CN115444820B - Skin external preparation with beautifying effect and raw materials, preparation and application thereof - Google Patents
Skin external preparation with beautifying effect and raw materials, preparation and application thereof Download PDFInfo
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- CN115444820B CN115444820B CN202211077170.0A CN202211077170A CN115444820B CN 115444820 B CN115444820 B CN 115444820B CN 202211077170 A CN202211077170 A CN 202211077170A CN 115444820 B CN115444820 B CN 115444820B
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- cyclodextrin
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- hydroxypropyl
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- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
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Abstract
The present disclosure provides a skin external agent with a cosmetic effect, and raw materials, preparation and applications thereof. The nanometer composition comprises a nanometer micelle formed by entrapment of hydrophobic drugs by amphiphilic block copolymers; the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (0.5-4); wherein the amphiphilic block copolymer is hydrophilic polysaccharide modified by hydrophobic property; the hydrophobic modifier used in the hydrophobic modification process includes a hydrophobic ester modifier and/or a hydrophobic polyester modifier. Compared with the commonly used nano-carrier at present, the nano-composition prepared by the method has higher stability and drug entrapment capacity, and the transdermal effect and the slow release effect of the drug are ideal. Compared with petroleum-based polymers, the amphiphilic block copolymer adopted by the present disclosure has ideal biocompatibility and is easy to degrade.
Description
Technical Field
The disclosure belongs to the technical field of cosmetics, and particularly relates to a skin external agent with a beautifying effect, and raw materials, preparation and application thereof.
Background
The nanometer medicine is one new kind of medicine preparation capable of promoting transdermal permeation, and its core technology is nanometer carrier technology. The drug is loaded in the nano carrier with the particle size of 10-1000 nm by the nano carrier technology for percutaneous delivery, so that the drug permeation capacity of the drug through biological membranes can be effectively increased, the drug release speed can be regulated, the bioavailability of the drug can be obviously improved, the solubility and water dispersibility of the poorly soluble drug can be improved, the stability of the photosensitive or thermosensitive drug can be improved, and the drug has good application prospects in the fields of skin disease treatment, skin care, cosmetology and the like.
Specific forms of nanocarriers generally include nanovesicles, liposomes, microemulsions/nanoemulsions, nanocrystals, lipid crystals, polymeric nanomicelles, inorganic nanocarriers, and the like. At present, liposome and nanoemulsion are commonly used as nano carriers, but the liposome and nanoemulsion are often used as carriers for coating hydrophobic drugs, so that the problems of low encapsulation rate, insufficient stability, abrupt drug release and the like are caused. The polymer nano micelle as a novel nano carrier has various advantages, such as adjustable polymer structure and micelle performance, solubilization of hydrophobic drugs, improvement of drug stability and the like. However, the traditional petroleum-based polymer micelle nano-carrier has poor biocompatibility, is not degradable, has single polymer wall material selection, and has limited lifting degree although improving the entrapment capacity of hydrophobic drugs, and has unsatisfactory drug release effect and transdermal effect.
Therefore, there is a need in the art to develop a biodegradable nano-drug with good stability, high drug encapsulation efficiency and high transdermal drug delivery rate, and slow drug release.
Disclosure of Invention
The technical problem to be solved by the present disclosure is to overcome the defects of the prior art that the nano-carrier has low encapsulation rate and loading rate for hydrophobic drugs, and insufficient stability, resulting in burst release of the drugs, poor biocompatibility, unsatisfactory transdermal effect of the drugs, non-degradability, etc., and to provide a skin external preparation with cosmetic efficacy, and raw materials, preparation and application thereof. The present disclosure employs amphiphilic block copolymers with biocompatibility to self-assemble in solvents to form nanomicelles by dialysis. Compared with the commonly used nano carriers (liposome and nanoemulsion), the nano composition prepared by the method has higher stability and drug entrapment capacity, and the transdermal effect and the slow release effect of the drug are ideal. Compared with petroleum-based polymers, the amphiphilic block copolymer adopted by the present disclosure has ideal biocompatibility and is easy to degrade.
The present disclosure solves the above technical problems by adopting the following technical scheme:
the present disclosure provides a nano composition comprising a nano micelle formed by entrapping a hydrophobic drug by an amphiphilic block copolymer; the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (0.5-4);
Wherein the amphiphilic block copolymer is a hydrophobically modified hydrophilic polysaccharide; the hydrophobic modifier used in the hydrophobic modification process comprises a hydrophobic ester modifier and/or a hydrophobic polyester modifier.
In some embodiments, the mass of the amphiphilic block copolymer and the hydrophobic drug is preferably 1: (0.5 to 2), more preferably 1: (1-2).
In some embodiments, the average particle size of the nanocomposite may be generally from 100 to 200nm, preferably from 100 to 150nm.
In some embodiments, the hydrophilic polysaccharide may include hydroxypropyl-beta-cyclodextrin and/or chitosan. Wherein the degree of substitution of hydroxyl in the hydroxypropyl-beta-cyclodextrin is 14.
In some embodiments, the hydrophobic drug may be an active ingredient having cosmetic efficacy in the cosmetic field, and may generally include at least one of a whitening active ingredient, an anti-aging active ingredient, an anti-inflammatory active ingredient, and a skin barrier protecting active ingredient.
In some embodiments, the hydrophobic drug may include a linear hydrophobic drug and/or a benzene ring-containing hydrophobic drug.
Wherein the linear hydrophobic drug may include at least one of retinol, squalane, and decarboxylated carnosine.
Wherein the benzene ring-containing hydrophobic drug may include a single benzene ring-containing hydrophobic drug and/or a multi-benzene ring-containing hydrophobic drug. The hydrophobic drug containing a single benzene ring may include at least one of tocopherol, 4-butylresorcinol, and methyl salicylate. The multi-benzene ring containing hydrophobic drug may include glabridin and/or phenethyl resorcinol.
In some embodiments, the hydrophobic ester modifier may include benzyl p-nitrochloroformate.
In some embodiments, the hydrophobic polyester-based modifier may include polycaprolactone and/or polylactic acid.
Wherein the polymerization degree of the polycaprolactone can be 2-6.
Wherein the polymerization degree of the polylactic acid can be 2-6.
In a preferred embodiment, when the hydrophobic modifier is benzyl p-nitrochloroformate, the amphiphilic block copolymer is benzyl p-nitrochloroformate modified hydrophilic polysaccharide, and the hydrophobic drug is a benzene ring-containing hydrophobic drug, more preferably a multi-benzene ring-containing hydrophobic drug. In the research and development process, when the hydrophobic modifier in the amphiphilic block copolymer is benzyl p-nitro chloroformate, the hydrophobic modifier is favorable for containing the benzene ring hydrophobic drugs, and is more favorable for containing the multi-benzene ring hydrophobic drugs.
In a more preferred embodiment, when the amphiphilic block copolymer comprises the benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin and/or benzyl p-nitrochloroformate modified chitosan, the hydrophobic drug comprises a multi-benzene ring-containing hydrophobic drug.
In a preferred embodiment, when the hydrophobic modifier is benzyl p-nitro chloroformate, the preparation method of the amphiphilic block copolymer comprises the following steps: and in inert atmosphere and solvent, the hydrophilic polysaccharide, the benzyl p-nitrochloroformate and the connector react under the action of a catalyst A and a condensing agent to prepare the benzyl p-nitrochloroformate modified hydrophilic polysaccharide. The reaction includes an esterification reaction and an amidation reaction, as known to those skilled in the art.
Wherein the solvent may include N, N-dimethylformamide as conventionally used in the art.
Wherein the linker may be a compound having at least one amino group and one carboxyl group at the end group, preferably at least one of 4-aminobutyric acid, 5-aminopentanoic acid and beta-aminopropionic acid, more preferably 4-aminobutyric acid.
Wherein the condensing agent may comprise at least one of dicyclohexylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and diisopropylcarbodiimide, preferably dicyclohexylcarbodiimide.
Wherein the catalyst A may comprise 4-dimethylaminopyridine.
Wherein the temperature of the reaction may be 25 to 50 ℃, for example 30 ℃.
Wherein the reaction time may be 24 to 72 hours, for example 48 hours.
Wherein the reaction may further comprise dialysis and/or drying operations after completion of the reaction. The MWCO of the dialysis bag at dialysis can be 500-3500 Da, preferably 1000Da. The dialysis time may be 48 to 72 hours. The drying method may be a freeze-drying method conventionally used in the art.
