CN115400084B

CN115400084B - NO-releasable liposome and preparation method and application thereof

Info

Publication number: CN115400084B
Application number: CN202211040209.1A
Authority: CN
Inventors: 马栋; 李国巍; 邢辉; 程兆怡; 胡云峰
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2022-08-29
Filing date: 2022-08-29
Publication date: 2023-07-25
Anticipated expiration: 2042-08-29
Also published as: CN115400084A

Abstract

The invention discloses a liposome capable of releasing NO, a preparation method and application thereof, belonging to the technical field of biomedical engineering materials; the NO-releasable liposomes comprise a phospholipid and a novel ionic NO donor material; the liposome can realize the dilating of capillary blood vessels at the administration part, quickening the blood flow rate and changing the blood microcirculation system of local tissues due to transdermal and nitric oxide release, thereby realizing the effect of high-efficiency permeation of the medicine to the skin; the invention selects the cholesterol modified by cations as a novel NO donor, so that the cholesterol NO donor is assembled with lecithin to participate in the liposome forming process. The NO donor molecule is used as a part of a liposome structure, is used as a liposome skeleton molecule, and takes part in liposome assembly to obtain the liposome releasing nitric oxide, which is used as a transdermal drug delivery carrier, so that the purposes of effective load of various drugs and efficient permeation of skin can be realized.

Description

NO-releasable liposome and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical engineering materials, and particularly relates to a liposome capable of releasing NO, and a preparation method and application thereof.

Background

Transdermal drug delivery systems (Transdermal drug delivery system, TDDS) are novel formulations for delivery to the skin surface where the drug is delivered through the layers of the skin at a rate to achieve effective blood levels by capillary blood entry into the systemic blood circulation of the body, for systemic or local therapeutic action. Compared with oral administration, intravenous injection and other modes, the transdermal administration has the advantages of avoiding the first pass effect of stomach and intestine, reducing the fluctuation of blood concentration, avoiding the irritation of medicine to stomach and intestine, having no wound in administration, having strong compliance and the like. However, skin is a difficult-to-penetrate barrier for most drugs, and many drugs are difficult to penetrate into skin or the penetration amount is difficult to reach therapeutic levels, so that promotion of transdermal penetration of drugs by appropriate methods is critical for research into TDDS. Among techniques for promoting transdermal penetration of drugs, nanotechnology is an effective means, and some drugs difficult to penetrate transdermally can enter the human body with the assistance of nanocarriers and exert drug effects. Thus, nanotechnology has become an effective passive transdermal delivery modality developed in recent years.

The liposome is a spheroid formed by dispersing phospholipid and other amphiphilic substances in water and encapsulating one or more layers of concentric lipid bilayer membranes, can encapsulate hydrophilic or lipophilic medicaments, and has wide application in the aspects of vein, skin, lung, oral administration and the like. The use of liposomes as carrier material for transdermal administration has the following advantages: 1. the lipid bilayer can promote the drug to enter into the stratum corneum or epidermis lipid, and increase the retention and residence time of the drug in the skin; 2. the skin targeting effect is achieved, and adverse reactions caused by systemic absorption of the medicine can be avoided; 3. after being wrapped, the stability and the durability of the medicine are improved; 4. nontoxic, non-irritating, safe to apply, etc. (Journal of Drug Delivery Science and technology.2014,24, 245-250). However, aiming at the tight brick wall structure of the skin, how to further improve the drug permeation capacity of the liposome to the skin and achieve the maximization of the drug treatment effect has become a key problem of the transdermal drug delivery preparation of the liposome.

