CN115400084A

CN115400084A - Liposome capable of releasing NO, and preparation method and application thereof

Info

Publication number: CN115400084A
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: 2022-11-29
Anticipated expiration: 2042-08-29
Also published as: CN115400084B

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 penetrate skin and release nitric oxide, so that the vasodilatation of capillary vessels at the administration part can be realized, the blood flow rate is accelerated, and the blood microcirculation system of local tissues is changed, thereby realizing the effect of high-efficiency permeation of the medicine to the skin; the invention selects the cholesterol modified by cation as a novel NO donor, thereby leading the cholesterol NO donor to be assembled with the lecithin and participate in the process of forming the liposome. The NO donor molecule is used as a part for constructing a liposome structure, so that the NO donor molecule is used as a liposome skeleton molecule, and the nitric oxide releasing liposome obtained by participating in liposome assembly is used as a transdermal delivery carrier, thereby realizing the purposes of effective loading of various types of medicines and high-efficiency permeation of skin.

Description

Liposome capable of releasing NO, 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 System (TDDS) is a novel preparation that is administered on the surface of the skin, and the drug penetrates through the layers of the skin at a certain speed, enters the systemic blood circulation of the human body from the capillary vessels to achieve effective blood concentration, and realizes systemic or local therapeutic action. Compared with oral administration, intravenous injection and other modes, transdermal administration has the advantages of avoiding first pass effect with gastrointestinal tract, reducing fluctuation of blood concentration, avoiding stimulation of medicine to gastrointestinal tract, having no trauma during administration, having strong compliance and the like. However, the skin is a difficult permeable barrier to most drugs, and many drugs are difficult to penetrate into the skin or the amount of penetration reaches a therapeutic level, so that it is essential to study TDDS to promote the transdermal penetration of drugs by proper methods. Among the technologies for promoting the transdermal penetration of drugs, nanotechnology is an effective means, and some drugs which are difficult to penetrate through skin can enter the human body and exert the drug effect with the assistance of a nano-carrier. Therefore, nanotechnology has become an effective passive transdermal delivery method developed in recent years.

The liposome is a spheroid which is formed by dispersing phospholipid and other amphiphilic substances in water and sealing one or more layers of concentric lipid bilayer membranes, can wrap hydrophilic or lipophilic medicaments, and is widely applied to the aspects of intravenous administration, skin administration, lung administration, oral administration and the like. The use of liposomes as a carrier material for transdermal administration has the following advantages: 1. the lipid bilayer can promote the drug to enter stratum corneum or epidermal lipid, and increase the retention amount and retention time of the drug in the skin; 2. has skin targeting effect, and can avoid adverse reaction caused by systemic absorption of the medicine; 3. the stability and the durability of the medicine are improved after the coating; 4. no toxicity, no irritation, and safety in application (Journal of Drug Delivery Science and technology.2014,24, 245-250). However, aiming at the 'brick wall' structure with compact skin, how to further improve the drug penetration capability of the liposome to the skin and realize the maximization of the drug treatment effect has become a key problem of the transdermal liposome drug delivery preparation.

NO has important regulatory roles in various physiological and pathological processes, such as promotion of vasodilation, and acceleration of blood flow rate. Rao (Frontiers in Bioengineering and Biotechnology.2020,8, 00578) and Li (biomaterials.2020, 241, 119904) et al found that the production of equimolar to nanomolar concentrations of NO is catalyzed by nitric oxide synthase, thereby achieving vasodilatory modulation of blood vessels, increasing coronary blood flow velocity and effectively inhibiting platelet aggregation and adhesion. However, NO as a highly active gas molecule has the problems of poor stability, low loading capacity, high release rate and the like, and NO can not effectively enter the skin to further play a role in a 'brick wall' structure with compact skin. Mark (ACS Biomate. Sci. Eng.2017,3, 2136-2143) and other people encapsulate various cationic NO donors in the liposome, and finally a series of stable NO liposomes are obtained. Experimental results show that the release period of the NO micromolecules encapsulated in the liposome can reach about 48 hours, and the NO micromolecules can also exist in buffer solution or serum for a long time. Takuma (International Journal of pharmaceuticals.2019, 565, 481-487) et al entrap diethylenetriamine as a small NO molecule donor inside liposomes to obtain releasable NO liposomes. Although research results show that the NO liposome can realize the sustained release of NO and realize the effective accumulation at the focus part through blood circulation, thereby effectively treating diseases. However, the NO liposome can not realize the stable loading of NO micromolecules, and certain NO micromolecules leak in the circulation process of body fluid, so that the effective concentration of NO for treating the focus part can be realized by multiple times of administration. Therefore, how to realize the high-efficiency loading and sustained release of the liposome on NO micromolecules and prevent and treat drug leakage becomes the key for further realizing the 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, a preparation method and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

