CN114225046A

CN114225046A - Antibacterial/healing-promoting dual-drug delivery system, drug composition, preparation method and application thereof

Info

Publication number: CN114225046A
Application number: CN202111563204.2A
Authority: CN
Inventors: 马晓溦; 朱奎; 沈建忠; 赵谦; 陈莹娴; 韩军华
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-03-25

Abstract

The invention provides an antibacterial/healing-promoting dual-drug delivery system, a drug composition, a preparation method and an application thereof. The outer layer of the antibacterial/pro-healing dual drug delivery system comprises a hydrophilic segment, the inner core comprises a hydrophobic segment, and the MMP-9 responsive peptide is embedded between the hydrophilic segment and the hydrophobic segment. The antibacterial/healing-promoting dual-drug delivery system provided by the invention can respond to the environment of chronic wounds which are difficult to heal, so that hydrophobic drugs are enriched at the wounds, and can promote the hydrophobic drugs to enter cells to effectively kill pathogenic bacteria in the cells; in addition, the double-drug delivery system also comprises an EGFR targeting peptide connected to the tail end of the hydrophilic chain segment, which can increase the endocytosis of the drug delivery system by epithelial cells, thereby increasing the amount of the drug entering the epithelial cells and leading the drug to be capable of effectively eliminating bacteria invading the epithelial cells.

Description

Antibacterial/healing-promoting dual-drug delivery system, drug composition, preparation method and application thereof

Technical Field

The invention relates to the technical field of medicinal chemistry, in particular to an antibacterial/healing-promoting dual-drug delivery system, a medicinal composition, a preparation method and application thereof.

Background

Trauma is the destruction of tissues or organs caused by mechanical factors added to the human body, and is often accompanied by the damage of important organs, more blood or plasma loss, severe anoxia, acidosis, massive tissue necrosis, and the like. These intense stimuli contribute to tissue degeneration, thereby accelerating the rate of metabolic changes, leading to severe complications such as shock, acute renal failure, sepsis, and the like. In the face of such wounds, treatment and repair of the damaged area is required after the rescue has been performed. Infection and adhesion are often accompanied in the process of wound tissue healing, and antibiotics, anti-adhesion membranes and hemostatic products are generally adopted to act on the wound to solve the problems. Most of the anti-adhesion and hemostatic products comprise polylactic acid, chitosan, starch, gelatin and other components, and the components can play a certain role in promoting the healing of body tissues.

In addition, bacterial infections continue to be a significant health hazard for humans, and serious bacterial infections can cause death. The skin is the natural barrier of the body and protects various tissues and organs of the body from bacterial infections. However, when the skin is wounded, the skin is easily infected by bacteria, and staphylococcus aureus which is common in chronic wounds difficult to heal invades epithelial cells to cause repeated infection of the bacteria. There are some reports in the past literature on drug delivery systems and pharmaceutical compositions that promote wound healing.

CN101982202A discloses a medical hydrogel dressing and a preparation method thereof. The hydrogel is prepared by taking 10-30% of starch and 2-15% of water-soluble polymer as raw materials by weight, taking guanidine salt polycondensate as an antibacterial agent, adding a cross-linking agent, and reacting at 40-80 ℃. Although the raw materials are low in cost, the used chemical cross-linking agent has certain toxicity and is easy to cause harm to human bodies, and the hydrogel dressing is difficult to thoroughly eliminate pathogenic bacteria infected in cells.

CN112941913A discloses a hydrogel for treating chronic difficult-to-heal wound and a preparation method thereof. The hydrogel is composed of methacrylate, methacrylic acid, wood vinegar tea polyphenol mixed solution, a cross-linking agent, polyethylene glycol monomethyl ether diacrylate and TEMPO oxidized cellulose nano fibrils. Although the invention can accelerate the healing of the wound to a certain extent by supplying healing molecules required for the wound, the hydrogel can not be maintained on the wound surface for a long time and has low bioavailability.

CN113081951 discloses a hydrogel for chronic wound healing and a preparation method thereof. The hydrogel is an interpenetrating network structure polymer hydrogel formed by mixing hyaluronic acid connected with matrix metalloproteinase sensitive polypeptide and aldehyde group modified glucan. The medicine is released by utilizing the cleavage effect of the enzyme on a specific polypeptide sequence, so that the intelligent regulation and control effect is realized; however, the polypeptide specifically recognized by the MMP-2 enzyme needs to be modified into hyaluronic acid containing the polypeptide, so that the steps are complicated, and the preparation process is complex.