Wherein, when the hydrophilic polysaccharide is the hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the benzyl p-nitrochloroformate may be 1: (14.7 to 17.5), for example, 1:16.
wherein when the hydrophilic polysaccharide is the hydroxypropyl- β -cyclodextrin, the molar ratio of the hydroxypropyl- β -cyclodextrin to the linker may be 1: (14.7 to 17.5), for example, 1:16.
wherein when the hydrophilic polysaccharide is the hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the condensing agent may be 1: (14.7 to 17.5), for example, 1:15.
wherein when the hydrophilic polysaccharide is the hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the catalyst a may be 1: (1.47 to 1.75), for example, 1:1.5.
Wherein, when the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan to the benzyl p-nitro chloroformate may be 1: (10.5-16).
Wherein when the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan to the linker may be 1: (10.5-16).
Wherein, when the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan and the condensing agent may be 1: (10.5-15).
Wherein, when the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan to the catalyst a may be 1: (1.05-1.5).
In a preferred embodiment, when the hydrophobic modifier is the polycaprolactone, the preparation method of the amphiphilic block copolymer comprises the following steps: in inert atmosphere, the hydrophilic polysaccharide and epsilon-caprolactone undergo ring-opening polymerization reaction under the action of a catalyst B to prepare the polycaprolactone modified hydrophilic polysaccharide.
Wherein the catalyst B may comprise stannous octoate. The catalyst B may comprise 0.5% to 2%, preferably 1% by mass of the epsilon-caprolactone.
Wherein the temperature of the ring-opening polymerization reaction may be 100 to 150 ℃, for example 130 ℃.
Wherein the time of the ring-opening polymerization reaction may be 6 to 24 hours, for example, 12 hours.
Wherein, the operation of ring-opening polymerization reaction can be further followed by purification and/or drying.
The purification comprises the following steps: CH is added into the material after the ring-opening polymerization reaction 2 Cl 2 To the premisesAnd (3) completely dissolving the material after the ring-opening polymerization reaction, and dripping the dissolved material into n-hexane to prepare the purified polycaprolactone modified hydrophilic polysaccharide.
Wherein, when the hydrophilic polysaccharide is hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the epsilon-caprolactone may be 1: (33.6 to 100.8), for example, 1:50.
wherein when the hydrophilic polysaccharide is chitosan, the molar ratio of the chitosan to the epsilon-caprolactone may be 1: (48-144), for example 1:50.
in a preferred embodiment, when the hydrophobic modifier is the polylactic acid, the preparation method of the amphiphilic block copolymer comprises the following steps: in inert atmosphere, the hydrophilic polysaccharide and D, L-lactide are subjected to ring-opening polymerization reaction under the action of a catalyst C to prepare the polylactic acid modified hydrophilic polysaccharide.
Wherein the catalyst C may comprise stannous octoate. The catalyst C may be 0.5 to 2% by mass, preferably 1% by mass of the D, L-lactide.
Wherein the temperature of the ring-opening polymerization reaction may be 100 to 150 ℃, for example 130 ℃.
Wherein the time of the ring-opening polymerization reaction may be 6 to 24 hours, for example, 12 hours.
Wherein, when the hydrophilic polysaccharide is hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin and the D, L-lactide may be 1: (30-100.8).
Wherein, when the hydrophilic polysaccharide is chitosan, the molar ratio of the chitosan and the D, L-lactide may be 1: (30-144).
In a preferred embodiment, the amphiphilic block copolymer comprises at least one of polycaprolactone-modified hydroxypropyl- β -cyclodextrin, polylactic acid-modified hydroxypropyl- β -cyclodextrin, p-nitro-chloroformate-modified hydroxypropyl- β -cyclodextrin, polycaprolactone-modified chitosan, polylactic acid-modified chitosan and p-nitro-chloroformate-modified chitosan, preferably p-nitro-chloroformate-modified hydroxypropyl- β -cyclodextrin and/or p-nitro-chloroformate-modified chitosan.
Wherein the number average molecular weight of the polycaprolactone-modified hydroxypropyl-beta-cyclodextrin can be 3000 to 11000, such as 6169.
Wherein the polylactic acid modified hydroxypropyl-beta-cyclodextrin may have a number average molecular weight of 4000 to 14000, such as 5407.
Wherein the number average molecular weight of the benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin may be 4000 to 14000, such as 5332.
Wherein the polycaprolactone-modified chitosan may have a number average molecular weight of 5000-18000, such as 6320.
Wherein the polylactic acid modified chitosan may have a number average molecular weight of 5000 to 23000, for example 5558.
Wherein the number average molecular weight of the benzyl p-nitrochloroformate-modified chitosan may be 3000-8000, for example 5483.
The present disclosure also provides a method of preparing the nanocomposite as described above, comprising the steps of:
(1) The amphiphilic block copolymer, the hydrophobic drug and the solvent are mixed to prepare a material A; the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (0.5-4);
(2) And (3) dropwise adding the material A into water under the condition of stirring, performing ultrasonic treatment and dialysis to obtain the nano composition.
In the step (1), the solvent may include at least one of an alkane solvent, a ketone solvent, a furan solvent, an amide solvent, a sulfoxide solvent, and an alcohol solvent.
Wherein the alkane solvent may comprise methylene chloride.
Wherein the ketone solvent may include acetone.
Wherein the furan-based solvent may comprise tetrahydrofuran.
Wherein the amide solvent may include N, N' -dimethylformamide.
Wherein the sulfoxide solvent may include dimethyl sulfoxide.
Wherein the alcohol solvent may comprise at least one of 1, 3-propanediol, glycerol, 1, 4-butanediol, pentanediol, 1, 2-hexanediol, ethylene glycol, pentaerythritol, dipropylene glycol, and diethylene glycol, and preferably 1, 3-propanediol.
In step (1), the mixing may be carried out under stirring conditions as is conventional in the art, and the stirring speed may be 30 to 300rpm, for example 150rpm. The stirring time may be 10 to 60 minutes, for example 30 minutes.
In step (1), the total mass of the amphiphilic block copolymer and the hydrophobic drug may be 1% to 30%, preferably 6% to 15%, for example 8% by mass of the solvent.
In step (1), the mass of the amphiphilic block copolymer and the hydrophobic drug is preferably 1: (0.5 to 2), more preferably 1: (1-2).
In the step (2), the stirring speed may be 30 to 300rpm.
In the step (2), the dropping speed may be 0.5 to 2mL/min, preferably 0.5 to 1mL/min.
In step (2), the frequency of the ultrasound may be 300 to 900W, preferably 500 to 700W.
In the step (2), the time of the ultrasonic treatment may be 10 to 60 minutes, preferably 30 minutes.
In step (2), the dialysis bag used for the dialysis may have a MWCO cut-off of 500-5000 Da, preferably 1000-3000 Da.
In step (2), the dialysis medium may be water.
In step (2), the dialysis time may be 24 to 72 hours, for example 48 hours.
In the step (2), the dialysis operation may further include a filtration operation.
Wherein the pore diameter of the filter membrane used for filtering can be 0.2-0.8 mu m.
In the present disclosure, the inert atmosphere may include nitrogen and/or argon as is conventionally used in the art.
The present disclosure also provides a nanocomposite prepared by the method of preparing a nanocomposite as described above.
The disclosure also provides an application of the nano composition as a raw material in preparing a skin external agent.
The present disclosure also provides a skin external agent having a cosmetic effect, which includes the nano composition as described above.
The nano composition is a raw material for preparing the skin external preparation with the cosmetic effect.
In some embodiments, the nano composition may be used as at least one of a whitening active ingredient, an anti-aging active ingredient, an anti-inflammatory active ingredient, and a skin protecting active ingredient in the skin external preparation having a cosmetic effect.
In some embodiments, the external preparation for skin with cosmetic effect may further comprise at least one of emulsion stabilizer, surfactant, antifreezing agent and solvent.
The emulsion stabilizer may include emulsion stabilizers conventionally used in the art, and preferably includes caprylic/capric triglyceride and/or caprylic/capric glyceride polyglycerin-10 esters.
The surfactant may include surfactants conventionally used in the art, and preferably includes at least one of poloxamer 188, polysorbate-80 and polyglyceryl-10 laurate.
The antifreeze may include, among others, antifreeze conventionally used in the art, preferably glycerin.
The solvent may include a solvent conventionally used in the art, and preferably includes at least one of 1, 3-propanediol, 1, 2-hexanediol, and water.
Wherein, the weight part ratio of the hydrophobic drug to the emulsion stabilizer in the nano composition can be 1: (0.2 to 10), preferably 1:1.5.
wherein, the weight part ratio of the hydrophobic drug to the surfactant in the nano composition can be 1: (0.67 to 22.5), preferably 1:2.1.
wherein, the weight part ratio of the hydrophobic drug to the antifreezing agent in the nano composition can be 1: (0.48 to 7.5), preferably 1:1.3.