NO has important regulatory roles in various physiological and pathological processes, such as promoting vasodilation, accelerating blood flow rate. Rao (Frontiers in Bioengineering and biotechnology.2020,8,00578) and Li (biomaterials.2020, 241, 119904) et al have found that the catalytic production of nanomolar concentrations of NO by nitric oxide synthase effects diastolic regulation of blood vessels, increases coronary blood flow velocity and effectively inhibits platelet aggregation and adhesion. However, NO, as a high-activity gas molecule, has problems of poor stability, low load, fast release rate, etc., and cannot effectively enter the interior of skin due to a tight brick wall structure of skin, thereby further playing a role. Mark (ACS biomatter. Sci. Eng.2017,3, 2136-2143) et al encapsulate a variety of cationic NO donors in liposomes, resulting in a series of stable NO liposomes. Experimental results show that the release period of NO small molecules encapsulated in liposome can reach about 48 hours, and the NO small molecules can also exist in buffer solution or serum stably for a long time. Takuma (International Journal of pharmaceuticals.2019, 565, 481-487) et al encapsulate diethylenetriamine as a small molecule donor of NO within liposomes to provide a releasable NO liposome. Although research results show that the NO liposome can realize the sustained release of NO, and can realize the effective aggregation at the focus part through blood circulation, thereby effectively treating diseases. However, the NO liposome cannot realize stable loading of NO small molecules, and certain NO small molecules leak in the body fluid circulation process, so that the effective concentration of NO on treatment of focus positions is realized through repeated administration. Therefore, how to realize the high-efficiency load of the liposome on NO small molecules, release for a long time and prevent and treat drug leakage becomes the key of further realizing clinical application of the NO liposome.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a liposome capable of releasing NO, and a preparation method and application thereof.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the preparation method of the novel ionic NO donor material comprises the following steps:

(1) Dissolving a cationic polymer in an organic solvent A, adding cholesterol chloroformate and a catalyst triethylamine, reacting at 0-4 ℃, heating to 5-35 ℃, continuing to react, concentrating after the reaction is finished, adding hydrochloric acid into the concentrate for redissolution, washing, settling and drying to obtain cholesterol modified by the cationic polymer, wherein the molar ratio of the cationic polymer to the cholesterol chloroformate is 1:1-4;

(2) Dissolving the cholesterol modified by the cationic polymer obtained in the step (1) in an organic solvent B, then adding sodium methoxide to provide an alkaline environment to continue dissolving, introducing inert gas after dissolving, and then introducing NO gas to react to obtain the novel ionic NO donor material.

Sodium methoxide provides an alkaline environment on the one hand and sodium ions in sodium methoxide can achieve charge balance with electronegative NO groups on the other hand.

Preferably, in the step (1), the cationic polymer comprises triethylene diamine, pentaethylene hexamine, hyperbranched polyethyleneimine with molecular weight of 600-25000 and chitosan with molecular weight of 1000-50000; the organic solvent A comprises methylene dichloride; the mass volume ratio of the cholesterol chloroformate to the organic solvent A is (1-4) g:10 mL; the mol ratio of the triethylamine to the cholesterol chloroformate is 1:0.1-0.5; the concentration of the hydrochloric acid is 0.1-0.4M; the mass volume ratio of the concentrate to the hydrochloric acid is (2-4) g to (10-20) mL; the reaction time at the temperature of 0-4 ℃ is 10-30 min, and the continuous reaction time is 10-14 h; in the step (2), the mass ratio of the cholesterol modified by the cationic polymer to the sodium methoxide is 1 (0.1-0.5); the organic solvent B comprises a mixed solution consisting of methanol and tetrahydrofuran according to the volume ratio of 1:0.1-0.5; the mass volume ratio of the cholesterol modified by the cationic polymer to the methanol is (0.5-1) g to 10mL; the temperature of the reaction of the introduced NO gas is 5-35 ℃ and the time is 3-7 days.

The second technical scheme of the invention is as follows: a novel ionic NO donor material prepared according to the preparation method described above.

The novel ionic NO donor material prepared by the invention improves the chemical structure of the existing ionic NO donor material, so that the novel ionic NO donor material can participate in liposome formation/liposome nanostructure construction.

The third technical scheme of the invention: a liposome capable of releasing NO comprising a phospholipid and the novel ionic NO donor material described above.

The technical scheme of the invention is as follows: the preparation method of the NO-releasable liposome comprises the following steps: and dissolving phospholipid and the novel NO donor material in an organic solvent C, and evaporating to remove the organic solvent to obtain a film, namely the NO-releasable liposome.