The methods for promoting wound healing disclosed in the above documents have the problems of high biological toxicity of the preparation, complicated steps, short maintenance time of the drug in the wound, low bioavailability and the like.

Therefore, it is a problem to be solved at present how to provide a drug delivery system and a drug composition with simple synthesis steps, and to prolong the maintenance time of the drug in a chronic wound, so as to enhance the bioavailability of the drug at the wound site, so that pathogenic bacteria infected in cells can be completely eliminated, and the healing of the wound can be promoted as soon as possible.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an antibacterial/healing-promoting dual-drug delivery system, a drug composition, a preparation method and an application thereof. The antibacterial/healing-promoting dual-drug delivery system can prolong the retention time of the drug at the wound, respond to the environment of the chronic wound which is difficult to heal, promote the drug to enter cells, kill bacteria in the cells, improve the bioavailability of the drug and further improve the treatment effect of the drug; in addition, the antibacterial/healing promoting dual-drug delivery system can be combined with EGFR to increase the endocytosis of epithelial cells to the drug delivery system, so that the amount of the drug entering the epithelial cells is increased, and the drug can effectively eliminate bacteria invading the epithelial cells.

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

in a first aspect, the present invention provides an antibacterial/healing dual drug delivery system, the outer layer of which comprises a hydrophilic segment, the inner core comprises a hydrophobic segment, and an MMP-9 responsive peptide is embedded between the hydrophilic segment and the hydrophobic segment.

In the present invention, the MMP-9 responsive peptide is embedded between the hydrophilic segment and the hydrophobic segment by forming covalent bonds with the hydrophilic polymer, the hydrophobic compound, respectively.

Preferably, an amide bond is formed between the MMP-9 responsive peptide and the hydrophilic polymer, and a carbon-nitrogen bond is formed between the MMP-9 responsive peptide and the hydrophobic compound.

In the invention, the carboxyl of the MMP-9 response peptide and the hydrophilic polymer modified by amino are subjected to amide condensation reaction to generate an amide bond; the secondary amino group of the proline of the MMP-9 response peptide is used as a nucleophilic reagent to generate Michael addition reaction with the hydrophobic compound modified by maleimide, and a carbon-nitrogen bond is generated. The MMP-9 responsive peptide is embedded between the hydrophilic segment and the hydrophobic segment by forming an amide bond with the hydrophilic polymer, forming a carbon-nitrogen bond with the hydrophobic compound.

Preferably, the molar ratio of the MMP-9 responsive peptide, hydrophilic polymer, and hydrophobic compound is (0.7-0.9): (1-1.2):1, and may be, for example, 0.7:1:1, 0.7:1.1:1, 0.7:1.2:1, 0.8:1:1, 0.8:1.1:1, 0.8:1.2:1, 0.9:1:1, 0.9:1.1:1, 0.9:1.2:1, etc., preferably 0.8:1.1: 1.

In the invention, under the condition that the hydrophilic polymer and the hydrophobic compound are relatively excessive, the MMP-9 response peptide can be ensured to be completely connected with the hydrophilic polymer and the hydrophobic compound, so that the modification efficiency of the MMP-9 response peptide on the amphiphilic polymer is improved.

Preferably, the amino acid sequence of the MMP-9 response peptide includes any one of PVGLIG, PLGLAG or GPLGVR, preferably PVGLIG.

Preferably, the hydrophilic polymer comprises methoxy polyethylene glycol amine (mPEG-NH)₂) Any one or combination of at least two of succinamide polyethylene glycol amine (PEG-NHS) or methoxy polyethylene glycol maleimide (mPEG-Mal), preferably methoxy polyethylene glycol amine (mPEG-NH)₂)。

In the invention, the methoxypolyethylene glycol amine is a non-ionic water-soluble polymer, which is not only non-toxic and harmless to human bodies, but also can further increase the dissolution rate of the medicament and improve the bioavailability of the medicament.

Preferably, the number average molecular weight of the hydrophilic polymer is 1000-4000, which may be, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, etc., preferably 2000.

Preferably, the hydrophobic compound comprises polycaprolactone-maleimide (PCL-Mal), aminocaprolactam (PCL-NH)₂) Distearoylphosphatidylethanolamine-maleimide (DSPE-Mal) or aminodistearoylphosphatidylethanolamine (DSPE-NH)₂) Any one or a combination of at least two of them, preferably distearoylphosphatidylethanolamine-maleimideAmine (DSPE-Mal).