Wherein, the weight part ratio of the hydrophobic drug to the solvent in the nano composition can be 1: (1-40), preferably 1:7.4.
on the basis of conforming to the common knowledge in the art, the preferred conditions can be arbitrarily combined to obtain the preferred examples of the disclosure.
Reagents and materials for the present disclosure are commercially available.
The positive progress effect of the present disclosure is: the present disclosure employs amphiphilic block copolymers with biocompatibility to self-assemble in solvents to form nanomicelles by dialysis. Compared with the commonly used nano carriers (liposome and nanoemulsion), the nano composition adopted by the present disclosure has higher stability and drug entrapment capacity, and the drug slow release effect and transdermal capacity are more ideal. Compared with petroleum-based polymers, the amphiphilic block copolymers employed in the present disclosure have better biocompatibility.
Drawings
The present disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are included to provide a further illustration of the preferred embodiments of the disclosure and to explain the principles and advantages of the disclosure, are incorporated in and form a part of the specification along with the detailed description that follows. Wherein:
FIG. 1 is a transmission electron micrograph of the nanocomposite obtained in example 1, example 2 and example 7;
FIG. 2 is a graph showing the comparison of the cumulative drug release rate with time of the nanocomposites prepared in examples 1 to 6;
FIG. 3 is a graph showing the comparison of the cumulative drug release rate with time of the nanocomposites prepared in examples 1 and 7 to 9;
FIG. 4 is a graph showing the cumulative drug release rate versus time of the nanocomposites prepared in example 1, examples 11 to 13 and comparative example 9;
FIG. 5 is a graph showing the cumulative drug release rate versus time of the nanocomposites prepared in example 1 and comparative examples 1 to 6;
FIG. 6 is a graph showing the comparison of the in vitro cytotoxicity results of nanocarriers without the hydrophobic drug coated in example 1, comparative example 7, and comparative example 8;
FIG. 7 is a graph comparing cytotoxicity results of the nano-compositions prepared in example 1, comparative example 7, and comparative example 8 and phenethyl resorcinol in vitro.
Detailed Description
The present disclosure is further illustrated by way of examples below, but is not thereby limited to the scope of the examples described. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Example 1
(1) Preparation of benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin: hydroxypropyl-beta-cyclodextrin (1.375 g, 0.001mol) with the substitution degree of 14, benzyl p-nitrochloroformate (3.803 g,0.016 mol), 4-aminobutyric acid (1.605 g,0.016 mol), dicyclohexylcarbodiimide (3.177 g,0.015 mol) and 4-dimethylaminopyridine (0.188 g,0.0015 mol) are added into a reaction bottle, 30mL of anhydrous N, N-dimethylformamide is added, the reaction system is kept at 30 ℃ under the protection of nitrogen, the reaction is carried out for 48 hours, after the reaction, the reacted material is transferred into a dialysis bag with the molecular weight cut-off MWCO of 1000Da, deionized water is used for dialysis for 48 hours, and the materials in the dialysis bag are subjected to freeze drying to prepare the benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin with the number average molecular weight Mn of 5332;
(2) Adding the prepared 1g of benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin and 2g of phenethyl resorcinol into 20g of 1, 3-propanediol, magnetically stirring for 30min at a stirring speed of 150rpm, and completely dissolving the benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin and the phenethyl resorcinol to prepare a material A; and (3) dropwise adding the material A into 77g of deionized water under the condition of stirring, wherein the stirring rotating speed is 150rpm, the dropwise adding speed is 1mL/min, performing ultrasonic treatment for 30min under the condition of 500W after the dropwise adding is finished, transferring the ultrasonic material into a dialysis bag with the MWCO of 1000, dialyzing in the deionized water for 48h, and filtering by an organic filter membrane with the pore diameter of 0.45 mu m after the dialyzing is finished to prepare the nano composition.
Example 2
Compared with the example 1, the difference is only that the polycaprolactone-modified hydroxypropyl-beta-cyclodextrin is prepared in the step (1), the benzyl p-nitro chloroformate-modified hydroxypropyl-beta-cyclodextrin in the step (2) is replaced by the same amount of polycaprolactone-modified hydroxypropyl-beta-cyclodextrin, and other condition parameters are the same as those in the example 1, so that the nano composition is prepared;
the preparation method of the polycaprolactone modified hydroxypropyl-beta-cyclodextrin comprises the following steps: hydroxypropyl-beta-cyclodextrin (1.375 g,0.001 mol) with a substitution degree of 14 and epsilon-caprolactone (5.745 g,0.05 mol) are sequentially added into a reaction bottle, stannous octoate (0.057 g,0.14 mmol) is added, the reaction system is kept at 130 ℃ for reaction for 12 hours under the protection of nitrogen, and after the reaction is finished, 50mL of CH is added into the reacted material 2 Cl 2 And (3) until the materials are completely dissolved, dropwise adding the dissolved materials into 500mL of n-hexane for purification, collecting solid precipitate, and drying the solid precipitate to obtain the polycaprolactone modified hydroxypropyl-beta-cyclodextrin, wherein the number average molecular weight Mn is 6169, and the polymerization degree of the polycaprolactone is 3.
Example 3
Compared with the example 1, the difference is only that the polylactic acid modified hydroxypropyl-beta-cyclodextrin prepared in the step (1) is replaced by the same amount of polylactic acid modified hydroxypropyl-beta-cyclodextrin in the step (2), and other condition parameters are the same as those of the example 1, so that the nano composition is prepared;
The preparation method of the polylactic acid modified hydroxypropyl-beta-cyclodextrin comprises the following steps: hydroxypropyl-beta-cyclodextrin (1.375 g,0.001 mol) with a substitution degree of 14 and D, L-lactide (4.84 g,0.03 mol) are sequentially added into a reaction bottle, stannous octoate (0.048 g,0.12 mmol) is added, the reaction system is kept at 130 ℃ for reaction for 12 hours under the protection of nitrogen, and after the reaction is finished, 50mL of CH is added into the reacted material 2 Cl 2 Until the materials are completely dissolved, dripping the dissolved materials into 500mL of n-hexane for purification, and collecting the solid sedimentDrying the solid precipitate to obtain polylactic acid modified hydroxypropyl-beta-cyclodextrin, wherein the number average molecular weight Mn is 5407, and the polymerization degree of polylactic acid is 2.
Example 4
Compared with the example 1, the difference is that the benzyl p-nitro chloroformate modified chitosan prepared in the step (1) is replaced by the benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin in the step (2) with the same amount of benzyl p-nitro chloroformate modified chitosan, and other condition parameters are the same as those in the example 1, so that the nano composition is prepared;
the preparation method of the p-nitro benzyl chloroformate modified chitosan comprises the following steps: chitosan (1.526 g,0.001 mol), benzyl p-nitrochloroformate (3.803 g,0.016 mol), 4-aminobutyric acid (1.605 g,0.016 mol), dicyclohexylcarbodiimide (3.177 g,0.015 mol) and 4-dimethylaminopyridine (0.188 g,0.0015 mol) are added into a reaction flask, 30mL of anhydrous N, N-dimethylformamide is added, the reaction system is kept at 30 ℃ for reaction for 48 hours under the protection of nitrogen, after the reaction is finished, the reacted materials are transferred into a dialysis bag with MWCO of 1000Da, deionized water is used for dialysis for 48 hours, and the materials in the dialysis bag are subjected to freeze drying to prepare benzyl p-nitrochloroformate modified chitosan, and the number average molecular weight Mn of the benzyl p-nitrochloroformate modified chitosan is 5483.
Example 5
Compared with the example 1, the difference is only that the polycaprolactone-modified chitosan prepared in the step (1) is replaced by the same amount of polycaprolactone-modified chitosan by benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin in the step (2), and other condition parameters are the same as those of the example 1, so that the nano composition is prepared;
the preparation method of the polycaprolactone modified chitosan comprises the following steps: sequentially adding chitosan (1.526 g,0.001 mol) and epsilon-caprolactone (5.745 g,0.05 mol) into a reaction bottle, adding stannous octoate (0.057 g,0.14 mmol), reacting for 12h at 130 ℃ under the protection of nitrogen, and adding 50mL of CH into the reacted material after the reaction 2 Cl 2 Until the materials are completely dissolved, dripping the dissolved materials into 500mL of n-hexane for purification, collecting solid precipitate, drying the solid precipitate to obtain polycaprolactone modified chitosan, and obtaining the number average fractionThe Mn molecular weight was 6320 and the degree of polymerization of polycaprolactone was 5.