Preferably, the phospholipid comprises soybean lecithin, and the mass ratio of the phospholipid to the novel ionic NO donor material is 1: (0.05-1); the organic solvent C comprises a mixed solution of methanol and chloroform with the volume ratio of 1:1-4, a mixed solution of methanol and tetrahydrofuran with the volume ratio of 1:1-2, a mixed solution of methanol and toluene with the volume ratio of 1:2-6, a mixed solution of methanol and ethanol with the volume ratio of 1:1-3 or a mixed solution of methanol and ethyl acetate with the volume ratio of 1:2-4; the dosage of the methanol in the organic solvent C is calculated by adding 0.1-0.5 g of soybean lecithin into each 10mL, namely the mass volume ratio of the soybean lecithin to the methanol in the organic solvent C is (0.1-0.5) g to 10mL.

The fifth technical scheme of the invention is as follows: the application of the liposome capable of releasing NO in the preparation of transdermal administration preparations.

The sixth technical scheme of the invention: a transdermal formulation comprising a NO-releasing liposome as described above and an efficacy molecule.

Preferably, the efficacy molecule comprises one or more of sodium hyaluronate, acyclovir, nicotinamide, ceramide, and an amino acid.

The seventh technical scheme of the invention: the preparation method of the transdermal administration preparation comprises the following steps: dissolving the liposome capable of releasing NO in a composite solvent, adding the functional molecules, evaporating to remove the solvent to obtain a film, then introducing inert gas, vacuum drying overnight at room temperature, adding phosphate buffer solution or water into the film for ultrasonic hydration for 10-20 min at 0-4 ℃ to obtain a liposome suspension, namely the transdermal administration preparation.

Preferably, the composite solvent comprises a mixed solution consisting of methanol and chloroform according to the volume ratio of 1:1-4; the mass volume ratio of the liposome capable of releasing NO to the composite solvent is (0.05-0.2) g:10 mL; the mass ratio of the efficacy molecule to the liposome capable of releasing NO is 1 (25-100); the ultrasonic condition is 50-70 KHz and 100W; the mass volume ratio of the film to the phosphate buffer solution or water is 0.03g to (1-2) mL.

Eighth technical scheme of the invention: the preparation method of the transdermal administration preparation comprises the following steps: dissolving the functional molecules, the phospholipid and the novel ionic NO donor material in a composite solvent, carrying out ultrasonic treatment for 5-10 times, each time for 5-10 min, then evaporating the solvent at room temperature under reduced pressure to obtain a film, then introducing inert gas, carrying out vacuum drying overnight at room temperature, adding phosphate buffer solution or water into the film at 0-4 ℃ for ultrasonic hydration for 10-20 min, and obtaining liposome suspension, namely the transdermal administration preparation.

Preferably, the composite solvent comprises a mixed solution consisting of methanol and chloroform according to the volume ratio of 1:1-4; the mass volume ratio of the methanol to the phospholipid is (0.1-0.5) g to 10mL, and the phospholipid comprises soybean lecithin; the mass ratio of the phospholipid to the novel ionic NO donor material is 1:0.2-0.5, and the mass ratio of the functional molecule to the novel ionic NO donor material is 1 (10-20); the ultrasonic condition is 50-70 KHz and 100W; the mass volume ratio of the film to the phosphate buffer solution or water is 0.03g to (1-2) mL.

Compared with the prior art, the invention has the following beneficial effects:

compared with the traditional liposome, the novel transdermal drug delivery carrier is obtained by combining the ionic NO donor and the liposome, and the liposome can relax capillaries at a drug delivery part, quicken blood flow rate and change the blood microcirculation system of local tissues due to the releasable nitric oxide, so that the effect of high-efficiency permeation of the drug to the skin is realized, and the liposome is hopeful to become a break-through for improving the permeation capacity of the liposome transdermal drug delivery preparation.

The invention selects cholesterol modified by cations as NO donor, so that the cholesterol NO donor is assembled with lecithin to participate in the liposome forming process. The NO donor molecule is used as a part of a liposome structure, is used as a liposome skeleton molecule, and takes part in liposome assembly to obtain the liposome capable of releasing nitric oxide, which is used as a transdermal delivery carrier, and rhodamine is utilized to simulate a functional small molecule to promote permeation. Can realize the effective load of functional small molecules and the efficient permeation of skin.

