Calcium cross-linked drug-loaded liposome gel and preparation method thereof

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a calcium cross-linking drug-loaded liposome gel and a preparation method thereof. By adding calcium ions into the drug-loaded liposome, under the action of the calcium ions, glutathione-modified low acyl gellan gum is cross-linked outside the liposome to form a protective film, and then the calcium cross-linking drug-loaded liposome gel is formed. Compared with the drug-loaded liposome, the stability of the encapsulation rate of the calcium cross-linking liposome gel is significantly increased, and the calcium cross-linking liposome gel is non-toxic.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a calcium cross-linked drug-loaded liposome gel and a preparation method thereof. BACKGROUND

[0002] In the field of nanomedicine, liposomes are used as drug delivery carriers. Since liposomes have low toxicity and immunogenicity, the components (such as phospholipids) are highly biocompatible and biodegradable, can increase the drug concentration in the body and protect the drug from degradation, and the liposomes are easy to be modified with various ligands and functional molecules, and can be used for targeted delivery of drugs and gene drugs. However, according to some studies, there are still some shortcomings in using liposomes as drug delivery carriers, such as poor physical and chemical stability, difficult to withstand large mechanical stress, etc. SUMMARY

[0003] In order to overcome the problems in the prior art, the present application provides a calcium cross-linked drug-loaded liposome gel and a preparation method thereof. Under the action of calcium ions in the liposome, glutathione-modified low-acyl gellan gum is cross-linked into a gel outside the liposome, realizing the stability of the liposome membrane and effective control of drug release.

[0004] In order to achieve the purpose of the present application, the technical solution adopted is as follows: a calcium cross-linked drug-loaded liposome gel, comprising a drug encapsulated in the inside of a liposome and glutathione-modified low-acyl gellan gum wrapped outside the liposome.

[0005] Preferably, the drug encapsulated in the inside of the liposome is a drug capable of being administered orally, by injection, ophthalmically or nasally (such as doxorubicin hydrochloride, galanthamine hydrobromide, etc.).

[0006] Preferably, the molecular weight of the glutathione-modified low-acyl gellan gum is 12-14 kDa.

[0007] Preferably, the glutathione-modified low-acyl gellan gum is obtained by grafting modification of low-acyl gellan gum with glutathione after activation of carboxyl groups.

[0008] The preparation method of the calcium cross-linked drug-loaded liposome gel comprises the following steps:

[0009] (1) Dissolve a prescribed amount of drug in 5% glucose solution, then add a certain amount of calcium chloride, and control the concentration of calcium chloride to be between 1-10 mM, preferably 5 mM; then add a lipid film, and shake in a shaking bed for 30±10 min to make the lipid film redissolve; then perform water bath ultrasonic treatment for 10±5 min to make the solution transparent; then use an ultrasonic cell crusher to perform ultrasonic treatment on the obtained transparent solution for 6 s, pause for 6 s, and repeat for 6 min to form a dispersion of the drug-loaded liposome;

[0010] (2) The dispersion of drug-loaded liposomes prepared in step (1) is subjected to solid-liquid separation to remove the external calcium chloride solution, and then re-dissolved using a 5% glucose solution;

[0011] (3) The drug-loaded liposome solution in step (2) is incubated with glutathione-modified low-acyl gellan gum for 30 min ± 10 min, and under the action of calcium ions inside the liposomes, the glutathione-modified low-acyl gellan gum is cross-linked into a gel outside the liposomes and encapsulates the liposomes; the excess glutathione-modified low-acyl gellan gum solution in the liposome gel dispersion is removed by solid-liquid separation, and finally the above-mentioned precipitate after centrifugation is re-dispersed in a solution using a 5% glucose solution to obtain calcium cross-linked drug-loaded liposome gel wrapped with glutathione-modified low-acyl gellan gum solution outside.

[0012] Preferably, the lipid film in step (1) is prepared by the following method: phospholipid material and cholesterol are weighed separately, and a chloroform mixed solution of phospholipid and cholesterol is prepared, the molar ratio of phospholipid to cholesterol being 1:1-2, then a rotary evaporator is used to rotary evaporate at room temperature to form the lipid film; wherein the phospholipid material is any one of soybean lecithin or DOTAP phospholipid.