Example 6
Compared with the embodiment 1, the method is characterized in that the polylactic acid modified chitosan prepared in the step 1 is replaced by the same amount of polylactic acid modified chitosan by benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin in the step 2, and other condition parameters are the same as those of the embodiment 1, so that the nano composition is prepared;
The preparation method of the polylactic acid modified chitosan comprises the following steps: sequentially adding chitosan (1.526 g,0.001 mol) and D, L-lactide (4.84 g,0.03 mol) into a reaction bottle, adding stannous octoate (0.048 g,0.12 mmol), reacting for 12h at 130 ℃ under the protection of nitrogen, and adding 50mL of CH into the reacted material after the reaction 2 Cl 2 And (3) until the materials are completely dissolved, dropwise adding the dissolved materials into 500mL of n-hexane for purification, collecting solid precipitate, and drying the solid precipitate to obtain polylactic acid modified chitosan with the number average molecular weight Mn of 5558 and the polylactic acid polymerization degree of 3.
Example 7
The difference compared with example 1 is that the hydrophobic drug phenethyl resorcinol in step (2) is replaced with an equivalent amount of 4-butyl resorcinol, and other conditions are the same as in example 1, to prepare the nano composition.
Example 8
The difference compared with example 1 is that the hydrophobic drug phenethyl resorcinol in step (2) is replaced with an equal amount of tocopherol, and other conditions are the same as in example 1, to prepare a nano composition.
Example 9
The difference compared with example 1 is that the hydrophobic drug phenethyl resorcinol in step (2) is replaced with an equal amount of methyl salicylate, and other conditions and parameters are the same as in example 1, so as to prepare the nano composition.
Example 10
The difference from example 1 is that the hydrophobic drug phenethyl resorcinol in step (2) is replaced with the same amount of glabridin, and other conditions are the same as in example 1, to prepare the nano composition.
Example 11
The difference compared with example 1 is only that in step (2), 1g of benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin and 2g of phenethyl resorcinol are replaced with 2g of benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin and 1g of phenethyl resorcinol, and the amount of deionized water is 77g, and other condition parameters are not changed, so that the nano composition is prepared.
Example 12
The difference compared with example 1 is that in the step (2), 2g of phenethyl resorcinol is replaced with 1g of phenethyl resorcinol, the addition amount of deionized water is 78g, and other condition parameters are not changed, so that the nano composition is prepared.
Example 13
The difference compared with example 1 is that in the step (2), 2g of phenethyl resorcinol is replaced with 4g of phenethyl resorcinol, the addition amount of deionized water is 75g, and other condition parameters are not changed, so that the nano composition is prepared.
Example 14
(1) The preparation method of the polycaprolactone modified hydroxypropyl-beta-cyclodextrin comprises the following steps: hydroxypropyl-beta-cyclodextrin (1.375 g,0.001 mol) with a substitution degree of 14 and epsilon-caprolactone (5.745 g,0.05 mol) are sequentially added into a reaction bottle, stannous octoate (0.057 g,0.14 mmol) is added, the reaction system is kept at 130 ℃ for reaction for 12 hours under the protection of nitrogen, and after the reaction is finished, 50mL of CH is added into the reacted material 2 Cl 2 The materials are completely dissolved, the dissolved materials are dripped into 500mL of n-hexane for purification, solid precipitate is collected, and the solid precipitate is dried to obtain polycaprolactone modified hydroxypropyl-beta-cyclodextrin with the number average molecular weight Mn of 6169;
(2) Adding the prepared 1g of polycaprolactone-modified hydroxypropyl-beta-cyclodextrin and 2g of phenethyl resorcinol into 35g of dimethyl sulfoxide, magnetically stirring for 30min at a stirring speed of 150rpm, and preparing a material A after the polycaprolactone-modified hydroxypropyl-beta-cyclodextrin and the phenethyl resorcinol are completely dissolved; and (3) dropwise adding the material A into 77g of deionized water under the condition of stirring, wherein the stirring rotation speed is 300rpm, the dropwise adding speed is 0.5mL/min, performing ultrasonic treatment for 30min under the condition of 700W after the dropwise adding is finished, transferring the ultrasonic material into a dialysis bag with the MWCO of 3000, dialyzing in the deionized water for 48h, and filtering by an organic filter membrane with the pore diameter of 0.45 mu m after the dialyzing is finished to obtain the nano composition.
During the development process, the efficacy of the nano composition prepared in this example was found to be more similar to that of example 2.
Example 15
(1) The preparation method of the polycaprolactone modified hydroxypropyl-beta-cyclodextrin comprises the following steps: hydroxypropyl-beta-cyclodextrin (1.375 g,0.001 mol) with a substitution degree of 14 and epsilon-caprolactone (5.745 g,0.05 mol) are sequentially added into a reaction bottle, stannous octoate (0.057 g,0.14 mmol) is added, the reaction system is kept at 130 ℃ for reaction for 12 hours under the protection of nitrogen, and after the reaction is finished, 50mL of CH is added into the reacted material 2 Cl 2 The materials are completely dissolved, the dissolved materials are dripped into 500mL of n-hexane for purification, solid precipitate is collected, and the solid precipitate is dried to obtain polycaprolactone modified hydroxypropyl-beta-cyclodextrin with the number average molecular weight Mn of 6169;
(2) Adding the prepared 1g of polycaprolactone-modified hydroxypropyl-beta-cyclodextrin and 2g of decarboxylated carnosine into 50g of diethylene glycol, magnetically stirring for 30min at a stirring speed of 150rpm, and preparing a material A after the polycaprolactone-modified hydroxypropyl-beta-cyclodextrin and the decarboxylated carnosine are completely dissolved; and (3) dropwise adding the material A into 77g of deionized water under the condition of stirring, wherein the stirring rotating speed is 150rpm, the dropwise adding speed is 2mL/min, performing ultrasonic treatment for 30min under the condition of 300W after the dropwise adding is finished, transferring the ultrasonic material into a dialysis bag with the MWCO of 1000, dialyzing in the deionized water for 48h, and filtering by an organic filter membrane with the pore diameter of 0.45 mu m after the dialyzing is finished, so as to obtain the nano composition.
During the development process, the efficacy of the nano composition prepared in this example was found to be more similar to that of example 8.
Example 16
(1) Preparation of benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin: hydroxypropyl-beta-cyclodextrin (1.375 g, 0.001mol) with the substitution degree of 14, benzyl p-nitrochloroformate (3.803 g,0.016 mol), 4-aminobutyric acid (1.605 g,0.016 mol), dicyclohexylcarbodiimide (3.177 g,0.015 mol) and 4-dimethylaminopyridine (0.188 g,0.0015 mol) are added into a reaction bottle, 30mL of anhydrous N, N-dimethylformamide is added, the reaction system is kept at 30 ℃ for 48 hours under the protection of nitrogen, after the reaction, the reacted materials are transferred into a dialysis bag with the molecular weight MWCO of 1000Da, deionized water is used for dialysis for 48 hours, and the materials in the dialysis bag are subjected to freeze drying to prepare the benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin with the number average molecular weight Mn of 5332;
(2) Adding the prepared 1g of p-nitro benzyl chloroformate modified hydroxypropyl-beta-cyclodextrin, 0.5g of phenethyl resorcinol, 0.5g of glabridin, 0.5g of tocopherol and 0.5g of deacidified carnosine into 20g of 1, 3-propanediol, magnetically stirring for 30min, wherein the stirring speed is 150rpm, and completely dissolving to obtain a material A; and (3) dropwise adding the material A into 77g of deionized water under the condition of stirring, wherein the stirring rotating speed is 150rpm, the dropwise adding speed is 1mL/min, performing ultrasonic treatment for 30min under the condition of 500W after the dropwise adding is finished, transferring the ultrasonic material into a dialysis bag with the MWCO of 1000, dialyzing in the deionized water for 48h, and filtering by an organic filter membrane with the pore diameter of 0.45 mu m after the dialyzing is finished to prepare the nano composition.
During the development process, the efficacy of the nano composition prepared in this example was found to be more similar to that of example 7.
Application example 1
The preparation method of the skin external preparation with the beauty effect comprises the following steps:
adding the prepared 1.875g of benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin, 5g of phenethyl resorcinol, 1g of tocopherol, 1g of glabridin and 0.5g of deacidified carnosine into 20g of 1, 3-propanediol, magnetically stirring for 30min at a stirring speed of 150rpm, and preparing a material A after the benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin and the phenethyl resorcinol are completely dissolved; dropwise adding the material A into 47.625g of deionized water under the condition of stirring, wherein the stirring rotating speed is 150rpm, the dropwise adding speed is 1mL/min, performing ultrasonic treatment for 30min under the condition of 500W after the dropwise adding, transferring the ultrasonic material into a dialysis bag with the MWCO of 1000, dialyzing in the deionized water for 48h, and filtering by an organic filter membrane with the aperture of 0.45 mu m after the dialyzing is finished to prepare a nano composition;
The prepared nano composition is mixed with 6g of caprylic/capric triglyceride, 5g of 1, 3-propylene glycol, 1g of 1, 2-hexanediol, 10g of glycerol, 3g of poloxamer 188, 8g of polysorbate-80, 5g of polyglycerol-10 laurate and 5g of caprylic/capric glyceride polyglycerol-10 ester to prepare the skin external preparation with the beautifying effect.