According to the invention, on the premise of utilizing the effect of NO on vasodilation regulation, NO small molecules are embedded into a liposome structure to become a component part in the liposome, so that a stable liposome structure with high loading and long-term release of NO is obtained, and the blood circulation is further accelerated through NO permeation, so that the skin permeation capacity of the liposome wrapping the drug is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing the construction principle of NO-loaded liposomes of example 4;

FIG. 2 is a nuclear magnetic resonance spectrum of polyethyleneimine modified cholesterol Cho-PEI prepared in step a of example 1;

FIG. 3 is an infrared spectrum of the NO donor material Cho-PEI/NONOate prepared in example 1;

in fig. 4, A, B, C is a physical image, a particle size image and an SEM image of the liposome suspension prepared in example 4, respectively;

FIG. 5 is a graph showing the dynamic process of NO release in a liposome suspension prepared in example 3 under simulated physiological conditions;

FIG. 6 is a graph showing the permeation effect of the liposome suspension prepared in example 4 on the skin of mice, wherein A is a live imaging graph of the liposome suspension PEI/NONOate applied for 1h, and B is a live imaging graph of the anatomical thigh tissue of the liposome suspension PEI/NONOate applied for 1 h;

FIG. 7 shows the amounts of active gas molecules NO in the Lipo@PEI/NONONOate liposomes obtained by measurement in example 10 over different time periods;

in fig. 8, (a) is a schematic diagram of the construction principle of the liposome material rhob@pei/nonoate@lipo in example 11; (B) Liposome suspensions RhoB@Lipo@PEI/NONOate and RhoB@PEI/NONOate@Lipo in example 11 were used for 1h as a live imaging map and a live imaging map of anatomical thigh tissue.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

In the following examples, the preparation method of the anhydrous dichloromethane comprises the following operation steps: adding calcium hydride into dichloromethane, stirring for 6-24 hours, and then distilling at normal pressure to obtain anhydrous dichloromethane, wherein the adding amount of the calcium hydride is 1-2 g per 500mL of dichloromethane; the anhydrous methanol, tetrahydrofuran, chloroform and absolute ethanol are also obtained after the treatment;

in the following examples, the purity of the acetone used was ACS, which was 99.5% or more; the purity of the diethyl ether adopted is AR 98%; the number average molecular weight of the polyethyleneimine used is 600; the purity of the rhodamine B adopted is ACS which is more than or equal to 99.5 percent; cholesterol purity was AR,98%;

in the following examples, room temperature is 10 to 35 ℃.

The description will not be repeated below.

Example 1

NO donor material: preparation of polyethyleneimine modified cholesterol (Cho-PEI/NONOate):

a. under the condition of ice-water bath, dissolving Polyethyleneimine (PEI) with the number average molecular weight of 600 in anhydrous dichloromethane, taking triethylamine as a catalyst, adding cholesterol chloroformate with the molar ratio of 1:1 to PEI, wherein the mass volume ratio of the cholesterol chloroformate to the anhydrous dichloromethane is 1g to 10mL, the molar ratio of the triethylamine to the cholesterol chloroformate is 1:0.1, reacting for 20min at 0-4 ℃, then raising the temperature to room temperature (25 ℃), continuing to react for 12h, concentrating under reduced pressure, re-dissolving the obtained concentrate by using 10mL of hydrochloric acid solution with the concentration of 0.1M, washing with dichloromethane, settling with acetone, and drying in vacuum at the room temperature of 25 ℃ to obtain white solid, namely polyethyleneimine modified cholesterol Cho-PEI.

b. C, dissolving the Cho-PEI obtained in the step a in a mixed solution consisting of anhydrous methanol and tetrahydrofuran according to the volume ratio of 1:0.1 at room temperature of 25 ℃, wherein the mass volume ratio of the Cho-PEI to the anhydrous methanol is 0.5 g:10 mL, dissolving for 5min, adding dry sodium methoxide for continuous dissolution, wherein the mass ratio of the Cho-PEI to the sodium methoxide is 1:0.1, stabilizing for 30min, and then placing in a high-pressure reaction kettle for sealing and detecting the air tightness; high-purity nitrogen (20 psi) is introduced into the reaction kettle for 10min, air in the reaction kettle is removed, NO gas (80 psi) is then introduced into the reaction kettle, and the reaction is carried out for 3 days at 25 ℃. After the reaction was completed, NO was discharged with 20psi of high purity nitrogen, and the reaction vessel was opened after continuing to vent for 30 minutes to take out the reaction product. Washing for 2 times by diethyl ether sedimentation, and drying at room temperature in vacuum to obtain the novel ionic NO donor material Cho-PEI/NONOate.