In the present invention, the antibacterial/pro-healing dual drug delivery system further comprises an amphiphilic polymer modified with an EGFR-targeting peptide located at the end of the hydrophilic segment of the amphiphilic polymer.

In the present invention, an EGFR targeting peptide is further included in the antibacterial/pro-healing dual drug delivery system, and the EGFR targeting peptide is linked to the end of the hydrophilic segment in the amphiphilic polymer. The addition of the EGFR targeting peptide can increase the endocytosis of the epithelial cells to the drug delivery system, thereby increasing the amount of the drug entering the epithelial cells and enabling the drug to more effectively eliminate bacteria invading the epithelial cells.

Preferably, the loading amount of the EGFR targeting peptide modified amphiphilic polymer accounts for 11.3-22.7% of the total mass of the antibacterial/healing promoting dual drug delivery system, and may be, for example, 11.3%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 22.7%, etc.

Preferably, the amino acid sequence of the EGFR targeting peptide comprises KYFPPLALYNPTEYFY or YHWYGYTPQNVI, preferably YHWYGYTPQNVI.

Preferably, the amphiphilic polymer comprises any one or a combination of at least two of distearoylphosphatidylethanolamine-polyethylene glycol-amino, polycaprolactone-polyethylene glycol-amino, or polylactic glycolic acid-polyethylene glycol-amino, preferably distearoylphosphatidylethanolamine-polyethylene glycol;

preferably, the hydrophilic segment of the amphiphilic polymer has a number average molecular weight of 1000-4000, which may be, for example, 1000, 2000, 3000, 4000, etc., preferably 2000.

In a second aspect, the present invention provides a method of preparing an antibacterial/healing promoting dual drug delivery system according to the first aspect, the method of preparing the antibacterial/healing promoting dual drug delivery system comprising the steps of: the MMP-9 response peptide is firstly reacted with hydrophobic compounds and then reacted with hydrophilic polymers to prepare the antibacterial/healing promoting dual-drug delivery system.

In the invention, MMP-9 response peptide reacts with hydrophobic compound, one end of the MMP-9 response peptide is connected with the phospholipid end of the hydrophobic compound through covalent bond; and then reacting with a hydrophilic polymer to enable the other end of the MMP-9 response peptide to be connected to the hydrophilic polymer through covalent bond, thus preparing the antibacterial/healing promoting dual-drug delivery system.

In the present invention, the preparation method of the antibacterial/healing promoting dual drug delivery system comprises the following steps:

(a) dissolving MMP-9 response peptide in an organic solvent A to obtain a solution A; dissolving a hydrophobic compound in an organic solvent B to obtain a solution B;

(b) mixing the solution A and the solution B obtained in the step (a), reacting, and removing a solvent to obtain an intermediate product;

(c) mixing the intermediate product obtained in the step (b), a hydrophilic polymer and an organic solvent C, and reacting to obtain the MMP-9 response peptide modified polymer.

Preferably, in step (a), the organic solvent a comprises any one of methanol, ethanol or acetone or a combination of at least two thereof, preferably methanol.

Preferably, in step (a), the organic solvent B comprises any one or a combination of at least two of chloroform, methanol or ethyl acetate, preferably chloroform.

Preferably, in step (b), the reaction temperature is 30-50 ℃, for example, 30 ℃, 32 ℃, 34 ℃, 36 ℃, 40 ℃, 42 ℃, 46 ℃, 48 ℃, 50 ℃ and the like; the reaction time is 3 to 5 hours, and may be, for example, 3 hours, 3.2 hours, 3.4 hours, 3.6 hours, 3.8 hours, 4 hours, 4.2 hours, 4.4 hours, 4.6 hours, 4.8 hours, 5 hours, or the like.

Preferably, in step (C), the organic solvent C comprises any one or a combination of at least two of chloroform, methanol or ethyl acetate, preferably chloroform.

Preferably, in step (c), the reaction temperature is 30-50 ℃, for example, 30 ℃, 32 ℃, 34 ℃, 36 ℃, 40 ℃, 42 ℃, 46 ℃, 48 ℃, 50 ℃ and the like; the reaction time is 3 to 5 hours, and may be, for example, 3 hours, 3.2 hours, 3.4 hours, 3.6 hours, 3.8 hours, 4 hours, 4.2 hours, 4.4 hours, 4.6 hours, 4.8 hours, 5 hours, or the like.