Comparative example 1
The only difference compared to example 1 is that 1g of benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin was replaced with 1g of polyurethane (petroleum-based polymer, not having biocompatibility), and other condition parameters were unchanged, to prepare a nanocomposite.
Comparative example 2
The only difference compared to example 1 is that 1g of benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin was replaced with 1g of beta-cyclodextrin, and other condition parameters were unchanged, to prepare a nano composition.
Comparative example 3
The only difference compared to example 1 is that 1g of benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin was replaced with 1g of hydroxypropyl-beta-cyclodextrin, and other condition parameters were unchanged, to prepare a nano composition.
Comparative example 4
Compared with example 1, the difference is that 1g of p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin is replaced by 1g of N-alkylation modified chitosan (the preparation method of the N-alkylation modified chitosan refers to the preparation of N-alkylation chitosan/bletilla striata polysaccharide composite material, guo Liangliang and the like, the application chemistry is 2020,49 (03), 632-637), and other condition parameters are unchanged, so that the nano composition is prepared.
Comparative example 5 (no dialysis procedure)
The only difference compared to example 1 is the operation of step (2), specifically: adding 1g of benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin and 2g of phenethyl resorcinol into 20g of 1, 3-propanediol, magnetically stirring for 30min at a stirring speed of 150rpm, and completely dissolving the benzyl p-nitro chloroformate modified hydroxypropyl-beta-cyclodextrin and the phenethyl resorcinol to obtain a material A; and (3) dropwise adding the material A into 77g of deionized water under the stirring condition, wherein the stirring rotating speed is 150rpm, the dropwise adding speed is 1mL/min, and after the dropwise adding is finished, carrying out ultrasonic treatment under the condition of 500W for 30min, and filtering through an organic filter membrane with the pore diameter of 0.45 mu m after ultrasonic treatment to obtain the nano composition.
Comparative example 6
The only difference compared to example 1 is the operation of step (2), specifically: adding 1g of p-nitro benzyl chloroformate modified hydroxypropyl-beta-cyclodextrin into 20g of 1, 3-propanediol, magnetically stirring for 30min at a stirring speed of 150rpm, and obtaining a material B after complete dissolution; dropwise adding the material B into 77g of deionized water under the condition of stirring, wherein the stirring rotating speed is 150rpm, the dropwise adding speed is 1mL/min, performing ultrasonic treatment for 30min under the condition of 500W after the dropwise adding is finished, transferring the ultrasonic material into a dialysis bag with the MWCO of 1000, and performing dialysis in the deionized water for 48h to prepare a blank polymer micelle solution; 2g of phenethyl resorcinol is added into 10g of acetone, after complete dissolution, the mixture is added into a blank polymer micelle solution, and the mixture is magnetically stirred for 30min under the condition of 150rpm, and after the acetone volatilizes, the nano composition is obtained.
Comparative example 7
0.9g of soybean lecithin, 0.1g of cholesterol and 2g of phenethyl resorcinol are added into 20g of 1, 3-propanediol, and the mixture is magnetically stirred for 3 hours at 60 ℃ at a stirring speed of 150rpm to obtain a mixed solution; removing 1, 3-propanediol from the above mixed solution by rotary evaporation (40 ℃ C., rotation speed of 250 rpm); after the 1, 3-propanediol is completely removed, adding the mixture into 77g of deionized water, carrying out ultrasonic dispersion treatment for 30min, wherein the ultrasonic power is 500w, and filtering the ultrasonic material through an organic phase filter membrane with the pore diameter of 0.45 mu m to obtain liposome solution of the wrapped phenethyl resorcinol.
Comparative example 8
2.0g of phenethyl resorcinol and 15.0g of caprylic/capric triglyceride are mixed and heated to 85 ℃ until the phenethyl resorcinol is completely dissolved, and then added into 20g of 1, 3-propanediol to obtain an oil phase component; and (3) dropwise adding 63g of deionized water into the oil phase component through a peristaltic pump at the speed of 1mL/min, shearing at a high speed under the condition that the shearing speed is 15000rpm after the dropwise adding is finished, homogenizing at a high pressure under the condition of 1000bar, and carrying out high-pressure homogenizing circulation treatment for 5 times to obtain the nano emulsion coated with the phenethyl resorcinol.
Comparative example 9
The difference compared with example 1 is that in the step (2), 2g of phenethyl resorcinol is replaced with 5g of phenethyl resorcinol, the addition amount of deionized water is 74g, and other condition parameters are not changed, so that the nano composition is prepared.
Effect example 1
The particle sizes of the samples prepared in examples 1-13 and comparative examples 1-9 above were characterized using a dynamic light scattering particle size analyzer (DLS) model BI-200SM, brookhaven Instruments Corporation, USA, and the results are shown in Table 1.
The samples (test solutions) prepared in examples 1 to 13 and comparative examples 1 to 9 were subjected to encapsulation efficiency measurement.
Encapsulation efficiency determination method: 200 mu L of the liquid to be detected is taken, the liquid to be detected is subjected to ultrafiltration centrifugation (9000 rpm,30 min) to obtain filtrate, and 5 mu L of the filtrate is taken to measure the content of the hydrophobic drug in the filtrate by a high performance liquid chromatograph (HPLC, shimadzu), namely the content of the free hydrophobic drug which is not coated in the liquid to be detected. And adding a methanol solution into the liquid to be measured according to the liquid to be measured: methanol=1:9 (v/v) ultrasonic demulsification is carried out for 30min, after filtration by a 0.45 mu m organic phase filter membrane, 5 mu L of sample solution is taken, and the content of the hydrophobic drugs is measured by a high performance liquid chromatograph (HPLC, shimadzu), namely the total content of the hydrophobic drugs in the liquid to be measured. The Encapsulation Efficiency (EE) of the nanocarriers to the hydrophobic drug was calculated according to formula 1. The analytical column used in the HPLC system was a nonpolar C18 column, the mobile phase was acetonitrile, the flow rate was 1.0mL/min, the column temperature of the chromatographic column was 30deg.C, three parallel experiments were performed for each group, and the arithmetic average value was obtained for the experimental results, as shown in Table 1.
EE(%)=(1-C 1 /C 0 ) X 100% (formula 1);
C 1 indicating the concentration of the uncoated free hydrophobic drug in the test solution; c (C) 0 Indicating the total concentration of the hydrophobic drugs in the liquid to be tested after methanol ultrasonic demulsification.
Samples of examples 1, 2 and 7 were taken and dropped onto a 200 mesh copper mesh for a transmission electron microscope coated with a carbon film, and left standing at room temperature until the solvent was completely volatilized, and the microscopic morphology of the nanocarrier was characterized by using a transmission electron microscope of the type HT700, hitachi, ltd, japan, with an acceleration voltage of 120kV, and a comparative graph shown in FIG. 1, wherein (A), (B) and (C) in FIG. 1 correspond to those of examples 1, 2 and 7, respectively, to prepare a transmission electron microscope graph of the nanocapsules.
TABLE 1
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As is apparent from the comparison between the results of example 1 and comparative example 1, the particle size of the nano-micelle obtained by encapsulation of the hydrophobic drug with the amphiphilic block copolymer having biocompatibility of example 1 of the present disclosure is smaller and the encapsulation efficiency is higher than that of the petroleum-based polymer.
As is clear from the comparison between the results of example 1 and comparative examples 2 to 4, when the hydrophobic drug is coated with beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and N-alkylated modified chitosan, the obtained nano-micelle has a larger particle size and a lower encapsulation efficiency. The particle size of the nano micelle prepared by the embodiment of the disclosure can be controlled between 100nm and 200nm, and the encapsulation efficiency of the nano micelle is higher.
As is clear from comparing the results of example 1 and comparative example 5, the step of the dialysis treatment affects the particle size and encapsulation efficiency of the nano-micelle. The encapsulation efficiency of the nano micelle which is not subjected to dialysis treatment is only 62.4%, and the encapsulation efficiency is obviously reduced.
As is apparent from the comparison between the results of example 1 and comparative example 6, the preparation method has a large influence on the particle size and encapsulation efficiency of the finally prepared nano-micelle. The test result shows that the nano micelle prepared by the method disclosed by the invention has smaller particle size and larger encapsulation efficiency, and is more suitable for the field of cosmetics.