[0013] Preferably, the shaking time in step (1) is 30 min in a shaking table, and the ultrasonic time in a water bath is 10 min.

[0014] Preferably, the solid-liquid separation method in step (2) is centrifugal separation, the speed of the centrifuge used is 13000 r / min, the centrifugal time is 30 min, and the centrifugal number is at least 1.

[0015] Preferably, the preparation method of the glutathione-modified low-acyl gellan gum solution in step (3) is as follows: the glutathione-modified low-acyl gellan gum is added to a solvent, heated to 80 ± 10℃, and adjusted to a pH of 6 or higher to completely dissolve it. The glutathione-modified low-acyl gellan gum has pH responsiveness, and cannot be dissolved in the solvent when the pH is less than 6. After complete dissolution, the pH can be adjusted to the corresponding value according to the actual needs. The pH should not be adjusted to strong acidity or strong alkalinity, otherwise the gelation performance will be affected.

[0016] Preferably, the solvent used in the preparation method of the glutathione-modified low-acyl gellan gum is purified water or a 5% glucose solution.

[0017] Preferably, the solid-liquid separation method in step (3) is centrifugal separation, the speed of the centrifuge used is 13000 r / min, the centrifugal time is 30 min, and the centrifugal number is at least 2.

[0018] Compared with the prior art, the application has the following beneficial technical effects: under the action of calcium ions inside the liposome, glutathione modified low acyl k-carrageenan is cross-linked outside the liposome to form a protective film, and then a calcium cross-linked drug-loaded liposome gel is formed, which has better stability and more obvious sustained release effect of drug release compared with the unmodified drug-loaded liposome. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The influence of the concentration of calcium chloride solution on the particle size of the drug-loaded liposome gel;

[0020] Figure 2 The influence of the incubation time of glutathione modified low acyl k-carrageenan and drug-loaded liposome on the particle size of the drug-loaded liposome gel;

[0021] Figure 3 The potential characterization of the drug-loaded liposome and the drug-loaded liposome gel;

[0022] Figure 4 The particle size investigation of the low-temperature storage stability of the drug-loaded liposome and the drug-loaded liposome gel;

[0023] Figure 5 The encapsulation efficiency investigation of the low-temperature storage stability of the drug-loaded liposome and the drug-loaded liposome gel;

[0024] Figure 6 The in vitro drug release of the calcium cross-linked liposome gel, the DOX-loaded liposome, the DOX-loaded gel and free DOX;

[0025] Figure 7 The cytotoxicity study of the drug-loaded liposome and the drug-loaded liposome gel. DETAILED DESCRIPTION

[0026] The application is not limited to the following specific embodiments, and those skilled in the art can implement the application in other various specific embodiments according to the content disclosed in the application, or any simple changes or modifications made by using the design structure and ideas of the application also fall within the protection scope of the application. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0027] The application is further described in detail below in combination with the embodiments:

[0028] A calcium cross-linked drug-loaded liposome gel comprises a drug encapsulated in a liposome and glutathione modified low acyl k-carrageenan wrapped outside the liposome. The drug loaded in the liposome is generally a drug capable of being administered orally, ophthalmically or nasally (hydrochloric acid doxorubicin is taken as an example in the following embodiments).

[0029] The glutathione-modified low acyl gellan gum solution used in the following examples was prepared by the following method:

[0030] Step 1: Preparation of glutathione-modified low acyl gellan gum (GSH-GG)

[0031] The carboxyl groups of the low acyl gellan gum were activated at 20-30°C. Specifically, the carboxyl activator 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) was added to the aqueous solution of low acyl gellan gum to activate the carboxyl groups in the low acyl gellan gum. After the activation was completed, glutathione was added and the pH was adjusted to between 5.0 and 6.0, and the reaction was continued for about 3 hours. After the reaction was completed, the product was purified by dialysis in a N2 environment, with the dialysis temperature controlled at about 4°C, and the molecular weight cut-off was 12-14 kDa. Finally, the product was freeze-dried to obtain the glutathione-modified low acyl gellan gum.

[0032] In the above, the low acyl gellan gum is obtained by removing the O-acyl groups from natural gellan gum (i.e., high acyl gellan gum) by alkaline treatment, followed by filtration and purification. The low acyl gellan gum is commercially available and is commonly used in the food and medical fields.