Preferably, in step (c), the reaction also requires the addition of a catalyst and/or a coupling agent.

Preferably, the catalyst comprises any one or a combination of at least two of 4-Dimethylaminopyridine (DMAP), 4- (dimethylamino) pyridine-N-oxide (DMAPO) or polypyrrole (PPY), preferably DMAP.

Preferably, the molar ratio of the catalyst to the hydrophobic compound is (0.05-0.2):1, and may be, for example, 0.05:1, 0.08:1, 0.1:1, 0.12:1, 0.14:1, 0.16:1, 0.18:1, 0.2:1, and the like.

Preferably, the coupling agent comprises any one or a combination of at least two of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), Dicyclohexylcarbodiimide (DCC) or dimethyl carbonate (DMC), preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide.

Preferably, the molar ratio of the coupling agent to the hydrophobic compound is (1-3):1, and may be, for example, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, and the like.

Preferably, in step (c), the reaction is followed by a post-treatment, which comprises concentration, reconstitution, filtration, dialysis and drying in sequence.

Preferably, the reconstitution employs water.

Preferably, a 0.22 μm needle filter is used for the filtration.

Preferably, the cut-off molecular weight of the dialysis bag used for dialysis is 3000-4000Da, such as 3000Da, 3100Da, 3200Da, 3300Da, 3400Da, 3500Da, 3600Da, 3700Da, 3800Da, 3900Da, 4000Da, etc.; the dialysis time is 20-40h, for example, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, etc.

Preferably, the drying is freeze drying.

In the invention, PVGLIG is used as MMP-9 response peptide, methoxypolyethylene glycol amine is used as a hydrophilic polymer, distearyl phosphatidyl ethanolamine-maleimide is used as a hydrophobic compound, and the synthetic route of the polymer modified by the MMP-9 response peptide is shown as follows:

wherein mPEG-NH₂The number average molecular weight of (a) is 1000-4000, and may be, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, etc.

In the invention, PVGLIG is used as MMP-9 response peptide, methoxy polyethylene glycol amine is used as a hydrophilic polymer, polycaprolactone-maleimide is used as a hydrophobic compound, and the synthetic route of the polymer modified by the MMP-9 response peptide is shown as follows:

wherein, the number average molecular weight of PCL-Mal is 3000-3500, such as 3000, 3100, 3200, 3300, 3400, 3500, etc.; mPEG-NH₂The number average molecular weight of (a) is 1000-4000, and may be, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, etc.

In the invention, the antibacterial/healing-promoting dual-drug delivery system further comprises an EGFR targeting peptide modified amphiphilic polymer, and the preparation method of the antibacterial/healing-promoting dual-drug delivery system comprises the following steps: and mixing the MMP-9 response peptide modified polymer with the EGFR targeting peptide modified amphiphilic polymer to obtain the antibacterial/healing promoting dual-drug delivery system.

Preferably, the weight ratio of the MMP-9 responsive peptide modified polymer to the EGFR targeting peptide modified amphiphilic polymer is (3-7):1, and may be, for example, 3:1, 4:1, 5:1, 6:1, 6.5:1, 7:1, etc.

Preferably, the preparation method of the EGFR targeting peptide modified amphiphilic polymer comprises the following steps: and mixing the EGFR targeting peptide, the amphiphilic polymer and a solvent for reaction to obtain the amphiphilic polymer modified by the EGFR targeting peptide.

Preferably, the molar ratio of the EGFR targeting peptide to the amphiphilic polymer is 1 (4-8), and may be, for example, 1:4, 1:5, 1:6, 1:7, 1:8, etc.

Preferably, the solvent comprises any one of acetonitrile, N-dimethylformamide or methanol or a combination of at least two thereof, preferably a combination of acetonitrile and N, N-dimethylformamide.

Preferably, the volume ratio of acetonitrile to N, N-dimethylformamide is (0.8-1.2):1, and may be, for example, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, etc., preferably 1: 1.

Preferably, the reaction time is 20-28h, for example, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h and the like; the reaction temperature is 25-35 deg.C, for example, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C, 31 deg.C, 32 deg.C, 33 deg.C, 34 deg.C, 35 deg.C, etc.