As is evident from comparing the results of example 1 and comparative examples 7 and 8, the kind of nanocarriers has a large influence on the particle size and encapsulation efficiency of the final product. Compared with the traditional liposome and nanoemulsion, the amphiphilic block copolymer of the specific type disclosed by the disclosure has smaller particle size and higher encapsulation rate when being used for wrapping hydrophobic drugs such as retinol, squalane, tocopherol, decarboxylated carnosine, 4-butylresorcinol, methyl salicylate, glabridin, phenethyl resorcinol and the like.
As is clear from the comparison of the results of examples 1 to 6 and comparative example 4, the kind of amphiphilic block copolymer affects the particle size and encapsulation efficiency of the nano-micelle. Test results show that the benzyl p-nitro chloroformate modified water-soluble polysaccharide has stronger coating capability on hydrophobic drugs, and the particle size of the prepared nano micelle is smaller. Comparative example 4 although the hydrophilic polysaccharide was also hydrophobically modified, the modifier thereof was not a hydrophobic ester modifier and a hydrophobic polyester modifier, and the coating ability of the prepared nano micelle to the hydrophobic drug was significantly inferior to the examples of the present disclosure.
As is clear from comparison of the results of examples 1 and examples 7 to 10, the type of hydrophobic drug has a certain effect on the particle size and encapsulation efficiency of the nano-micelle. Examples 1, 7-8 and 10 are the entrapment of benzyl p-nitrochloroformate modified hydroxypropyl-beta-cyclodextrin on phenethyl resorcinol, 4-butyl resorcinol, tocopherol, methyl salicylate and glabridin, respectively, showing that the encapsulation rates of the samples of examples 1 and 10 were close and high (94.3% and 93.7%, respectively) and the particle sizes of the corresponding samples were small (100.2 nm and 118.4nm, respectively). It is hypothesized that the molecular structure of the medicine is related to that phenethyl resorcinol and glabridin are in a double benzene ring structure, 4-butyl resorcinol, tocopherol and methyl salicylate are in a single benzene ring structure, and the benzyl p-nitro chloroformate modified hydrophilic polysaccharide is easier to coat the hydrophobic medicine containing the double benzene ring structure. In the research and development process, the encapsulation rate of the benzyl p-nitro chloroformate modified hydrophilic polysaccharide on the hydrophobic drug containing the benzene ring structure is obviously higher than that of the benzyl p-nitro chloroformate modified hydrophilic polysaccharide on the linear-structure hydrophobic drug.
As is clear from comparing the results of examples 1, 11 to 13 and comparative example 9, as the mass ratio of the amphiphilic block copolymer to the hydrophobic drug increases, the particle size of the prepared nano micelle changes, the encapsulation efficiency tends to increase and then decrease, and when the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1:2, the encapsulation efficiency of the sample is 94.3% at most; when the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1:5, the particle size of the polymer micelle is increased sharply, and the encapsulation efficiency is decreased sharply. Therefore, the present disclosure is suitable for controlling the mass ratio of amphiphilic block copolymer to hydrophobic drug at 1:0.5-4, and the effect is optimal when controlling at 1:1-2.
Effect example 2
5mL of the samples prepared in examples 1 to 13 and comparative examples 1 to 9 were placed in dialysis bags (MWCO 3500-4000 Da), respectively, the dialysis bags were sealed, the samples were transferred to a drug eluting apparatus, and PBS buffer solution (495 mL) containing 1% Tween-20 was added. The temperature was set at 37℃and the stirring speed was 150rpm, 5mL of each sample was taken at regular intervals, and 5mL of PBS buffer containing 1% Tween-20 was simultaneously added. The concentration of the drug in the sample solution was measured by high performance liquid chromatography (HPLC, shimadzu) to obtain the drug release profile of the nanocarrier, and the results are shown in table 2 and fig. 2 to 5.
TABLE 2
The results of the drug release experiments of examples 1 to 6 are shown in FIG. 2. As can be seen from fig. 2, the sample of example 1 has the slowest drug release rate, followed by examples 2 and 4. The results show that the nano-micelles prepared in examples 1-6 of the present disclosure have ideal slow release and release effects.
The results of the drug release experiments of example 1 and examples 7 to 9 are shown in FIG. 3. As can be seen from fig. 3, the kind of the entrapped drug affects the release effect of the drug. The slow release effect of the entrapped phenethyl resorcinol is best, and the slow release effect of the entrapped methyl salicylate is relatively poor.
The results of the drug release experiments of example 1, examples 11 to 13 and comparative example 9 are shown in fig. 4. As can be seen from fig. 4, the mass ratio of the amphiphilic block copolymer to the hydrophobic drug affects the drug release effect. When the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1:2, the slow release effect of the drug is the best; when the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1:5, the slow release effect of the drug is the worst.
The results of the drug release experiments of example 1 and comparative examples 1 to 6 are shown in FIG. 5. As can be seen from fig. 5, the hydrophobic modification treatment before the hydrophilic polysaccharide encapsulation, the preparation method of the nanocarriers, the dialysis treatment, the kinds of the nanocarriers, and the like affect the slow release effect of the drug. The sustained-release effect of example 1 is obviously better than that of comparative examples 1 to 6, which shows that in the present disclosure, the sustained-release effect of the specific type of amphiphilic block copolymer coated with the hydrophobic drug is obviously better than that of the liposome and nanoemulsion, the sustained-release effect of the hydrophilic polysaccharide after hydrophobic modification is better than that of the hydrophilic polysaccharide without hydrophobic modification treatment, and the sustained-release effect of the nanomicelle prepared by the preparation method of the present disclosure is better than that of the nanomicelle prepared by other methods.
Effect example 4
The effect of the nano-micelles prepared in examples 1 to 6 and comparative examples 1 to 8 on the transdermal absorption (permeation promotion) ability of the hydrophobic drugs was evaluated using a transdermal absorption (permeation promotion) test.
The specific method comprises the following steps:
in vitro transdermal experiments, a vertical diffusion cell was used, and nude mice skin was used as a model (abdomen skin, removal of subcutaneous fat layer and blood vessel). The receiving solution was phosphate buffer solution (PBS solution). The skin patch was held between the supply and receiving wells with the skin layer facing upward and equilibrated for 20min. The sample liquid is added into the supply tank, and the receiving liquid is taken at 1h, 2h, 4h, 6h, 8h and 24 h. The content of phenethyl resorcinol in the receiving liquid was measured by high performance liquid chromatography (HPLC, shimadzu) and the cumulative amount of permeation per unit area was calculated therefrom, and the results are shown in table 3.
The calculation formula of the cumulative transmission per unit area on the skin sheet is as follows:
wherein Qn is the cumulative transmission per unit area (μg/cm) of the sample at time t 2 ) A is the penetration area, C n For the measurement of the t-time concentration, C i For the concentration measurement before time t, V is the total volume of the receiving liquid, V 0 Is the sampling volume.
TABLE 3 Table 3
From the results of table 3, it can be seen that both the kind and the preparation method of the amphiphilic block copolymer affect the transdermal absorption effect of the hydrophobic drug.
Examples 1 to 6 have better transdermal absorption effects in 1h, 4h, 8h and 24h than comparative examples 1 to 4, showing that the amphiphilic block copolymer of the specific type of the present disclosure has higher matching with the hydrophobic drug, thereby improving the transdermal absorption effect of the hydrophobic drug.
Example 1 shows that dialysis absorption effects at 1h, 4h, 8h and 24h are better than comparative example 5 and comparative example 6, indicating that dialysis treatment and nanoparticle addition order affect transdermal absorption effects of hydrophobic drugs. In the research and development process, the nano micelle prepared by adopting the preparation method disclosed by the invention to coat the hydrophobic drugs with the linear molecular structures such as retinol, squalane, tocopherol, decarboxylated carnosine and the like, coat the hydrophobic drugs with the single benzene ring molecular structures such as 4-butylresorcinol, methyl salicylate and the like, and coat the hydrophobic drugs with the multi-benzene ring molecular structures such as glabridin, phenethyl resorcinol and the like has good transdermal absorption effect.
Example 1 shows that the transdermal absorption effect at 1h, 4h, 8h and 24h are better than those of comparative examples 7 and 8, and the variety of nanocarriers affects the transdermal absorption effect of the hydrophobic drug. The transdermal absorption effect of the nanoparticle prepared by coating the hydrophobic drug with the amphiphilic block copolymer of the specific type adopted by the present disclosure is obviously superior to that of the conventional nanoparticle.
Effect example 5
(1) The biocompatibility of blank example 1, blank comparative example 7, blank comparative example 8 was evaluated using an in vitro cytotoxicity test.