[0033] Step 2: Preparation of glutathione-modified low acyl gellan gum aqueous solution (glutathione-modified low acyl gellan gum aqueous solutions with mass concentrations of 0.1%, 0.2%, 0.5%, and 1% were prepared)

[0034] According to the desired concentration, the required glutathione-modified low acyl gellan gum was added to purified water, heated to about 80°C, and the pH was adjusted to above 6 to completely dissolve the product. The glutathione-modified low acyl gellan gum has pH responsiveness, and cannot be dissolved in the solvent when the pH is less than 6. After complete dissolution, the pH can be adjusted to the corresponding value according to the actual requirements. The pH should not be adjusted to strong acidity or strong alkalinity, otherwise the gelation performance will be affected.

[0035] Example 1

[0036] The calcium cross-linked drug-loaded liposome gel and its preparation method comprise the following steps:

[0037] (1) Dissolve the prescribed amount of doxorubicin hydrochloride (DOX) in 5% glucose solution, then add a certain amount of calcium chloride, and control the concentration of calcium chloride at 5 mM; then add it to the lipid film, shake it in a shaking bed for 30±10 min to make it re-dissolve, then perform water bath ultrasonic treatment for 10±5 min to make the solution transparent, then use an ultrasonic cell crusher to perform ultrasonic treatment on the transparent solution for 6 s, with 6 s intervals, and repeat this for 6 min to form a dispersion of DOX-loaded liposomes.

[0038] (2) The dispersion of the DOX-loaded liposome is subjected to solid-liquid separation to remove the calcium chloride solution outside the liposome. The solid-liquid separation is centrifugation at a speed of 13000 r / min for 30 min, and the centrifugation is repeated at least once. Subsequently, the precipitated lipids are redissolved in 5% glucose solution.

[0039] (3) The DOX-loaded liposome solution is slowly and uniformly added to the glutathione-modified low-acyl gellan gum solution prepared in advance, and incubated for 30 min±10 min. The incubated liposome gel solution is subjected to solid-liquid separation to remove the excess glutathione-modified low-acyl gellan gum. The solid-liquid separation is centrifugation at a speed of 13000 r / min for 30 min, and the centrifugation is repeated at least twice. Finally, the precipitate is redispersed in 5% glucose solution to obtain the DOX-loaded liposome solution coated with glutathione-modified low-acyl gellan gum.

[0040] The lipid film in step (1) is prepared by the following method: a certain amount of soybean lecithin and cholesterol with a molar ratio of 1:1, or DOTAP phospholipid and cholesterol with a molar ratio of 1:2, are weighed, respectively, and then the phospholipid material and cholesterol are dissolved in chloroform to prepare phospholipid chloroform solution and cholesterol chloroform solution, respectively. Then the two solutions are uniformly mixed, and finally, a rotary evaporator is used to evaporate the solvent at room temperature to form a lipid film.

[0041] The effect of calcium chloride solution concentration on the particle size of the drug-loaded liposome gel is studied. The calcium chloride solutions with concentrations of 1 mM, 2 mM, 4 mM, 8 mM, 10 mM and 20 mM are prepared, and the other steps are the same as in Example 1. As shown in Figure 1 the particle size of the calcium cross-linked drug-loaded liposome gel is basically the same when the concentration of the calcium chloride solution is between 1 mM and 10 mM. However, when the concentration of the calcium chloride solution is 20 mM, the particle size of the drug-loaded liposome gel increases significantly.

[0042] The method for measuring the encapsulation efficiency is as follows: firstly, a standard curve of doxorubicin hydrochloride is prepared, then the prepared doxorubicin hydrochloride-loaded liposome gel is measured for fluorescence intensity at a wavelength of Ex 480 nm and Em 590 nm, at this time, the measured is the fluorescence intensity of free doxorubicin hydrochloride, finally, the liposome is broken by using 1% Triton, and the fluorescence intensity is measured at a wavelength of Ex 480 nm and Em 590 nm, at this time, the measured is the total fluorescence intensity of doxorubicin hydrochloride. The encapsulation efficiency = (total concentration of doxorubicin hydrochloride - concentration of free doxorubicin hydrochloride) / total concentration of doxorubicin hydrochloride x 100%. The results are shown in Table 1, compared with the liposome, the encapsulation efficiency of the calcium cross-linked drug-loaded liposome gel does not change significantly, which proves that the drug loading amount and glutathione-modified low acyl gelling agent have little effect on the encapsulation efficiency. At the same time, it can be seen from Table 1 that, compared with the blank liposome, the stability of the calcium cross-linked drug-loaded liposome gel is obviously improved. Figure 4 and Figure 5 It can be seen that, compared with the blank liposome, the stability of the calcium cross-linked drug-loaded liposome gel is obviously improved.