Example 2

NO donor material: preparation of polyethylenimine as NO donor (PEI/NONOate):

dissolving dried polyethylenimine (PEI, with the number average molecular weight of 600) in a mixed solution consisting of absolute methanol and tetrahydrofuran according to the volume ratio of 1:0.1 at room temperature of 25 ℃, adding dried sodium methoxide for continuous dissolution after dissolving for 5min, wherein the mass ratio of PEI to sodium methoxide is 1:0.1, dissolving for 30min to obtain a mixed solution, adding the mixed solution into a high-pressure reaction kettle, sealing and detecting the air tightness. High-purity nitrogen (20 psi) is introduced into the reaction kettle for 10min, air in the reaction kettle is removed, NO gas (80 psi) is then introduced into the reaction kettle, and the reaction is carried out for 7 days at the room temperature of 25 ℃. After the reaction is finished, high-purity nitrogen with the pressure of 30 minutes and 50psi is introduced into the reaction kettle to discharge unreacted NO, and then the reaction kettle is opened to take out a reaction product. The reaction product was washed 2 times with anhydrous diethyl ether and dried in vacuo at 25℃for 24h to give the Polyethyleneimine (PEI) NO donor material PEI/NONOate which was stored in a desiccator at-4℃low temperature.

Example 3

Preparation of liposomes which release NO without transdermal delivery of the efficacy molecule:

the soybean lecithin and the NO donor material Cho-PEI/NONOate prepared in the example 1 are dissolved in a proper amount of mixed solvent consisting of anhydrous methanol and chloroform according to the volume ratio of 1:1 (wherein the mass ratio of the soybean lecithin to the Cho-PEI/NOate is 1:0.2; the mass-volume ratio of the soybean lecithin to the anhydrous methanol is 0.1 g:10 mL), and the ultrasonic treatment is carried out for 5 times in a short time under the conditions of 50KHz and 100W, and the ultrasonic treatment time is 5 minutes each time; then placing the liposome into a pear bottle, evaporating at room temperature under reduced pressure, removing the solvent, forming a uniform transparent film on the bottle wall, namely the liposome capable of releasing NO, introducing nitrogen, drying overnight at room temperature under vacuum, adding 5mL of Phosphate Buffer (PBS) (pH=7.4) into the dried film at 3 ℃, carrying out ultrasonic hydration for 10min to obtain milky white liposome suspension, namely the liposome capable of releasing NO, namely lipo@PEI/NONOate, and sealing at 4 ℃.

Example 4

Preparation of transdermal drug delivery preparation carrying efficacy molecule:

taking rhodamine B as a simulation efficacy molecule, weighing a certain mass, then mixing with soybean lecithin and the novel ionic NO donor material Cho-PEI/NONOate prepared in the embodiment 1, dissolving in a proper amount of mixed solvent consisting of anhydrous methanol and chloroform according to the volume ratio of 1:1 according to a certain proportion (wherein the mass ratio of the soybean lecithin to the chord-PEI/NOate to the rhodamine B is 1:0.2:0.01; the mass volume ratio of the soybean lecithin to the anhydrous methanol is 0.1 g:10 mL), carrying out ultrasonic treatment for 5 times in a short time, wherein the ultrasonic conditions are 50KHz and 100W, and the ultrasonic treatment time is 5min each time; then placing the liposome in a eggplant pear bottle, evaporating at room temperature under reduced pressure, removing a solvent, forming a uniform and transparent film on the bottle wall, namely, the drug-loaded liposome capable of releasing NO, introducing nitrogen, drying overnight at room temperature under vacuum, adding 5mL of Phosphate Buffer (PBS) (pH=7.4) into the dried film at the temperature of 3 ℃, and carrying out ultrasonic hydration for 10min to obtain milky red liposome suspension, namely, the liposome of NO loaded with functional molecules, which is a novel NO transdermal drug delivery preparation, is recorded as RhoB@lipo PEI/NOate, and is stored at the temperature of 4 ℃.