Preferably, the reaction is carried out under dark conditions.

Preferably, the reaction is carried out under a nitrogen atmosphere.

Preferably, the reaction also requires the addition of a coupling agent comprising N-hydroxysuccinimide (NHS) and/or 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), preferably N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide.

Preferably, the molar ratio of the EGFR-targeting peptide, N-hydroxysuccinimide, and 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 1 (15-25): 15-25), and may be, for example, 1:15:15, 1:17:16, 1:19:17, 1:20:21, 1:25:20, 1:24:21, 1:23:22, and the like.

Preferably, the reaction also requires the addition of N, N-Diisopropylethylamine (DIEA).

Preferably, the molar ratio of the EGFR-targeting peptide to N, N-diisopropylethylamine is 1 (0.15-0.25), and may be, for example, 1:0.15, 1:0.16, 1:0.17, 1:0.18, 1:0.19, 1:0.20, 1:0.21, 1:0.22, 1:0.23, 1:0.24, 1:0.25, etc.

Preferably, the reaction is further followed by a post-treatment, which comprises concentration, reconstitution, filtration, dialysis and drying in sequence.

Preferably, the reconstitution employs water, preferably ultrapure water.

Preferably, a 0.22 μm needle filter is used for the filtration.

Preferably, the drying is freeze drying.

In the invention, YHWYGYTPQNVI is used as EGFR targeting peptide, PCL-PEG-NH₂The synthesis route of the EGFR targeting peptide modified amphiphilic polymer is shown as follows:

wherein, the number average molecular weight of PCL is 3000-3500, such as 3000, 3100, 3200, 3300, 3400, 3500, etc.; the number average molecular weight of PEG is 1000-4000, for example, 1000, 2000, 3000, 4000, etc.

In the invention, YHWYGYTPQNVI is used as EGFR targeting peptide, DSPE-PEG-NH₂The synthesis route of the EGFR targeting peptide modified amphiphilic polymer is shown as follows:

wherein, the DSPE-PEG-NH₂The number average molecular weight of the PEG in the group (B) is 1000-4000, for example, 1000, 2000, 3000, 4000, etc.

As a preferred technical scheme, the preparation method of the antibacterial/healing promoting dual-drug delivery system comprises the following steps:

(I) preparation of MMP-9 responsive peptide modified polymers:

(a) dissolving MMP-9 response peptide into an organic solution A to obtain a solution A; dissolving a hydrophobic compound in the organic solution B to obtain a solution B;

(b) mixing the solution A and the solution B obtained in the step (a), reacting for 3-5h at 30-50 ℃, and removing the solvent to obtain an intermediate product;

(c) mixing the intermediate product obtained in the step (b), a hydrophilic polymer, an optional catalyst, an optional coupling agent and an organic solution C, reacting for 3-5h at 30-50 ℃, concentrating the reaction solution, redissolving with water, filtering by adopting a 0.22 mu m needle filter, dialyzing for 20-40h by using a dialysis bag with the molecular weight cutoff of 3000-4000Da, and freeze-drying to obtain the polymer modified by the MMP-9 response peptide;

(II) preparation of EGFR-targeting peptide modified amphiphilic Polymer:

mixing an EGFR targeting peptide, an amphiphilic polymer, an optional coupling agent, an optional N, N-diisopropylethylamine and a solvent, reacting for 20-28h at 25-35 ℃ under the dark condition and in the nitrogen atmosphere, concentrating the reaction solution, redissolving with water, filtering by adopting a 0.22 mu m needle filter, dialyzing for 20-40h by using a dialysis bag with the molecular weight cutoff of 3000-4000Da, and freeze-drying to obtain the amphiphilic polymer modified by the EGFR targeting peptide;

the molar ratio of the EGFR targeting peptide to the amphiphilic polymer is 1 (4-8);

(III) preparation of an antibacterial/healing promoting dual drug delivery system:

the MMP-9 response peptide modified polymer and the EGFR targeting peptide modified amphiphilic polymer are mixed according to the weight ratio of (3-7):1, so that the antibacterial/healing promoting dual-drug delivery system is obtained.

In a third aspect, the present invention provides a pharmaceutical composition comprising a hydrophobic drug and an antibacterial/healing promoting dual drug delivery system as described in the first aspect;

wherein the hydrophobic drug is located in the hydrophobic inner core of the antibacterial/pro-healing dual drug delivery system.