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

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

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

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

Preferably, in the step (1), the cationic polymer comprises triethylene diamine, pentaethylene hexamine, hyperbranched polyethyleneimine with the molecular weight of 600-25000 and chitosan with the molecular weight of 1000-50000; the organic solvent A comprises dichloromethane; the mass-volume ratio of the cholesteryl chloroformate to the organic solvent A is (1-4) g: 10mL; the molar ratio of the triethylamine to the cholesteryl 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 0-4 ℃ is 10-30 min, and the continuous reaction time is 10-14 h; in the step (2), the mass ratio of cholesterol modified by the cationic polymer to sodium methoxide is 1 (0.1-0.5); the organic solvent B comprises a mixed solution of methanol and tetrahydrofuran according to the volume ratio of 1 to (0.1-0.5); the mass volume ratio of the cholesterol modified by the cationic polymer to the methanol is (0.5-1) g: 10mL; the temperature of the NO gas introduced for reaction 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.

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 the formation of liposome/the construction of liposome nano-structure.

The third technical scheme of the invention is as follows: a liposome capable of releasing NO, which comprises phospholipid and the novel ionic NO donor material.

The fourth technical scheme of the invention is as follows: the preparation method of the liposome capable of releasing NO comprises the following steps: and (3) dissolving phospholipid and the novel NO donor material in an organic solvent C, and then 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 to (1-4), a mixed solution of methanol and tetrahydrofuran with the volume ratio of 1 to (1-2), a mixed solution of methanol and toluene with the volume ratio of 1 to (2-6), a mixed solution of methanol and ethanol with the volume ratio of 1 to (1-3) or a mixed solution of methanol and ethyl acetate with the volume ratio of 1 to (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 every 10mL, namely the mass volume ratio of the soybean lecithin to the methanol in the organic solvent C is (0.1-0.5) g: 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 drug delivery preparations.

The sixth technical scheme of the invention is as follows: a transdermal drug delivery formulation comprising the NO-releasable liposome and an efficacy molecule as described above.

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

The seventh technical scheme of the invention: a preparation method of the transdermal drug delivery 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, introducing inert gas, drying at room temperature overnight under vacuum, adding phosphate buffer solution or water into the film at 0-4 ℃, performing ultrasonic hydration for 10-20 min, adding phosphate buffer solution or water into the film, and hydrating to obtain liposome suspension, namely the transdermal drug delivery preparation.

Preferably, the composite solvent comprises a mixed solution 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: 10mL; the mass ratio of the efficacy molecules to the liposome capable of releasing NO is 1 (25-100); the ultrasonic conditions are 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.

The eighth technical scheme of the invention is as follows: a preparation method of the transdermal drug delivery preparation comprises the following steps: dissolving the functional molecule, phospholipid and the novel ionic NO donor material in a composite solvent, performing ultrasonic treatment for 5-10 times, each time for 5-10 min, performing reduced pressure evaporation at room temperature to remove the solvent to obtain a film, introducing inert gas, performing vacuum drying at room temperature overnight, and adding phosphate buffer solution or water into the film at 0-4 ℃ to perform ultrasonic hydration for 10-20 min to obtain liposome suspension, namely the transdermal drug delivery preparation.

