Tacrolimus liposome subcutaneous in-situ gel as well as preparation method and application thereof
A heptamethonium chloride-loaded liposome subcutaneous gel, stabilized by glutathione-modified low acyl gellan gum, addresses frequent dosing and toxicity issues in silver dermatitis treatment by providing targeted and stable drug delivery with enhanced therapeutic efficacy.
Patent Information
- Application Number
- CN202510563098.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-07-15
AI Technical Summary
The existing tacrolimus liposomes as drug delivery carriers have poor physical and chemical stability, which is difficult to withstand large mechanical stresses, and the drug is insufficient in the epidermal and dermis, resulting in insignificant effects in the treatment of psoriasis.
The liposome membrane is encapsulated by glutathione-modified low acyl gellan gum, combined with freeze-thaw circulation technology, and tacrolimus liposome subcutaneous in situ gel is prepared to improve drug encapsulation rate and release control.
The slow release of drugs at the affected area has been achieved, the targeted and therapeutic effect of treating psoriasis has been improved, the frequency of administration has been reduced, and the convenience and effectiveness of treatment have been enhanced.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a tacrolimus liposome subcutaneous in-situ gel, a preparation method thereof, and an application thereof. Background Art
[0002] During the treatment of psoriasis, most drugs need to be delivered through the skin, with frequent administration and large doses, and also having significant toxic effects. Therefore, changing the administration route is one of the methods to improve its efficacy and safety.
[0003] As a specific calcineurin inhibitor, the mechanism of action of tacrolimus in the treatment of psoriasis has been widely studied (such as in Document 1). Research shows that tacrolimus can effectively reduce psoriatic-like skin lesions by inhibiting Th17-related inflammation. The present invention prepares a tacrolimus liposome subcutaneous in-situ gel to precisely target psoriasis for treatment. Compared with the broad range of drug types in Comparative Document 1, the product of the present invention has a stronger treatment targeting property, can act more effectively on psoriasis lesions, and thus obtains a more significant treatment effect.
[0004] As a drug delivery carrier, liposomes have low toxicity, immunogenicity, high biocompatibility, and biodegradability. Using liposomes as a drug carrier to carry tacrolimus through skin pores can increase the retention amount of the drug in the epidermal layer and dermal layer. However, according to some studies, using liposomes as a drug delivery carrier still has some disadvantages, such as poor physical and chemical stability and difficulty in withstanding large mechanical stresses.
[0005] Document 1: Fan Chao, Xiang Huaguo, Chen Hongxiang. Tacrolimus treats imiquimod-induced psoriasis mouse model by inhibiting Th17 [J]. Journal of Huazhong University of Science and Technology (Medical Sciences), 2016, 45(05): 484 - 489 + 495. Summary of the Invention
[0006] In order to overcome the above problems in the prior art, the present invention provides a tacrolimus liposome subcutaneous in-situ gel and a preparation method thereof. By wrapping glutathione-modified low-acyl gellan gum outside the liposome loaded with tacrolimus, the stability of the liposome membrane and the effective control of drug release are achieved.
[0007] In order to achieve the object of the present invention, the technical solution adopted is: a tacrolimus liposome subcutaneous in-situ gel, which includes the drug tacrolimus lipid encapsulated inside the liposome and glutathione-modified low-acyl gellan gum wrapped outside the liposome.
[0008] The preparation method of the above-mentioned tacrolimus liposome subcutaneous in-situ gel includes the following steps:
[0009] (1) Dissolve tacrolimus, cholesterol, and soy lecithin in chloroform respectively. After mixing, rotate and evaporate to remove the solvent until a lipid film adheres to the bottle wall. Then add purified water to the system, ultrasonicate in a water bath, and then place it in a shaker to oscillate to form a dispersion.
[0010] (2) Immerse the dispersion obtained in step (1) in liquid nitrogen for 30 s for rapid freezing, and then thaw it by oscillating in a 37 °C water bath. Repeat the cycle 1 - 5 times (preferably 4 times) to obtain a dispersion of tacrolimus liposomes.