Preferably, the hydrophobic drug comprises a healing promoting drug and/or an antibacterial drug, preferably a combination of a healing promoting drug and an antibacterial drug.

In the present invention, the MMP-9 responsive peptide is linked to the hydrophobic segment by a carbon-nitrogen bond; the MMP-9 response peptide is connected with the hydrophilic chain segment through an amido bond; the EGFR targeting peptide is connected with the hydrophilic chain segment of the amphiphilic polymer; the hydrophobic inner core of the antibacterial/healing promoting dual drug delivery system entraps a healing promoting drug and an antibacterial drug.

In the process of wound tissue healing, bacterial infection is often accompanied, and staphylococcus aureus which is common in chronic wounds difficult to heal invades epithelial cells to cause repeated bacterial infection. Skin wounds often take a long time to fully recover without external intervention. In order to accelerate the time to wound healing, the pharmaceutical composition of the present invention employs a combination of antibacterial and healing-promoting drugs. Moreover, the invention adopts the nano-carrier to carry the antibacterial/healing-promoting double-effect medicine, so that the medicine can more easily enter the epithelial cells of the infected part, effectively kill pathogenic bacteria in the epithelial cells and improve the treatment effect of the medicine.

Preferably, the weight ratio of the healing promoting drug and the antibacterial drug is (0.5-2: 1), and may be, for example, 0.5:1, 0.7:1, 0.9:1, 1:1, 1.1:1, 1.3:1, 1.5:1, 1.7:1, 2:1, etc.

Preferably, the healing promoting drug comprises any one or a combination of at least two of curcumin, quercetin, cinnamaldehyde or soy isoflavone, preferably curcumin.

Preferably, the antibacterial drug comprises any one of rifampicin, vancomycin, linezolid or doxycycline or a combination of at least two thereof.

Preferably, the pharmaceutical composition is in the form of spherical micelles, the average particle size of which is 15-30nm, such as 15nm, 16nm, 20nm, 25nm, 28nm, 30nm, etc.

Preferably, the drug loading of the pharmaceutical composition is 4-10 wt%, e.g., can be 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt%, etc.

Preferably, the encapsulation efficiency of the pharmaceutical composition is 90-99.9 wt%, for example, may be 90 wt%, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, 97 wt%, 98 wt%, 99 wt%, 99.2 wt%, 99.4 wt%, 99.6 wt%, 99.9 wt%, etc.

Preferably, the pharmaceutical composition further comprises a temperature-sensitive hydrogel, and the drug-loaded micelle is uniformly dispersed in the temperature-sensitive hydrogel.

In the invention, the hydrogel is used as a dispersion medium of the medicine to cover the surface of the wound, so that the wound part is tightly attached.

The temperature-sensitive hydrogel is in a liquid state at a lower temperature, can be uniformly mixed with the drug-loaded micelle in the third aspect, and is simple to prepare; is liquid at lower temperature and gel at body temperature, and is convenient for administration. The hydrogel is nontoxic to human bodies, safe and reliable, can be slowly degraded, and reduces the administration frequency.

Preferably, the temperature-sensitive hydrogel comprises a polyoxyethylene polyoxypropylene ether block copolymer, preferably a combination of pluronic F127 and pluronic F68.

Preferably, the weight ratio of pluronic F127 and pluronic F68 is (16-22): (2-8), and may be, for example, 16:2, 16:3, 16:4, 16:5, 16:6, 16:7, 16:8, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 18:2, 18:3, 18:4, 18:5, 18:6, 18:7, 18:8, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 20:2, 20:3, 20:4, 20:5, 20:6, 20:7, 20:8, 21:2, 21:3, 21:4, 21:5, 21:6, 21:7, 21:8, 22:2, 22:3, 22:4, 22:5, 22:6, 22:7, 22:8, 22:7, 22:4, 22:6, 22:8, etc.

Preferably, the temperature-sensitive hydrogel also comprises water.

Preferably, the volume ratio of the total weight of pluronic F127 and pluronic F68 to water is 1mg (2-4.5) mL, and may be, for example, 1mg:2mL, 1mg:2.2mL, 1mg:2.4mL, 1mg:2.6mL, 1mg:2.8mL, 1mg:3mL, 1mg:3.2mL, 1mg:3.4mL, 1mg:3.6mL, 1mg:3.8mL, 1mg:4mL, 1mg:4.2mL, 1mg:4.4mL, 1mg:4.5mL, or the like.