On the basis of the preparation steps of example 1, comparative example 7 and comparative example 8, blank nanocarriers without drug loading were prepared, blank example 1, blank comparative example 7 and blank comparative example 8, respectively. The method comprises the following steps:
blank preparation of example 1: adding 1g of p-nitro benzyl chloroformate modified hydroxypropyl-beta-cyclodextrin into 20g of 1, 3-propanediol, magnetically stirring for 30min at a stirring speed of 150rpm, and preparing a mixed solution after complete dissolution; and (3) dropwise adding the mixed solution into 79g of deionized water under the condition of stirring, wherein the stirring rotating speed is 150rpm, the dropwise adding speed is 1mL/min, performing ultrasonic treatment for 30min under the condition of 500W after the dropwise adding, transferring the ultrasonic material into a dialysis bag with the MWCO of 1000Da, dialyzing in the deionized water for 48h, and filtering by an organic filter membrane with the aperture of 0.45 mu m after the dialyzing.
Preparation of blank comparative example 1: 1g of p-nitro-benzyl chloroformate-modified hydroxypropyl-beta-cyclodextrin in blank example 1 was replaced with 1g of polyurethane (petroleum-based polymer), and the remainder was unchanged.
Preparation of blank comparative example 7: adding 0.9g of soybean lecithin and 0.1g of cholesterol into 20g of 1, 3-propanediol, magnetically stirring for 3 hours at 60 ℃ at a stirring speed of 150rpm to obtain a mixed solution; removing 1, 3-propanediol from the above mixed solution by rotary evaporation (40 ℃ C., rotation speed of 250 rpm); after the 1, 3-propanediol is completely removed, adding the mixture into 79g of deionized water, carrying out ultrasonic dispersion treatment for 30min, wherein the ultrasonic power is 500w, and filtering the ultrasonic material by an organic phase filter membrane with the pore diameter of 0.45 mu m.
Preparation of blank comparative example 8: 15.0g of caprylic/capric triglyceride is added into 20g of 1, 3-propylene glycol to obtain an oil phase component; and (3) dripping 65g of deionized water into the oil phase component through a peristaltic pump at the speed of 1mL/min, performing high-speed shearing at the shearing speed of 15000rpm after dripping, and performing high-pressure homogenization at the shearing speed of 1000bar for 5 times.
In vitro cytotoxicity test using CCK-8 experimental method and mouse fibroblast (NIH-3T 3 cell) as model. NIH-3T3 cells (density 3000 cells/well) were seeded in 96-well plates, DMEM medium (10% FBS and 1% P/S) was added, and NIH-3T3 cells were placed at 37℃and 5% CO 2 Is incubated for 24h in a cell incubator. The experimental group was removed of the medium, replaced with a medium solution containing the above test sample, and the concentration of the medium solution containing the above sample was 3.7. Mu.g/mL, 11.11. Mu.g/mL, 33.33. Mu.g/mL, 100. Mu.g/mL and 300. Mu.g/mL, respectively (taking 3.7. Mu.g/mL of the medium solution containing blank example 1 as an example, meaning that the content of the product produced in blank example 1 contained in 1mL of the medium solution was 3.7. Mu.g), and the culture was continued in the cell incubator for 24 hours. The control group was removed from the medium and replaced with a new medium solution, and the culture was continued in the cell incubator for 24 hours. Blank groups were set, i.e., cells were not cultured in 96-well plates, and only blank medium was added. CCK-8 solvent (10. Mu.L/well) was added to the 96-well plate and incubated for another 4h. The absorbance at 450nm of the solution in each well was measured by a microplate reader and the viability of the NIH-3T3 cells was calculated using the following formula.
Wherein Asample represents the absorbance of the experimental group; acontrol represents the absorbance of the control group; ablank represents absorbance of the blank group.
In vitro cytotoxicity results of blank example 1, blank comparative example 7 and blank comparative example 8 are shown in fig. 6.
The results show that the cell viability of NIH-3T3 cells after 24h co-incubation with blank example 1 at each experimental concentration was higher than 95% and higher than that of NIH-3T3 cells after co-incubation with blank comparative example 1, blank comparative example 7 and blank comparative example 8, respectively. It is explained that the wall material used in example 1 of the present disclosure has good biocompatibility and is superior to the wall material used in comparative example 1, comparative example 7 and comparative example 8.
(2) The biocompatibility of example 1, comparative example 7, comparative example 8 and phenethyl resorcinol dispersions was evaluated using the same in vitro cytotoxicity test as described above.
Preparing a phenethyl resorcinol dispersion with a phenethyl resorcinol mass percentage of 2 wt%; the nanomicelles and phenethyl resorcinol dispersions prepared in example 1, comparative example 7, and comparative example 8 were prepared as test solutions having concentrations of 3.7 μg/mL, 11.11 μg/mL, 33.33 μg/mL, 100 μg/mL, and 300 μg/mL, respectively (test solution preparation method is the same as the blank example 1 medium solution preparation method), and in vitro cytotoxicity results are shown in FIG. 7.
The results show that the cell viability of the experimental group of example 1 was significantly higher than that of the experimental group of phenethyl resorcinol after 24h co-incubation of NIH-3T3 cells with each experimental concentration of example 1 and phenethyl resorcinol, indicating that the toxicity of phenethyl resorcinol can be significantly reduced by polymer encapsulation of example 1.
After NIH-3T3 cells were co-cultured with each of the experimental concentrations of example 1, comparative example 7 and comparative example 8 for 24 hours, the experimental group of example 1 had higher cell viability than the experimental group of comparative example 1, comparative example 7 and comparative example 8, demonstrating that the effect of example 1 on reducing the toxicity of phenethyl resorcinol was superior to that of comparative example 1, comparative example 7 and comparative example 8.
Finally, it is also noted that in this disclosure the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While the disclosure has been disclosed by the foregoing description of specific embodiments thereof, it will be understood that various modifications, improvements, or equivalents may be devised by those skilled in the art that will fall within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this disclosure.
Claims (23)
1. A method for preparing a nano composition, which is characterized in that the nano composition comprises nano micelle formed by entrapment of hydrophobic drugs by amphiphilic block copolymers;
wherein the amphiphilic block copolymer is a hydrophobically modified hydrophilic polysaccharide; the hydrophobic modifier used in the hydrophobic modification process is benzyl p-nitrochloroformate, polycaprolactone or polylactic acid; the hydrophilic polysaccharide is hydroxypropyl-beta-cyclodextrin or chitosan; the hydrophobic drug is a benzene ring-containing hydrophobic drug; the benzene ring-containing hydrophobic drug is a single benzene ring-containing hydrophobic drug and/or a multi-benzene ring-containing hydrophobic drug; the hydrophobic medicine containing the single benzene ring is at least one of tocopherol, 4-butyl resorcinol and methyl salicylate; the multi-benzene ring-containing hydrophobic drug is glabridin and/or phenethyl resorcinol;
the preparation method of the nano composition comprises the following steps:
(1) The amphiphilic block copolymer, the hydrophobic drug and the solvent are mixed to prepare a material A; the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (0.5-4);
(2) And (3) dropwise adding the material A into water under the condition of stirring, performing ultrasonic treatment and dialysis to obtain the nano composition.
2. The method of preparing a nanocomposite of claim 1, wherein the method of preparing satisfies at least one of the following conditions:
the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (0.5-2);
the average particle size of the nano composition is 100-200 nm.
3. The method of preparing a nanocomposite as claimed in claim 2, wherein the method of preparing satisfies at least one of the following conditions:
the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (1-2);
the average particle size of the nano composition is 100-150 nm.
4. The method of preparing a nanocomposite of claim 1, wherein the method of preparing satisfies at least one of the following conditions:
the polymerization degree of the polycaprolactone is 2-6;
the polymerization degree of the polylactic acid is 2-6.
5. The method of preparing a nano composition according to claim 1, wherein when the hydrophobic modifier is benzyl p-nitrochloroformate, the amphiphilic block copolymer is benzyl p-nitrochloroformate modified hydrophilic polysaccharide, and the hydrophobic drug is a benzene-ring-containing hydrophobic drug.
6. The method of preparing a nanocomposite as claimed in claim 5, wherein when the hydrophobic modifier is benzyl p-nitrochloroformate, the amphiphilic block copolymer is benzyl p-nitrochloroformate modified hydrophilic polysaccharide, and the hydrophobic drug is a multi-benzene ring-containing hydrophobic drug.
7. The method of preparing a nano composition according to claim 1, wherein when the hydrophobic modifier is benzyl p-nitrochloroformate, the method of preparing the amphiphilic block copolymer comprises the steps of: in inert atmosphere and solvent, the hydrophilic polysaccharide, the benzyl p-nitrochloroformate and the connector react under the action of a catalyst A and a condensing agent to prepare benzyl p-nitrochloroformate modified hydrophilic polysaccharide; the reaction includes an esterification reaction and an amidation reaction.