[0011] (3) After uniformly mixing the dispersion of tacrolimus liposomes obtained in step (2) with a glutathione - modified low - acyl gellan gum solution, stir and incubate at room temperature for 12 h to obtain a subcutaneous in - situ gel of tacrolimus liposomes.
[0012] Furthermore, in step (1), the mass ratio of cholesterol to soy lecithin is 1:3 - 7 (preferably 1:5).
[0013] Furthermore, in the subcutaneous in - situ gel of tacrolimus liposomes, the drug - lipid ratio (mass ratio of the total mass of tacrolimus, cholesterol, and soy lecithin) is 1:20.
[0014] Furthermore, in step (1), the rotation evaporation time is 30 min, the ultrasonic time in the water bath is 5 min, and the oscillation time in the shaker is 2 h.
[0015] Furthermore, in step (3), the mass concentration of the glutathione - modified gellan gum solution is 2%.
[0016] Furthermore, in step (3), the volume ratio of the dispersion of tacrolimus liposomes to the glutathione - modified gellan gum solution is 1:1.
[0017] Furthermore, in the subcutaneous in - situ gel of tacrolimus liposomes, the mass concentration of tacrolimus is 0.1 - 0.5%.
[0018] Administer the above subcutaneous in - situ gel by subcutaneous injection to psoriatic lesion skin, which is beneficial to the gelation of the drug subcutaneously at the affected area, thereby slowly releasing the drug.
[0019] The beneficial effects of the present invention are as follows:
[0020] The present invention prepares a subcutaneous in - situ gel of tacrolimus liposomes and applies it to subcutaneous injection for psoriatic lesion skin, which is beneficial to the gelation of the drug subcutaneously at the affected area, thereby slowly releasing the drug. The subcutaneous injection administration method has the advantage of drug slow - release, can effectively improve the problem of frequent drug administration for psoriasis, and provides a more convenient and effective treatment plan for patients. Brief Description of the Drawings
[0021] Figure 1 The average particle size and PDI of blank liposomes with different ratios of soybean lecithin to cholesterol in Example 1;
[0022] Figure 2 The encapsulation efficiency of tacrolimus liposomes at different drug-lipid ratios in Example 2;
[0023] Figure 3 The average particle size and PDI of tacrolimus liposomes at different drug-lipid ratios in Example 2
[0024] Figure 4 The encapsulation efficiency of tacrolimus liposomes at different numbers of freeze-thaw cycles in Example 2;
[0025] Figure 5 The comparison of the encapsulation efficiency of liposomes in Example 3 and Example 4;
[0026] Figure 6 The body weights of mice in different groups;
[0027] Figure 7 The spleen weights of mice in different groups;
[0028] Figure 8 The epidermal section diagrams of mice in different groups;
[0029] Figure 9 The epidermal thicknesses of mice in different groups;
[0030] Figure 10 The epidermal section diagrams of mice of liposome gel without tacrolimus and liposome hydrogel containing tacrolimus, wherein, a is the blank group; b is the control group, that is, only induced with 5% imiquimod ointment without giving drugs; c and d are the experimental groups, wherein c is given blank liposome gel without tacrolimus, and d is given 0.1% tacrolimus liposome subcutaneous in-situ gel;
[0031] Figure 11 The epidermal thicknesses of mice of liposome gel without tacrolimus and liposome hydrogel containing tacrolimus. Detailed implementation manners
[0032] The present invention will be described in detail below in conjunction with the embodiments.