In this example, the construction principle of the NO-loaded liposome is schematically shown in FIG. 1.

Example 5

1mg of the polyethyleneimine-modified cholesterol Cho-PEI obtained in step a of example 1 was dissolved in 1mL of deuterated chloroform (CDCl) ₃ ) The solution was clear, transparent, free of suspended matter or impurities, and was detected on a nuclear magnetic resonance apparatus (NMR-Bruker-300) using a clean, special nuclear magnetic resonance sample tube. Reference is made to deuterated solvent residual peaks: (deuterated chloroform 7.26 ppm). As a result of the experiment, as shown in FIG. 2, PEI formed an amide bond after reaction with cholesterol ester, and a peak was present around the chemical shift of 6.78ppm, as shown in FIG. 2 a, and the characteristic of cholesterol was that hydrogen atoms were shown at 5.2ppm and 4.2ppm, as shown in FIGS. 2 b and c, and the hydrogen atoms in PEI were mostly shown at d-g between 2 and 3 ppm. Indicating successful synthesis of Cho-PEI.

Example 6

The NO donor material Cho-PEI/NONOate obtained in example 1 was subjected to infrared spectrum characterization by potassium bromide tabletting method. The results are shown in FIG. 3, in redIn the external spectrum, the characteristic absorption peak of the ionic NO donor material Cho-PEI/-NONOate is 1250cm ^-1 Where it is indicated that the material has been successfully loaded with NO.

Example 7

1mg of the milky red liposome suspension of RhoB@Lipo@PEI/NONOate obtained in example 4 was dispersed in 1mL of pure water, and after ultrasonic dispersion was performed uniformly, the nanoparticle potential and the particle size were measured by using a Markov laser particle sizer, as shown in FIG. 4, A, B, C, (wherein A is a physical image of the liposome suspension, B is a particle size image, and C is an SEM image at 1000 x), and as can be seen from FIG. 4: the liposome has average particle diameter of about 600nm and uniform size, and can be stably suspended in solution.

Example 8

5mg of the lipo@PEI/NONOate milky white liposome suspension obtained in example 3 was weighed, dispersed in 25mL of citrate buffer (pH 7.4), centrifuged at 5000rpm for 5min at 37℃for different periods of time, 50. Mu.L of supernatant was mixed with 50. Mu.L of PBS buffer (pH=7.4), 100. Mu.L of Gris reagent (PEI/NOate and S-nitrosoglutathione were used to test NO content using Nanjing to build nitric oxide kit A013-2-1, L-arginine was used to build nitric oxide kit A012-1-1) and stored in a dark place for 15min, absorbance was measured using a microplate reader at 540nm wavelength, and a drug release profile was drawn. As shown in FIG. 5, the lipo@PEI/NONOate liposome suspension has a rapid NO release effect, the accumulated NO release amount can reach 60%, and the NO in the lipo@PEI/NONOate liposome can be released continuously over time for more than 50 hours. Thus, lipo@PEI/NONOate liposomes can achieve a long release of NO.

Example 9

5 μl of the liposome suspension rhob@lipo@pei/NONOate of example 4 with rhodamine B as a model efficacy molecule was weighed, smeared on the left leg (a position) of the hair-removed mouse, and the right leg was liposome prepared without adding Cho-PEI/NONOate with soybean lecithin and rhodamine B alone (B position), to simulate drug skin permeation, and after 1 hour of smearing, live imaging was performed, as shown in fig. 6 a. The thigh tissue was dissected after 1 hour and imaged in vivo to detect fluorescence intensity as shown in fig. 6B. As can be seen from fig. 6, for rhob@lipo@pei/NONOate liposomes, the fluorescence intensity was weaker in the left leg (position a) and stronger in the isolated thigh tissue (position a 1), indicating that rhodamine in the left leg penetrated the skin well into the thigh internal tissues. However, the fluorescence intensity of the right leg (position b) was strong, and the fluorescence intensity of the isolated thigh tissue (position b 1) was weak, indicating that rhodamine of the right leg remained on the skin surface all the time, and the liposome permeation effect was weak. Therefore, this experiment fully proves that NO has an excellent promoting effect on the permeation effect of liposomes.