8. The method of preparing a nanocomposite as claimed in claim 7, wherein when the hydrophobic modifier is benzyl p-nitrochloroformate, the method of preparing the amphiphilic block copolymer satisfies at least one of the following conditions:
the solvent comprises N, N-dimethylformamide;
the connector is a compound with at least one amino group and one carboxyl group as end groups;
The condensing agent comprises at least one of dicyclohexylcarbodiimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and diisopropylcarbodiimide;
the catalyst A comprises 4-dimethylaminopyridine;
the temperature of the reaction is 25-50 ℃;
the reaction time is 24-72 h;
the reaction further comprises dialysis and/or drying operation after the reaction is finished;
when the hydrophilic polysaccharide is the hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the benzyl p-nitrochloroformate is 1: (14.7-17.5);
when the hydrophilic polysaccharide is the hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the linker is 1: (14.7-17.5);
when the hydrophilic polysaccharide is the hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the condensing agent is 1: (14.7-17.5);
when the hydrophilic polysaccharide is the hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the catalyst a is 1: (1.47-1.75);
when the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan to the benzyl p-nitrochloroformate is 1: (10.5-16);
When the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan to the linker is 1: (10.5-16);
when the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan to the condensing agent is 1: (10.5-15);
when the hydrophilic polysaccharide is the chitosan, the molar ratio of the chitosan to the catalyst A is 1: (1.05-1.5).
9. The method of preparing a nano composition according to claim 8, wherein when the hydrophobic modifier is benzyl p-nitrochloroformate, the linker comprises at least one of 4-aminobutyric acid, 5-aminopentanoic acid, and β -aminopropionic acid.
10. The method of preparing a nanocomposite according to claim 1, wherein when the hydrophobic modifier is the polycaprolactone, the method of preparing the amphiphilic block copolymer comprises the steps of: in inert atmosphere, the hydrophilic polysaccharide and epsilon-caprolactone undergo ring-opening polymerization reaction under the action of a catalyst B to prepare the polycaprolactone modified hydrophilic polysaccharide.
11. The method of preparing a nanocomposite of claim 10, wherein when the hydrophobic modifier is the polycaprolactone, the method of preparing the amphiphilic block copolymer satisfies at least one of the following conditions:
The catalyst B comprises stannous octoate;
the catalyst B accounts for 0.5-2% of the epsilon-caprolactone in percentage by mass;
the temperature of the ring-opening polymerization reaction is 100-150 ℃;
the time of the ring-opening polymerization reaction is 6-24 hours;
the operation of ring-opening polymerization reaction is further followed by purification and/or drying operation;
when the hydrophilic polysaccharide is hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the epsilon-caprolactone is 1: (33.6-100.8);
when the hydrophilic polysaccharide is chitosan, the molar ratio of the chitosan to the epsilon-caprolactone is 1: (48-144).
12. The method of preparing a nanocomposite of claim 11, wherein when the hydrophobicity-modifying agent is the polycaprolactone, the amphiphilic blockIn the preparation process of the copolymer, the purification comprises the following steps: CH is added into the material after the ring-opening polymerization reaction 2 Cl 2 And (3) completely dissolving the material after the ring-opening polymerization reaction, and dripping the dissolved material into n-hexane to obtain the purified polycaprolactone modified hydrophilic polysaccharide.
13. The method of preparing a nano composition according to claim 1, wherein when the hydrophobic modifier is the polylactic acid, the method of preparing the amphiphilic block copolymer comprises the steps of: in inert atmosphere, the hydrophilic polysaccharide and D, L-lactide are subjected to ring-opening polymerization reaction under the action of a catalyst C to prepare the polylactic acid modified hydrophilic polysaccharide.
14. The method of preparing a nanocomposite of claim 13, wherein when the hydrophobic modifier is the polylactic acid, the method of preparing the amphiphilic block copolymer satisfies at least one of the following conditions:
the catalyst C comprises stannous octoate;
the catalyst C accounts for 0.5-2% of the mass of the D, L-lactide;
the temperature of the ring-opening polymerization reaction is 100-150 ℃;
the time of the ring-opening polymerization reaction is 6-24 hours;
when the hydrophilic polysaccharide is hydroxypropyl-beta-cyclodextrin, the molar ratio of the hydroxypropyl-beta-cyclodextrin to the D, L-lactide is 1: (30-100.8);
when the hydrophilic polysaccharide is chitosan, the molar ratio of the chitosan to the D, L-lactide is 1: (30-144).
15. The method of preparing a nanocomposite of claim 1, wherein the amphiphilic block copolymer comprises at least one of polycaprolactone-modified hydroxypropyl- β -cyclodextrin, polylactic acid-modified hydroxypropyl- β -cyclodextrin, benzyl p-nitrochloroformate-modified hydroxypropyl- β -cyclodextrin, polycaprolactone-modified chitosan, polylactic acid-modified chitosan, and benzyl p-nitrochloroformate-modified chitosan.
16. The method of preparing a nanocomposite of claim 15, wherein the method of preparing satisfies at least one of the following conditions:
the number average molecular weight of the polycaprolactone-modified hydroxypropyl-beta-cyclodextrin is 3000-11000;
the number average molecular weight of the polylactic acid modified hydroxypropyl-beta-cyclodextrin is 4000-14000;
the number average molecular weight of the p-nitro benzyl chloroformate modified hydroxypropyl-beta-cyclodextrin is 4000-14000;
the number average molecular weight of the polycaprolactone-modified chitosan is 5000-18000;
the number average molecular weight of the polylactic acid modified chitosan is 5000-23000;
the number average molecular weight of the p-nitro benzyl chloroformate modified chitosan is 3000-8000.
17. The method of any one of claims 1 to 16, wherein the method of preparing satisfies at least one of the following conditions:
in the step (1), the solvent comprises at least one of alkane solvents, ketone solvents, furan solvents, amide solvents, sulfoxide solvents and alcohol solvents;
in the step (1), the mixing is performed under the condition of stirring, the rotating speed of the stirring is 30-300 rpm, and the stirring time is 10-60 min;
In the step (1), the total mass of the amphiphilic block copolymer and the hydrophobic drug accounts for 1-30% of the mass of the solvent;
in the step (1), the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (0.5-2);
in the step (2), the stirring rotation speed is 30-300 rpm;
in the step (2), the dropping speed is 0.5-2 mL/min;
in the step (2), the ultrasonic frequency is 300-900W;
in the step (2), the ultrasonic time is 10-60 min;
in the step (2), MWCO of a dialysis bag used for dialysis is 500-5000 Da;
in the step (2), the dialysis medium is water;
in the step (2), the dialysis time is 24-72 hours;
in the step (2), the dialysis operation further comprises a filtration operation, wherein the pore diameter of a filter membrane adopted by the filtration is 0.2-0.8 mu m.
18. The method of preparing a nanocomposite of claim 17, wherein the method of preparing satisfies at least one of the following conditions:
in the step (1), the total mass of the amphiphilic block copolymer and the hydrophobic drug accounts for 6-15% of the mass of the solvent;
In the step (1), the mass ratio of the amphiphilic block copolymer to the hydrophobic drug is 1: (1-2);
in the step (2), the dropping speed is 0.5-1 mL/min;
in the step (2), the ultrasonic frequency is 500-700W;
in the step (2), the ultrasonic time is 30min;
in the step (2), the MWCO of the dialysis bag used for dialysis is 1000-3000 Da.
19. A nano-composition prepared by the method of preparing a nano-composition according to any one of claims 1 to 18.
20. Use of the nano composition according to claim 19 as a raw material for preparing a skin external preparation.
21. A cosmetic skin external preparation comprising the nano-composition according to claim 19.
22. The external preparation for skin according to claim 21, wherein the external preparation for skin having a cosmetic effect further comprises at least one of an emulsion stabilizer, a surfactant, an antifreezing agent and a solvent.
23. The skin external preparation according to claim 22, wherein the skin external preparation satisfies at least one of the following conditions:
the emulsion stabilizer comprises caprylic/capric triglyceride and/or caprylic/capric glyceride polyglycerol-10 ester;
The surfactant comprises at least one of poloxamer 188, polysorbate-80 and polyglycerol-10 laurate;
the antifreeze agent includes glycerin;
the solvent comprises at least one of 1, 3-propanediol, 1, 2-hexanediol and water;
the weight part ratio of the hydrophobic drug to the emulsion stabilizer in the nano composition is 1: (0.2-10);
the weight part ratio of the hydrophobic drug to the surfactant in the nano composition is 1: (0.67 to 22.5);
the weight part ratio of the hydrophobic drug to the antifreezing agent in the nano composition is 1: (0.48 to 7.5);
the weight part ratio of the hydrophobic drug to the solvent in the nano composition is 1: (1-40).
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