Preferably, the composite solvent comprises a mixed solution 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: 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 effective molecule to the novel ionic NO donor material is 1: 10-20; the ultrasonic conditions are 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 invention combines the ionic NO donor and the liposome to obtain a novel transdermal drug delivery carrier, and the liposome can release nitric oxide, so that the relaxation of capillary vessels at the drug delivery part, the blood flow rate is accelerated, and the blood microcirculation system of local tissues is changed, thereby realizing the effect of high-efficiency permeation of drugs to the skin, and being expected to become a breakthrough for improving the permeation capability of the liposome transdermal drug delivery preparation.

The invention selects the cholesterol modified by cation as NO donor, thus leading the cholesterol NO donor to be assembled with lecithin and participate in the process of forming liposome. The NO donor molecule is used as a part for constructing a liposome structure, so that the NO donor molecule is used as a liposome skeleton molecule, the nitric oxide-releasing liposome obtained by participating in liposome assembly is used as a transdermal delivery carrier, and rhodamine is used for simulating functional small molecules to promote permeation. Can realize the purposes of effective loading of functional small molecules and high-efficiency permeation of skin.

According to the invention, on the premise of utilizing the effect of NO on vasodilatation regulation, NO micromolecules are embedded into the liposome structure to form a component in the liposome, so that a stable liposome structure with high load and long-term NO release is obtained, and further, the blood circulation is accelerated through NO permeation, so that the skin permeation capability of the liposome for wrapping the medicine 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 required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

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

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

FIG. 3 is a chart of the infrared spectrum of Cho-PEI/NONONOate, the NO donor material prepared in example 1;

in FIG. 4, A, B and C are 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 of the liposome suspension prepared in example 3 under simulated human 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 graph showing the in vivo image taken after 1 hour of the PEI/NONONONOATE liposome suspension being applied, and B is a graph showing the in vivo image taken after 1 hour of the PEI/NONONONONONONOATE liposome suspension being applied, taken for dissecting the tissues of the thigh;

FIG. 7 is the content of active gas molecule NO in the lipo @ PEI/NONONONOATE liposomes in the hypodermal tissue and the thigh muscle tissue measured at different time periods in example 10;

FIG. 8, (A) is a schematic diagram showing the construction principle of the liposome material RhoB @ PEI/NONONOate @ lipo in example 11; (B) In example 11, the in vivo image of the liposome suspension RhoB @ Lipo @ PEI/NONONOate and RhoB @ PEI/NONONONONOate @ Lipo at application time of 1h and the in vivo image of the dissected thigh tissue were shown.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description 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.

Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is 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 open-ended terms that mean including but not limited to.

In the following examples, the preparation of anhydrous dichloromethane was carried out according to the following operating steps: adding calcium hydride into dichloromethane, stirring for 6-24 hours, and then distilling at normal pressure to obtain anhydrous dichloromethane, wherein the addition amount of the calcium hydride is calculated by adding 1-2 grams into every 500mL of dichloromethane; the anhydrous methanol, tetrahydrofuran, chloroform and anhydrous ethanol are obtained after the same treatment;

in the following examples, the purity of acetone used is ACS, which is not less than 99.5%; the purity of the adopted ether is AR,98%; the number average molecular weight of the polyethyleneimine used is 600; the purity of the adopted rhodamine B is ACS which is more than or equal to 99.5 percent; the purity of the cholesterol is AR,98%;

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

The description will not be repeated below.

