-2-stearoylphosphatidylcholine to combine through hydrogen bonds and hydrophobic forces to form a stable liposome structure, dipalmitoylphosphatidylcholine provides a basic phospholipid bilayer structure, 1-myristoyl-2-stearoylphosphatidylcholine causes the liposome to undergo structural changes at a specific temperature, thereby changing the fluidity and structure of the liposome membrane to form thermosensitive liposomes, diosmetin is encapsulated in the hydrophobic cavity of the thermosensitive liposomes to form diosmetin thermosensitive liposomes, protect active ingredients and improve the bioavailability thereof, and the results show that the encapsulation rate of the diosmetin thermosensitive liposomes is 89.89±0.0016%, the drug loading is 6.64%, the particle size is 88.31nm, the potential is -10.30mV, and the phase transition temperature is 53.04°C. At 37°C, the lignin thermosensitive liposome released only 44.5% in 12 hours, and the cumulative release remained at 55.2% after 36 hours. At 42°C, the cumulative release was 67.4% in 12 hours, and the cumulative release remained at 82.9% after 24 hours. At 53°C, equilibrium was reached after 8 hours, with a cumulative release rate of 83.8%, indicating that it can achieve significant drug release at the target temperature, achieve precise control of the drug release rate, enhance the therapeutic effect and reduce side effects, and has potential application value in drug delivery and treatment, especially in cases where it is necessary to control drug release and improve drug stability.

[0103] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for preparing dioscorea thermosensitive liposomes, characterized in that: The method comprises: placing diosmetin, dipalmitoylphosphatidylcholine and 1-myristoyl-2-stearoylphosphatidylcholine in an eggplant-shaped bottle at a mass ratio of 2-2.6:15-18:2 respectively, adding methanol to mix with the diosmetin, dissolving the solid under ultrasonic conditions, and performing reduced pressure rotary evaporation until the methanol reacts completely, wherein the dipalmitoylphosphatidylcholine and 1-myristoyl-2-stearoylphosphatidylcholine are bonded to the inner wall of the eggplant-shaped bottle through hydrogen bonds and hydrophobic forces to form a lipid film, and adding methanol to the bottle. Ultrapure water, demembrane hydration for 10 minutes, so that the lipid membrane swells to form a lipid bilayer structure to form liposomes. At the same time, the 1-myristoyl-2-stearoyl phosphatidylcholine causes the liposome to undergo a phase change to form a thermosensitive liposome. Dioscorea odorifera is encapsulated in the hydrophobic cavity of the thermosensitive liposome. The hydrated liposome is transferred to a beaker and dispersed completely under ultrasound. The liposome is repeatedly passed through the membrane with a liposome extruder at 50°C to make the particle size more uniform. The dioscorea odorifera thermosensitive liposome is obtained by low temperature storage.

2. The method for preparing the dioscorea thermosensitive liposome according to claim 1, wherein: The temperature of the rotary evaporation is 40°C to 50°C.

3. The method for preparing the dioscorea thermosensitive liposome according to claim 1, characterized in that: The temperature of the demoulding hydration is 40°C to 50°C.

4. The method for preparing the dioscorea thermosensitive liposome according to claim 1, characterized in that: The liposome was repeatedly passed through a 400 nm membrane 15 times and then through a 200 nm membrane 10 times using a liposome extruder.

5. A dioscorea thermosensitive liposome, characterized in that: The diosmetin thermosensitive liposome is prepared by the preparation method of the diosmetin thermosensitive liposome according to any one of claims 1 to 4, and the diosmetin thermosensitive liposome is delivered to the lesion area in the patient's body.

6. The diosmetin thermosensitive liposome according to claim 5, characterized in that: The phase transition temperature of the diosmetin thermosensitive liposome is 53.04°C.

Images