[0043] Table 1: Difference in encapsulation efficiency between the calcium cross-linked liposome gel and the liposome

[0044]

[0045] The zeta potential characterization test is performed using a Malvern laser particle size analyzer, and the results, as shown in Table 2, show that, compared with the liposome without wrapping glutathione gelling agent, the average zeta potential of the drug-loaded liposome gel wrapped with glutathione gelling agent prepared by using soy lecithin and cholesterol as raw materials for the lipid film becomes lower, and the average zeta potential of the drug-loaded liposome gel wrapped with glutathione gelling agent prepared by using DOTAP phospholipid and cholesterol as raw materials for the lipid film changes from positive to negative, which all indicates that the modification is successful. Figure 3

[0046] At the same time, the in vitro drug release of the calcium cross-linked liposome gel, the DOX-loaded liposome, the DOX-loaded gel and the free DOX within 50 h is tested respectively. The specific method is as follows: 2 ml of the calcium cross-linked DOX-loaded liposome gel is placed in a pre-treated dialysis bag (Mw = 14000 Da), the two ends are tightly tied, and is put into 30 ml of release medium which has been constant-temperature treated to 37℃, constant-temperature treated to 37℃ water bath is shaken at a constant speed of 100 r / min, 5 mL of release medium is collected at the specified time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 h) respectively, and at the same time, the same amount of release medium is supplemented, the fluorescence spectrophotometer method is used to determine the concentration of DOX in the sample. The free DOX solution, the DOX-loaded gel and the DOX-loaded liposome solution are also subjected to the determination of in vitro drug release according to the above experimental steps. The results are shown in Table 3. Figure 6 ​As shown, the glutathione gellan gel encapsulating the calcium-crosslinked drug-loaded liposome gel did not significantly affect the drug release capacity of the liposomes, and more than 70% of the drug could be released within 50 hours.

[0047] The preparation process of the free DOX solution is as follows: weigh an appropriate amount of DOX, add it to distilled water, and dissolve it by ultrasonication in a water bath to obtain the free DOX solution.

[0048] The preparation process of DOX-loaded gel is as follows: weigh an appropriate amount of DOX and add it to a 0.5% glutathione gel, and dissolve it by ultrasonication in a water bath to obtain DOX-loaded gel.

[0049] DOX-loaded liposomes were prepared using the traditional thin-film hydration method.

[0050] Depend on Figure 7 It can be seen that, compared with drug-loaded liposomes, the cell survival rate of calcium-crosslinked drug-loaded liposome gels with external glutathione gellan gum remains above 90%.

[0051] Preparation of glutathione-modified low-acyl gellan gel encapsulating unloaded liposomes:

[0052] (1) Dissolve a certain amount of calcium chloride in a 5% glucose solution and control the concentration of calcium chloride at 5mM; then add it to the lipid film and shake it in a shaker for 30±10min to re-dissolve it. Then, sonicate it in a water bath for 10±5min to make the solution transparent. Then, use an ultrasonic cell disruptor to sonicate the obtained transparent solution for 6s, with a 6s interval, and repeat this for 6min to form a dispersion of doxorubicin hydrochloride liposomes.

[0053] (2) The calcium chloride solution outside the liposomes was removed by solid-liquid separation. The solid-liquid separation method used was centrifugation, and the centrifuge speed was 13000 r / min, the centrifugation time was 30 min, and the centrifugation was performed at least once. Then, the lipid precipitate was re-dissolved using 5% glucose solution.

[0054] (3) The reconstituted liposomes were slowly and uniformly added to a pre-prepared glutathione-modified low-acyl gellan gel solution of a predetermined mass concentration, and incubated for 30 min ± 10 min. Subsequently, the incubated liposome gel solution was subjected to solid-liquid separation to remove excess glutathione-modified low-acyl gellan gel. The solid-liquid separation method used was centrifugation, with a centrifuge speed of 13000 r / min, a centrifugation time of 30 min, and at least two centrifugations. Finally, the precipitate was redispersed in the solution using a 5% glucose solution, resulting in a glutathione-modified low-acyl gellan gel solution containing drug-loaded liposomes.