Example 1

NO donor material: preparation of polyethyleneimine modified Cho-PEI/NONONOate:

a. dissolving Polyethyleneimine (PEI) with the number average molecular weight of 600 in anhydrous dichloromethane under the condition of ice-water bath, adding cholesteryl chloroformate with the molar ratio of 1: 1 to the PEI by using triethylamine as a catalyst, wherein the mass-volume ratio of the cholesteryl chloroformate to the anhydrous dichloromethane is 1 g: 10mL, the molar ratio of the triethylamine to the cholesteryl chloroformate is 1.

b. Dissolving the Cho-PEI obtained in the step a in a mixed solution composed of anhydrous methanol and tetrahydrofuran according to the volume ratio of 1:0.1 at room temperature and 25 ℃, adding dried sodium methoxide to continue dissolving after dissolving for 5min, wherein the mass ratio of the Cho-PEI to the sodium methoxide is 1; introducing high-purity nitrogen (20 psi) into the reaction kettle for 10min, removing air in the reaction kettle, introducing NO gas (80 psi), and reacting at 25 ℃ for 3 days. After the reaction is finished, discharging NO by using high-purity nitrogen with the pressure of 20psi, continuously ventilating for 30min, opening the reaction kettle, and taking out a reaction product. Washed 2 times with ether sedimentation and dried under vacuum at room temperature to give the novel ionic NO donor material Cho-PEI/NONONOATE.

Example 2

NO donor material: preparation of polyethyleneimine as NO donor (PEI/NONONAte):

dissolving dry polyethyleneimine (PEI, the number average molecular weight of 600) in a mixed solution composed of anhydrous methanol and tetrahydrofuran according to the volume ratio of 1:0.1 at room temperature and 25 ℃, adding dry sodium methoxide to continue dissolving after dissolving for 5min, wherein the mass ratio of PEI to sodium methoxide is 1. Introducing high-purity nitrogen (20 psi) into the reaction kettle for 10min, removing air in the reaction kettle, introducing NO gas (80 psi), and reacting at room temperature of 25 ℃ for 7 days. After the reaction is finished, introducing high-purity nitrogen gas for 30min and 50psi into the reaction kettle to discharge unreacted NO, opening the reaction kettle, and taking out a reaction product. Washing the reaction product with anhydrous ether for 2 times, vacuum drying at 25 deg.C for 24 hr to obtain Polyethyleneimine (PEI) NO donor material PEI/NONONONAte, and storing at-4 deg.C in a dryer.

Example 3

Preparing a transdermal administration preparation which is not loaded with functional molecules and can release NO:

soybean lecithin and the NO donor material Cho-PEI/NONONOate prepared in example 1 were dissolved in a proper amount of a mixed solvent composed of anhydrous methanol and chloroform in a volume ratio of 1: 1 (wherein the mass ratio of soybean lecithin to Cho-PEI/NONOate is 1:0.2; the mass-to-volume ratio of soybean lecithin to anhydrous methanol is 0.1 g: 10 mL), and subjected to ultrasonic treatment for 5 times in a short time under 50KHz and 100W conditions, wherein the ultrasonic treatment time is 5min; then placing the mixture into an eggplant pear bottle, decompressing and evaporating at room temperature, removing the solvent, forming a uniform and transparent film on the wall of the bottle, namely the liposome capable of releasing NO, introducing nitrogen, drying overnight at room temperature in vacuum, adding 5mL Phosphate Buffer Solution (PBS) (pH = 7.4) into the dried film at 3 ℃, and ultrasonically hydrating for 10min to obtain milky white liposome suspension, namely the liposome capable of releasing NO, which is marked as Lipo @ PEI/NONONONAte, and sealing and storing at 4 ℃.

Example 4

Preparing a transdermal administration preparation loaded with the efficacy molecules:

the method comprises the following steps of taking rhodamine B as a simulated efficacy molecule, weighing a certain mass of rhodamine B, then dissolving the rhodamine B, soybean lecithin and the novel ionic NO donor material Cho-PEI/NONONOate prepared in example 1 in a mixed solvent composed of an appropriate amount of anhydrous methanol and chloroform according to a volume ratio of 1: 1 (wherein the mass ratio of the soybean lecithin to the Cho-PEI/NONONOate to the rhodamine B is 1.2; then placing the mixture into an eggplant pear bottle, decompressing and evaporating at room temperature, removing a solvent, forming a uniform and transparent film on the wall of the bottle, namely a medicine-carrying liposome capable of releasing NO, introducing nitrogen, drying overnight at room temperature in vacuum, adding 5mL of Phosphate Buffer Solution (PBS) (pH = 7.4) into the dried film at 3 ℃, and carrying out ultrasonic hydration for 10min to obtain milky red liposome suspension, namely the liposome of NO loaded with efficacy molecules, which is a novel NO transdermal delivery preparation and is recorded as RhoB @ Lipo @ PEI/NONONAte, and sealing and storing at 4 ℃.