[0033] Example 1
[0034] Prepare blank liposomes, and the specific steps are as follows: Weigh soybean lecithin and cholesterol respectively, with a mass ratio of 3:1, 4:1, 5:1, 6:1, 7:1. Dissolve them in 1 mL of chloroform respectively. Among them, the mass of cholesterol is fixed at 16.67 mg. Rotate and evaporate at 37 °C for 30 min to remove the solvent and form a lipid film; then add 0.5 mL of purified water to the round-bottom flask, ultrasonicate in a water bath for 5 min, and then place it in a shaker and shake for 2 h to obtain a blank liposome dispersion. Analyze and measure it with a Malvern particle size analyzer, and the results are shown in Figure 1 . As Figure 1 shown, the aggregation index (PDI) of blank liposomes at different ratios is less than 0.3, indicating that the particle size distribution in the dispersion system is uniform. When the ratio is 5:1, the particle size is the smallest.
[0035] Prepare blank liposome gel, and the specific steps are as follows: Weigh 16.67 mg of cholesterol and 83.33 mg of soybean lecithin, dissolve them in 1 mL of chloroform respectively, and rotate and evaporate at 37 °C for 30 min; then add 0.5 mL of purified water to the round-bottom flask, ultrasonicate for 5 min, and then place it in a shaker and shake for 2 h to form a dispersion of blank liposomes; then weigh 20 mg of glutathione-modified low-acyl gellan gum, add it to 0.98 mL of purified water, and heat it to 80 °C to completely dissolve it to obtain a glutathione-modified low-acyl gellan gum solution with a mass concentration of 2%. Mix the diluted blank liposome dispersion and this gellan gum solution in a volume ratio of 1:1, and stir and co-incubate at room temperature for 12 h to obtain a liposome gel without tacrolimus.
[0036] Example 2
[0037] Weigh tacrolimus, cholesterol and soybean lecithin. Fix the masses of cholesterol and soybean lecithin at 16.67 mg and 83.33 mg respectively, and change the drug-lipid ratio (the mass ratio of the total mass of tacrolimus, cholesterol and soybean lecithin) to 1:5, 1:8, 1:10, 1:20, 1:30. Dissolve them in 1 mL of chloroform solution respectively. After mixing, rotate and evaporate at 37 °C for 30 min; then add 0.5 mL of purified water to the round-bottom flask, ultrasonicate for 5 min, and then place it in a shaker and shake for 2 h to form a dispersion of the tacrolimus liposomes with a mass concentration of 1%.
[0038] Precisely pipette 1 mL of the above-mentioned tacrolimus liposome dispersion into a pre-treated dialysis bag as the inner phase, and 20 mL of ethanol solution as the dialysis solution. Place it in a water bath shaker at 37 °C and shake at a speed of 200 rpm. After 8 h, dialysis reaches an equilibrium state. Take 1 mL of the dialysis solution, filter it with a 0.45 μm microporous filter membrane, and measure the content of the filtrate by HPLC method to calculate the amount of free drug (W f)。Meanwhile, precisely pipette 1 mL of the drug-loaded liposome solution into a 10 mL volumetric flask, demulsify with methanol and make up to the mark. After filtering the demulsified solution, determine the content by HPLC method and calculate the total amount of drug (W t ), and the encapsulation efficiency calculation formula is: (W t -W f ) / W t ×100%.
[0039] The results are shown in Figure 2 . As Figure 2 shown, with the increase of the drug-lipid ratio, the encapsulation efficiency of tacrolimus liposomes reaches the maximum at a drug-lipid ratio of 1:20.
[0040] Precisely pipette 1 mL of the above tacrolimus liposome solution, add 1 mL of distilled water for dilution, and measure the particle size with a Malvern laser particle size analyzer at a measurement temperature of 25 °C.
[0041] The results are shown in Figure 3 . As Figure 3 shown, compared with the blank liposomes (the blank liposomes with a ratio of soybean lecithin to cholesterol of 5:1 in Example 1, denoted as blank), when the drug-lipid ratio is 1:20, the tacrolimus liposome solution has a suitable particle size and is evenly distributed in the solution system.