[0055] In addition, the particle size of liposomes encapsulated in low-acyl gellan gel with different mass concentrations of glutathione and liposomes without glutathione-modified low-acyl gellan gel were studied. It was found that the particle size increased with the increase of drug loading, and the particle size of calcium-crosslinked drug-loaded liposome gel was larger than that of liposomes without external calcium crosslinking in low-acyl gellan gel.

[0056] Table 2: Particle size comparison of calcium-crosslinked glutathione-modified low-acyl gellan gum with different drug loading and mass concentrations.

[0057]

[0058] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a calcium-crosslinked drug-loaded liposome gel, characterized in that, Includes the following steps: (1) Dissolve the prescribed amount of drug in a 5% glucose solution, then add calcium chloride and control the concentration of calcium chloride between 1-10 mM; then add the lipid film and shake in a shaker for 30±10 min to re-dissolve the lipid film; then sonicate in a water bath for 10±5 min to make the solution clear, and obtain a clear solution; then use an ultrasonic cell disruptor to sonicate the obtained clear solution for 6 s, with a 6 s interval, and repeat this for 6 min to form a dispersion of the drug-loaded liposomes; (2) Remove the external calcium chloride solution from the drug-loaded liposome dispersion prepared in step (1) by solid-liquid separation, and then reconstitute it with 5% glucose solution; (3) The glutathione-modified low acyl gellan gel solution was incubated with the drug-loaded liposome solution in step (2) for 30±10 min. Then, the excess glutathione-modified low acyl gellan gel solution was removed by centrifugation. Finally, the precipitate after centrifugation was redispersed in the solution using 5% glucose solution to obtain a calcium cross-linked drug-loaded liposome gel with the glutathione-modified low acyl gellan gel solution on the outside. The lipid film described in step (1) is prepared by the following method: phospholipid material and cholesterol are weighed separately, and a chloroform mixed solution of phospholipid and cholesterol is prepared with a molar ratio of phospholipid to cholesterol of 1:1-2. Then, the solution is evaporated at room temperature using a rotary evaporator to form the lipid film. The phospholipid material is either soybean lecithin or DOTAP phospholipid. The preparation method of the glutathione-modified low-acyl gellan gum solution in step (3) is as follows: after adding the glutathione-modified low-acyl gellan gum to the solvent, heat it to 80±10℃ and adjust the pH to above 6 to make it completely dissolved; wherein, the solvent used in the preparation method of the glutathione-modified low-acyl gellan gum is either purified water or 5% glucose solution; the glutathione-modified low-acyl gellan gum is obtained by glutathione grafting modification and carboxyl activation of low-acyl gellan gum.

2. The method for preparing calcium-crosslinked drug-loaded liposome gel according to claim 1, characterized in that, Step (1) The shaking time in the shaker is 30 minutes, and the ultrasonic time in the water bath is 10 minutes.

3. The method for preparing calcium-crosslinked drug-loaded liposome gel according to claim 1, characterized in that, The solid-liquid separation method in step (2) is centrifugation, with the centrifuge speed being 13000 r / min, the centrifugation time being 30 min, and the centrifugation being performed at least once.

4. The method for preparing calcium-crosslinked drug-loaded liposome gel according to claim 1, characterized in that, The centrifuge used in step (3) has a rotation speed of 13000 r / min, a centrifugation time of 30 min, and a centrifugation number of at least 2 times.

5. The method for preparing calcium-crosslinked drug-loaded liposome gel according to claim 1, characterized in that, The glutathione-modified low-acyl gellan gum has a molecular weight of 12-14 kDa.

6. The calcium-crosslinked drug-loaded liposome gel prepared by the method according to any one of claims 1-5, characterized in that, This includes drugs encapsulated inside liposomes and glutathione-modified low-acyl gellan gum wrapped around the liposomes.

7. The calcium-crosslinked drug-loaded liposome gel according to claim 6, characterized in that, The drug encapsulated within the liposome is a drug that can be administered orally, by injection, through the eye, or through the nose.

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