In this example, the schematic construction of the NO-loaded liposomes loaded with the efficacy molecules is 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 ) In the method, the solution is clear, transparent and free of suspended matters or impurities, and is detected on a nuclear magnetic resonance instrument (NMR-Bruker-300) by using a clean nuclear magnetic resonance special sample tube. Reference to deuterated solvent residual peak: (deuterated chloroform 7.26 ppm). As shown in FIG. 2, PEI forms an amide bond after reacting with cholesterol ester, and has a peak around a chemical shift of 6.78ppm, as shown by a in FIG. 2, and hydrogen atoms in cholesterol are characterized by 5.2ppm and 4.2ppm as shown by b and c in FIG. 2, and most of the hydrogen atoms in PEI are between 2 and 3ppm as shown by d-g. Indicating the successful synthesis of Cho-PEI.

Example 6

The NO donor material Cho-PEI/NONONAte obtained in example 1 was characterized by IR spectroscopy using the potassium bromide pellet method. The results are shown in FIG. 3, where the ion type NO donor material Cho-PEI/-NONONAte has a characteristic absorption peak at 1250cm in the IR spectrum ^-1 Here, it is shown that the material has been successfully loaded with NO.

Example 7

1mg of the milky red liposome suspension of RhoB @ lipo @ PEI/NONONONONAte obtained in example 4 was dispersed in 1mL of pure water, and after uniform ultrasonic dispersion, the potential and particle size of the nanoparticles were measured by a Malvern laser particle sizer, and the results are shown in FIG. 4, wherein A is a real image of the liposome suspension, B is a particle size diagram, and C is an SEM image at 1000 times, as shown in FIG. 4: the liposome has an average particle size of about 600nm, is uniform in size, and can be stably suspended in a solution.

Example 8

5mg of the milky white liposome suspension of Lipo @ PEI/NONONONOate obtained in example 3 was weighed and 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 the supernatant was mixed with 50. Mu.L of PBS buffer (pH = 7.4), 100. Mu.L of Grignard reagent (PEI/NONOate and S-nitrosoglutathione were used for Nanjing to build a nitric oxide kit A013-2-1, L-arginine was used for Nanjing to build a nitric oxide kit A012-1 to test NO content) was added, the mixture was stored for 15min in the dark, and the absorbance was measured at 540nm using a microplate reader to plot the drug release curve. As shown in figure 5, the Lipo @ PEI/NONONONOATE liposome suspension has the effect of rapidly releasing NO within the first 10 hours, the accumulated release amount of NO can reach 60%, NO in the Lipo @ PEI/NONONONONOATE liposome can be continuously released along with the prolonging of time, and the release time can reach more than 50 hours. Thus, lipo @ PEI/NONONONAte liposomes can achieve a long release of NO.

Example 9

5 μ L of the liposome suspension RhoB @ Lipo @ PEI/NONONOate of example 4 using rhodamine B as the mimetic efficacy molecule was weighed out and applied to the left leg (position a) of a hair-removed mouse, and the right leg was a liposome prepared only from soybean lecithin and rhodamine B (position B) without adding Cho-PEI/NONOate, thereby mimicking drug skin permeation, and the living body was imaged after 1 hour of application, as shown in A of FIG. 6. The fluorescence intensity was detected after 1 hour by biopsy of thigh tissue and live imaging, as shown in fig. 6B. As can be seen from FIG. 6, for RhoB @ lipo @ PEI/NONONONOate liposome, the fluorescence intensity of the left leg (at position a) is weak, while the fluorescence intensity of isolated thigh tissue (at position a 1) is strong, indicating that rhodamine of the left leg permeates skin well and penetrates deep into the inner tissues of the thigh. However, the fluorescence intensity of the right leg (b position) is strong, and the fluorescence intensity of the isolated thigh tissue (b 1 position) is weak, which indicates that rhodamine in the right leg stays on the surface of the skin all the time, and the liposome penetration effect is weak. Therefore, this experiment sufficiently demonstrates that NO has an excellent promoting effect on the permeation effect of liposomes.