[0042] Take the tacrolimus liposome solution with a drug-lipid ratio of 1:20, immerse it in liquid nitrogen for rapid freezing for 30 s, and then place it in a 37 °C water bath for shaking and thawing for 1 - 2 min, and repeat this cycle, with the number of cycles being 0, 1, 2, 3, 4, 5 respectively. Measure its encapsulation efficiency again.
[0043] The results are shown in Figure 4 . As Figure 4 shown, the encapsulation efficiency of the liposomes without freeze-thaw cycles is 80.83%. In contrast, the encapsulation efficiency of the liposomes with 4 freeze-thaw cycles is significantly higher, at 88.51%.
[0044] The present invention adopts the freeze-thaw cycle technology and precisely controls the number of cycles to 4 times, and this operation has a significant effect on improving the drug encapsulation efficiency. It can be clearly seen from the experiment of Example 2 that when there is no freeze-thaw cycle, the encapsulation efficiency of the liposomes is 80.83%; while when 4 freeze-thaw cycles are carried out, the encapsulation efficiency is increased to 88.51%, as Figure 3 shown. A higher encapsulation efficiency means that more drugs can be effectively encapsulated in the liposomes, reducing the loss of drugs during storage and transportation. This not only improves the utilization efficiency of drugs, but also ensures that enough drugs are released to play a therapeutic role during use, strongly enhancing the therapeutic effect on psoriasis. Although the freeze-thaw cycle is a conventional means to improve the encapsulation efficiency in this field, in the present invention, by precisely controlling the number of cycles, an effect better than the conventional situation is achieved.
[0045] Example 3
[0046] Weigh 20 mg of glutathione-modified low-acyl gellan gum, add 0.98 mL of purified water, heat to 80 °C, and completely dissolve it to obtain a glutathione-modified low-acyl gellan gum solution with a mass concentration of 2%.
[0047] Weigh another 5 mg of tacrolimus, 16.67 mg of cholesterol, and 83.334 mg of soy lecithin, dissolve them separately in 1 mL of chloroform solution, mix them, and then rotate and evaporate at 37 °C for 30 min; then add 0.5 mL of purified water to the round-bottom flask, sonicate for 5 min, and then place it in a shaker and shake for 2 h to form a dispersion of the tacrolimus liposome with a mass concentration of 1%. Take this tacrolimus liposome dispersion, immerse it in liquid nitrogen and quickly freeze it for 30 s, and then place it in a 37 °C water bath and shake and thaw for 1 - 2 min, and repeat this cycle four times; dilute it with purified water to 0.4% and 0.2%, and then mix it with the above-mentioned glutathione-modified low-acyl gellan gum solution with a mass concentration of 2% according to a volume ratio of 1:1, and stir and co-incubate at room temperature for 12 h to obtain tacrolimus liposome in-situ gels with tacrolimus mass concentrations of 0.5%, 0.2%, and 0.1% respectively.
[0048] Example 4
[0049] Weigh 20 mg of glutathione-modified low-acyl gellan gum, add 0.98 mL of purified water, heat to 80 °C to completely dissolve it, and obtain a glutathione-modified low-acyl gellan gum solution with a mass concentration of 2%.
[0050] Weigh 5 mg of tacrolimus, 16.67 mg of cholesterol, and 83.33 mg of soy lecithin, dissolve them separately in 1 mL of chloroform solution to obtain a tacrolimus solution, a cholesterol solution, and a soy lecithin solution. Add 1 mL of 5% glucose solution with a mass concentration and 1 mL of 5 mM calcium chloride to the tacrolimus solution, and then add the cholesterol solution and the soy lecithin solution, and rotate and evaporate at 37 °C for 30 min; then add 0.5 mL of purified water to the round-bottom flask, sonicate for 5 min, and then place it in a shaker and shake for 2 h to form a dispersion of the tacrolimus liposome with a mass concentration of 1%; then mix it with the above-mentioned glutathione-modified low-acyl gellan gum solution with a mass concentration of 2% according to a volume ratio of 1:1, and stir and co-incubate at room temperature for 12 h to obtain a liposome gel.