Example 10

In order to verify that the active gas molecule NO in the lipo@PEI/NONOate liposome passes through skin tissues along with the liposome, enters muscle tissues and participates in the whole process of liposome transdermal, thereby effectively improving the permeation effect of the liposome. 10. Mu.L of the milky white liposome suspension lipo@PEI/NONOate prepared in example 3 was weighed, smeared on the left legs of three mice from which hair was removed, and after different periods of time (0.5, 1 and 2 hours), the rat thigh tissues were dissected, tissue homogenated extraction was performed by taking rat epidermis lower adipose tissues and muscle tissues of the same sites, and NO concentrations in the tissue homogenates were determined by referring to the corresponding procedure using a Kelvin nitric oxide assay kit (nitric acid reductase method). As shown in FIG. 7, after 0.5h, the presence of NO was detected in the underlying tissue of the skin, at which time the NO content of the thigh tissue was relatively low and only trace amounts of NO were present. Over time, after 1h, NO content in the skin underlying tissue and thigh tissue increased significantly. After 2 hours, the increase of NO content in the tissues below the skin is reduced, the NO content in the thigh tissues is continuously increased, and the increase degree is obvious. The experimental results fully prove that NO in the lipo@PEI/NONOate liposome suspension can effectively enter the inside of skin and effectively enter muscle tissues to participate in the whole process of liposome transdermal.

Example 11

The novel lipo@PEI/NONOate liposome is prepared by using rhodamine B as a simulation efficacy molecule and assembling cholesterol modified with cations as NO donors and lecithin, and the permeation effect of the liposome prepared by mixing the cholesterol, the lecithin and the NO donors on the skin is different. Taking the rhob@lipo PEI/NONOate liposome suspension obtained in example 4 as a control, weighing rhodamine B, soybean lecithin, cholesterol and the NO donor material PEI/NOate prepared in example 2, dissolving the rhodamine B, the soybean lecithin, the cholesterol and the NO donor material PEI/NOate in a proper amount in a mixed solvent consisting of anhydrous methanol and chloroform according to a volume ratio of 1:1 according to a certain proportion (wherein the content of each component of the soybean lecithin, the cholesterol, the PEI/NOate and the rhodamine B is consistent with the concentration of each component in example 4, and the concentration of the PEI/NOate is measured according to a Gris reagent, the mass volume ratio of the soybean lecithin to the anhydrous methanol is 0.1g to 10 mL), carrying out ultrasonic treatment for 5 times in a short time under the ultrasonic conditions of 50KHz and 100W, and the ultrasonic time of 5min each time; then placing the liposome into a eggplant pear bottle, evaporating at room temperature under reduced pressure, removing a solvent, forming a uniform and transparent film on the bottle wall, namely the drug-loaded liposome capable of releasing NO, introducing nitrogen, drying overnight at room temperature under vacuum, adding 5mL of Phosphate Buffer (PBS) (pH=7.4) into the dried film at the temperature of 3 ℃, carrying out ultrasonic hydration for 10min to obtain milky red liposome suspension, namely the liposome material RhoB@PEI/nonoate@lipo loaded with NO and rhodamine B (the construction principle schematic diagram of the liposome is shown in (A) in fig. 8), and sealing at the temperature of 4 ℃.

10. Mu.L of the liposome suspension RhoB@Lipo@PEI/NONOate obtained in example 4 and the RhoB@PEI/NOate@Lipo liposome of the mixed structure carrying NO and rhodamine B obtained above were applied to the left leg (c position) and the right leg (d position) of a hair-removed mouse, respectively, to simulate skin permeation of the drug, and after application, live imaging was performed for 1 hour, as shown in (B) of FIG. 8, c, d. After 1 hour, the thigh tissue was dissected and imaged in vivo to detect the fluorescence intensity, as shown in fig. 8 (B) c1 and d 1. As can be seen from fig. 8, for rhob@lipo@pei/NONOate liposomes, the fluorescence intensity was weaker in the left leg (c position) and stronger in the isolated thigh tissue (c 1 position), indicating better penetration of rhodamine in the left leg into the skin and deep into the thigh interior tissue. However, the fluorescence intensity of the right leg (d position) was strong, the fluorescence intensity of the isolated thigh tissue (d 1 position) was weak, indicating that rhodamine of the right leg remained on the skin surface all the time, and the liposome permeation effect was weak. Therefore, compared with the liposome which is constructed to wrap the NO donor and the efficacy molecule, the novel liposome RhoB@Lipo@PEI/NONONOate formed by taking the NO donor molecule as a part of constructing a liposome structure, taking the NO donor molecule as a liposome skeleton molecule and participating in liposome assembly and further wrapping the function molecule has the best penetration effect on skin.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims

1. A NO-releasable liposome comprising a phospholipid and an ionic NO donor material;

the preparation method of the ionic NO donor material comprises the following steps:

(1) Dissolving a cationic polymer in an organic solvent A, adding cholesterol chloroformate and a catalyst triethylamine, reacting at 0-4 ℃, heating to 5-35 ℃, continuing to react, concentrating after the reaction is finished, adding hydrochloric acid into the concentrate for redissolution, washing, settling and drying to obtain cholesterol modified by the cationic polymer, wherein the molar ratio of the cationic polymer to the cholesterol chloroformate is 1:1; the cationic polymer is hyperbranched polyethyleneimine with molecular weight of 600; the structural formula of the cationic polymer modified cholesterol is shown as follows:(2) Dissolving the cholesterol modified by the cationic polymer obtained in the step (1) in an organic solvent B, adding sodium methoxide for continuous dissolution, introducing inert gas after dissolution, and then introducing NO gas for reaction to obtain the ionic NO donor material;

the preparation method of the liposome capable of releasing NO comprises the following steps: dissolving phospholipid and the ionic NO donor material in an organic solvent C, and evaporating to remove the organic solvent to obtain a film, namely the liposome capable of releasing NO; the phospholipid is soybean lecithin, and the mass ratio of the phospholipid to the ionic NO donor material is 1:0.2.

2. The NO releasable liposome according to claim 1, wherein in step (1), the organic solvent a comprises dichloromethane; the mass volume ratio of the cholesterol chloroformate to the organic solvent A is (1-4) g:10 mL; the mol ratio of the triethylamine to the cholesterol chloroformate is 1:0.1-0.5; the concentration of the hydrochloric acid is 0.1-0.4M; the mass volume ratio of the concentrate to the hydrochloric acid is (2-4) g to (10-20) mL; reacting for 10-30 min at 0-4 ℃, then heating to 5-35 ℃, and continuing to react for 10-14 h; in the step (2), the mass ratio of the cholesterol modified by the cationic polymer to the sodium methoxide is 1:0.1-0.5; the organic solvent B comprises a mixed solution consisting of methanol and tetrahydrofuran according to the volume ratio of 1:0.1-0.5; the mass volume ratio of the cholesterol modified by the cationic polymer to the methanol is (0.5-1) g to 10mL; the temperature of the reaction of the introduced NO gas is 5-35 ℃ and the time is 3-7 days.

3. The NO-releasable liposome according to claim 1, wherein the organic solvent C comprises a mixed solution of methanol and chloroform in a volume ratio of 1:1 to 4, a mixed solution of methanol and tetrahydrofuran in a volume ratio of 1:1 to 2, a mixed solution of methanol and toluene in a volume ratio of 1:2 to 6, a mixed solution of methanol and ethanol in a volume ratio of 1:1 to 3, or a mixed solution of methanol and ethyl acetate in a volume ratio of 1:2 to 4; the amount of the methanol in the organic solvent C is 0.1-0.5 g of soybean lecithin per 10mL.

4. Use of a NO-releasable liposome according to claim 1 for the preparation of a transdermal formulation.

5. A transdermal formulation comprising the NO-releasable liposome of claim 1 and an efficacy molecule.

6. A method of preparing the transdermal formulation of claim 5, comprising the steps of: dissolving the liposome capable of releasing NO in a composite solvent, adding the efficacy molecules, evaporating to remove the solvent to obtain a film, drying under inert atmosphere, and adding phosphate buffer solution or water for hydration to obtain liposome suspension, namely the transdermal drug delivery preparation; the composite solvent comprises a mixed solution of methanol and chloroform according to the volume ratio of 1:1-4.