Example 10

In order to verify that active gas molecule NO in the Lipo @ PEI/NONONONAte liposome passes through skin tissues along with the liposome, enters muscle tissues and participates in the whole transdermal process of the liposome, so that the permeation effect of the liposome is effectively improved. 10 μ L of the milky white liposome suspension Lipo @ PEI/NONONONOate prepared in example 3 was weighed and applied to the left leg of three mice from which hairs were removed, respectively, after various time periods (0.5, 1 and 2 hours), the thigh tissue of the mice was dissected, the lower adipose tissue of the rat epidermis and the muscle tissue of the same site were taken to perform tissue homogenate extraction, and the NO concentration in the tissue homogenate was measured using a Kjeldahl nitric oxide detection kit (nitric acid reductase method) with reference to the corresponding procedure. The results of the experiment are shown in fig. 7, after 0.5h, the presence of NO was detected in the underlying tissue of the skin, when the NO content in the thigh tissue was relatively low, with only a slight amount of NO being present. With increasing time, the NO content in the skin underlying tissue and thigh tissue increased significantly after 1 h. After 2h, the increase of NO content in the skin lower layer tissue is weakened, and the NO content in the thigh tissue is continuously increased to a remarkable extent. The above experimental results fully prove that NO in the Lipo @ PEI/NONONOATE liposome suspension can effectively enter the skin, effectively enter muscle tissues and participate in the whole process of liposome transdermal penetration.

Example 11

The novel Lipo @ PEI/NONONOate liposome prepared by assembling cholesterol modified with cations as a NO donor and lecithin is different from the skin permeation effect of the liposome prepared by mixing cholesterol, lecithin and the NO donor by using rhodamine B as a simulated efficacy molecule and verifying the effect. Taking the RhoB @ Lipo PEI/NONONOate liposome suspension obtained in the example 4 as a reference, weighing the same mass of rhodamine B, soybean lecithin, cholesterol and NO donor material PEI/NONONOate prepared in the example 2, dissolving the rhodamine B and the soybean lecithin, cholesterol and PEI/NONONOate in a mixed solvent composed of anhydrous methanol and chloroform according to a certain proportion and a volume ratio of 1: 1 (wherein the content of each component of the soybean lecithin, the cholesterol, the PEI/NONONONOate and the rhodamine B is consistent with the concentration of each component in the example 4, and the PEI/NONOate concentration is determined according to a Grice reagent, the mass-volume ratio of the soybean lecithin to the anhydrous methanol is 0.1 g: 10 mL), and performing short-time ultrasound for 5 times under the conditions of 50KHz and 100W, wherein the ultrasound time is 5min each time; then placing the mixture into an eggplant pear bottle, decompressing and evaporating at room temperature, removing a solvent, forming a uniform and transparent film on the wall of the bottle, namely a drug-loaded liposome capable of releasing NO, introducing nitrogen, drying overnight at room temperature in vacuum, adding 5mL of Phosphate Buffer Solution (PBS) (pH = 7.4) into the dried film at 3 ℃, and ultrasonically hydrating for 10min to obtain milky red liposome suspension, namely obtaining a liposome material RhoB @ PEI/NONONONONAte @ lipo loading NO and rhodamine B (the construction principle schematic diagram of the liposome is shown in (A) in figure 8), and sealing at 4 ℃.