[0051] Measure the encapsulation efficiency of Examples 3 and 4, and the results are shown in Figure 5 , Example 4 is denoted as 0.5% TAC-Lipos-GG (Glu / CaCl2))
[0052] Thirty male C57BL / 6 mice (7 weeks old) were purchased. After adapting to the laboratory conditions, they were randomly divided into 6 groups, with 6 mice in each group.
[0053] Before the experiment started, an area of about 4 cm was shaved on the back of each mouse. 2 in area.
[0054] After the experiment started, except for the blank group, the remaining mice were smeared with commercially available You Shili 5% imiquimod ointment on the back every day to establish a psoriasis model. The dose was 120 mg / day. After 4 h, the mice were weighed and administered at a dose of 40 mg / cm 2 / mouse.
[0055] The control group was not given drugs. The remaining four groups, namely the experimental groups, were respectively given 0.1% tacrolimus liposome gel prepared in Example 3 (applied once a day), 0.2% tacrolimus liposome gel (subcutaneously injected once every two days), 0.5% tacrolimus liposome gel (subcutaneously injected once), and 0.5% tacrolimus liposome gel prepared in Example 4 (subcutaneously injected once), and were respectively labeled as the control group,
[0056] 0.1% TAC-Lipos-GG, 0.2% TAC-Lipos-GG, 0.5% TAC-Lipos-GG,
[0057] 0.5% TAC-Lipos-GG (Glu / CaCl2).
[0058] The experimental period was 5 days. On the last day of the experiment, the mice were euthanized, and their spleens and back skin sections were taken.
[0059] The experimental results are shown in Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 .
[0060] As Figure 7 shown, compared with the blank group, the spleen weight of the control group was significantly thickened, indicating that 5% imiquimod ointment was effective in induction. The spleen weights of the experimental group mice were reduced to varying degrees compared with the control group, indicating that they had certain therapeutic effects.
[0061] As Figure 8 、 Figure 9 shown, S1-S6 are the blank group, the control group, the 0.1% TAC-Lipos-GG group, the 0.2% TAC-Lipos-GG group, the 0.5% TAC-Lipos-GG group, and the 0.5% TAC-Lipos-GG (Glu / CaCl2) group respectively. Compared with the blank group, the epidermal thickness of the control group was significantly thickened, indicating that 5% imiquimod ointment was effective in induction.
[0062] Compared with the control group, the epidermal thickness of the three experimental groups, namely the 0.1% TAC-Lipos-GG group, 0.2% TAC-Lipos-GG group, and 0.5% TAC-Lipos-GG group, decreased significantly, by 89.12%, 81.83%, and 83.93% respectively, indicating that tacrolimus liposomal subcutaneous in-situ gel has an obvious improvement effect on the epidermal thickening caused by psoriasis. Moreover, there was no significant difference between the two groups administered by subcutaneous injection and the group applying 0.1% tacrolimus liposomal subcutaneous in-situ gel daily, demonstrating the advantage of its drug sustained release, and thus can improve the problem of frequent drug administration in the treatment of psoriasis.
[0063] The results of the liposomal gel without tacrolimus prepared in Example 1 in animal experiments are shown in Figure 10 、 Figure 11 . Among them, a is the blank group; b is the control group, that is, only induced with 5% imiquimod ointment without drug administration; c and d are the experimental groups. Among them, c is given the liposomal gel without tacrolimus in Example 1 (the mass ratio of soybean lecithin to cholesterol is 5:1), and d is given the 0.1% tacrolimus liposomal subcutaneous in-situ gel in Example 4. The administration method is daily application, and the experimental period is 5 days. It can be seen that there is no therapeutic effect on psoriasis in animal experiments, which clearly highlights the key role of tacrolimus in the treatment of psoriasis, and further proves the pertinence and necessity of selecting tacrolimus as the drug in the present invention.