The drug skin permeation was simulated by applying 10. Mu.L of the liposome suspension RhoB @ PEI/NONONONOATE obtained in example 4 and the RhoB @ PEI/NONONONONONONOATE @ Lipo liposome having the mixed structure of NO and rhodamine B to the left leg (position c) and the right leg (position d) of the mouse from which hair was removed, respectively, and the living body was imaged 1 hour after the application, as shown by c and d in the graph (B) of FIG. 8. The fluorescence intensity was measured by biopsy of the thigh tissue after 1 hour, as shown by c1 and d1 in the graph (B) of FIG. 8. As can be seen from FIG. 8, for RhoB @ Lipo @ PEI/NONONOATE liposome, the fluorescence intensity of the left leg (at position c) is weaker, while the fluorescence intensity of the isolated thigh tissue (at position c 1) is stronger, which indicates that rhodamine of the left leg better permeates the skin and goes deep into the thigh internal tissue. However, the fluorescence intensity of the right leg (d position) is strong, and the fluorescence intensity of the isolated thigh tissue (d 1 position) is weak, which indicates that rhodamine in the right leg stays on the surface of the skin all the time, and the liposome penetration effect is weak. Therefore, compared with the construction of the liposome for wrapping the NO donor and the functional molecule, the NO donor molecule is used as a part for constructing the structure of the liposome and is used as a liposome skeleton molecule to participate in the assembly of the liposome, and the functional molecule is further wrapped to form the novel liposome with the optimal permeation effect on the skin of RhoB @ Lipo @ PEI/NOnoate.

The embodiments of the present invention have been described in detail, but the embodiments are only examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions for the present invention are within the scope of the present invention for those skilled in the art. Accordingly, equivalent alterations and modifications are intended to be included within the scope of the present invention, without departing from the spirit and scope of the invention.

Claims

1. A preparation method of a novel ionic NO donor material is characterized by comprising the following steps:

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

2. The method according to claim 1, wherein in the step (1), the cationic polymer comprises triethylene diamine, pentaethylene hexamine, hyperbranched polyethyleneimine having a molecular weight of 600 to 25000, and chitosan having a molecular weight of 1000 to 50000; the organic solvent A comprises dichloromethane; the mass volume ratio of the cholesteryl chloroformate to the organic solvent A is (1-4) g: 10mL; the molar ratio of the triethylamine to the cholesteryl 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 0-4 ℃ is 10-30 min, and the continuous reaction time is 10-14 h; in the step (2), the mass ratio of cholesterol modified by the cationic polymer to sodium methoxide is 1: 0.1-0.5; the organic solvent B comprises a mixed solution 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: 10mL; the temperature of the NO gas introduced for reaction is 5-35 ℃, and the time is 3-7 days.

3. A novel ionic NO donor material prepared according to the preparation method of claim 1 or 2.

4. A liposome capable of releasing NO, comprising a phospholipid and the novel ionic NO donor material of claim 3.

5. The method for preparing NO-releasable liposomes of claim 4, comprising the steps of: and (3) dissolving phospholipid and the novel NO donor material in an organic solvent C, and then evaporating to remove the organic solvent to obtain a film, namely the NO-releasable liposome.

6. The method of claim 5, wherein the phospholipid comprises soy 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 amount of the methanol in the organic solvent C is calculated by adding 0.1-0.5 g of soybean lecithin into every 10mL.

7. Use of the NO-releasable liposome of claim 4 for the preparation of a transdermal delivery formulation.

8. A transdermal formulation comprising the NO-releasing liposome of claim 4 and an efficacy molecule.

9. A method of preparing a transdermal drug delivery formulation according to claim 8, comprising the steps of: dissolving the liposome capable of releasing NO in a composite solvent, adding the functional molecule, evaporating to remove the solvent to obtain a film, drying in an inert atmosphere, and adding a phosphate buffer solution or water for hydration to obtain a liposome suspension, namely the transdermal administration preparation.

10. A method of preparing a transdermal drug delivery formulation according to claim 8, comprising the steps of: dissolving the functional molecule, phospholipid and the novel ionic NO donor material of claim 3 in a composite solvent, evaporating to remove the solvent to obtain a film, drying in an inert atmosphere, and adding phosphate buffer solution or water for hydration to obtain liposome suspension, namely the transdermal drug delivery preparation.