[0064] Although the liposomal gel prepared in Example 4 has an encapsulation efficiency and a therapeutic effect in animal experiments comparable to those of the product of the present invention, due to the addition of glucose and calcium chloride, the complexity of the preparation process is increased, and more impurities may be introduced, which is not conducive to product quality control and industrial production.
[0065] The solution of the present invention simplifies the gel preparation system compared with the solution of Example 4, reduces the impurities introduced due to the addition of extra components, and reduces the product quality risk. Secondly, it avoids the interference of ionic strength that calcium chloride may cause to the stability of liposomes. During the preparation and storage of liposomes, changes in ionic strength may cause changes in the structure of the liposomal membrane, thereby affecting the stability of liposomes. From the results of Example 2 and Example 4 of the present invention, the absence of glucose and calcium chloride did not have an adverse effect on gel formation, drug encapsulation efficiency, and the final therapeutic effect. On the contrary, by simplifying the formula, the quality stability of the product is improved, and problems such as liposome aggregation and drug leakage are less likely to occur during storage, which better meets the requirements of industrial production for product quality consistency and stability.
[0066] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.
Claims
1. A tacrolimus liposome subcutaneous in-situ gel, characterized in that, It includes liposomes, the drug tacrolimus encapsulated inside the liposomes, and glutathione-modified low-acyl gellan gum wrapped outside the liposomes; the liposomes are prepared from cholesterol and soybean lecithin.
2. The preparation method of the tacrolimus liposome subcutaneous in-situ gel according to claim 1, wherein, It includes the following steps: (1) Dissolve tacrolimus, cholesterol, and soybean lecithin separately in chloroform. After mixing, rotate and evaporate to remove the solvent to obtain a lipid film; add purified water, perform ultrasonic treatment in a water bath, and then place it in a shaker for oscillation to form a dispersion of drug-loaded liposomes. (2) Subject the dispersion of the drug-loaded liposomes to freeze-thaw treatment to obtain a dispersion of tacrolimus liposomes. (3) Mix the dispersion of tacrolimus liposomes with a glutathione-modified low-acyl gellan gum solution, and rotate and stir at room temperature for 12 h to obtain a subcutaneous in-situ gel of tacrolimus liposomes with glutathione-modified low-acyl gellan gum wrapped outside.
3. The preparation method of the tacrolimus liposome subcutaneous in-situ gel according to claim 2, characterized in that: In step (1), the mass ratio of cholesterol to soybean lecithin is 1:3 - 7. The mass ratio of the total mass of tacrolimus, cholesterol, and soybean lecithin is 1:
20.
4. The preparation method of the tacrolimus liposome subcutaneous in-situ gel according to claim 2, characterized in that: In step (1), the rotation evaporation time is 30 min, the ultrasonic treatment time in the water bath is 5 min, and the oscillation time in the shaker is 2 h.
5. The preparation method of the tacrolimus liposome subcutaneous in-situ gel according to claim 2, wherein: In step (3), the mass concentration of the glutathione-modified gellan gum solution is 2%.
6. The preparation method of the tacrolimus liposome subcutaneous in-situ gel according to claim 2, wherein: In step (3), the volume ratio of the dispersion of tacrolimus liposomes to the glutathione-modified gellan gum solution is 1:
1.
7. The preparation method of the tacrolimus liposome subcutaneous in-situ gel according to claim 2, characterized in that: In step (3), the mass concentration of tacrolimus in the subcutaneous in-situ gel of tacrolimus liposomes is 0.1 - 0.5%.
8. The preparation method of tacrolimus liposome subcutaneous in-situ gel according to claim 2, characterized in that: The specific steps of the freeze-thaw treatment in step (2) are: quickly freeze in liquid nitrogen for 30 s, and then thaw by oscillation in a 37°C water bath, and repeat the cycle 1 - 5 times.