Preferably, the temperature-sensitive hydrogel is prepared by the following preparation method: and mixing the pluronic F127 and the pluronic F68 with water, and stirring to obtain the temperature-sensitive hydrogel.

Preferably, the stirring temperature is 4-10 ℃, for example, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃ and the like; the stirring time is 36-72h, for example, 36h, 40h, 44h, 48h, 52h, 56h, 60h, 64h, 68h, 72h and the like.

In a fourth aspect, the present invention provides a method for preparing the pharmaceutical composition according to the third aspect, the method for preparing the pharmaceutical composition comprises the following steps: and (3) coating the hydrophobic drug in a hydrophobic core of the antibacterial/healing promoting dual-drug delivery system by a film dispersion method to prepare the drug-loaded micelle.

Preferably, the thin film dispersion method comprises the following specific steps: mixing the hydrophobic drug, the antibacterial/healing promoting double-drug delivery system and an organic solvent D, and performing rotary evaporation to form a film in which the hydrophobic drug and the amphiphilic polymer are uniformly dispersed; and mixing the hydrophobic drug and the film with the amphiphilic polymer uniformly dispersed with water for hydration, and carrying out self-assembly on the polymer to obtain the drug-loaded micelle.

Preferably, the organic solvent D includes any one or a combination of at least two of tetrahydrofuran, chloroform, methanol, or acetone, preferably a combination of chloroform and methanol.

Preferably, the volume ratio of chloroform to methanol is (0.5-2):1, and may be, for example, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, etc., preferably 1: 1.

Preferably, the weight ratio of the hydrophobic drug and the antibacterial/healing promoting dual drug delivery system is 1 (5-20), and may be, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc., preferably 1: 10.

Preferably, the rotary evaporation temperature is 30-40 ℃, for example can be 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃ etc.; the rotating speed of the rotary evaporation is 60-100r/min, for example, 60r/min, 65r/min, 70r/min, 75r/min, 80r/min, 85r/min, 90r/min, 95r/min, 100r/min and the like; the time of the rotary evaporation is 0.5 to 1.5h, and may be, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, or the like.

Preferably, the volume of water required for hydrating the hydrophobic drug and amphiphilic polymer uniformly dispersed film is 1-4mL, and may be, for example, 1mL, 1.3mL, 1.6mL, 1.9mL, 2mL, 2.4mL, 2.6mL, 2.8mL, 3mL, 3.1mL, 3.3mL, 3.5mL, 3.7mL, 3.9mL, 4mL, etc.

Preferably, the temperature of the hydration is 55-70 ℃, for example, 55 ℃, 57 ℃, 59 ℃, 61 ℃, 63 ℃, 65 ℃, 67 ℃, 70 ℃ and the like; the hydration time is 0.5-1h, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, etc.

Preferably, the hydration is followed by sonication and/or filtration.

Preferably, the power of the ultrasound is 250-350W, such as 250W, 260W, 270W, 280W, 290W, 300W, 310W, 320W, 330W, 340W, 350W and the like; the ultrasonic treatment time is 5-30min, such as 5min, 7min, 9min, 11min, 13min, 15min, 17min, 19min, 21min, 23min, 25min, 27min, 29min, 30min, etc.

Preferably, the filtration is performed using a 0.22 μm aqueous membrane.

Preferably, the pharmaceutical composition further comprises a temperature-sensitive hydrogel, and the preparation method of the pharmaceutical composition comprises the following steps: and mixing the drug-loaded micelle and the temperature-sensitive hydrogel to obtain the drug composition containing the temperature-sensitive hydrogel.

Preferably, the volume ratio of the drug-loaded micelle to the temperature-sensitive hydrogel is (0.8-1.2):1, and for example, can be 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, and the like.

Preferably, the mixing temperature is 4-10 ℃, for example, can be 4 degrees, 5 degrees, 6 degrees, 7 degrees, 8 degrees, 9 degrees, 10 degrees C.

As a preferred technical scheme, the preparation method of the pharmaceutical composition comprises the following steps:

(I) preparing a drug-loaded micelle:

mixing hydrophobic drug, antibacterial/healing promoting double-drug delivery system and organic solvent, and performing rotary evaporation to form a film with uniformly dispersed hydrophobic drug and amphiphilic polymer; mixing the hydrophobic drug and the film with the amphiphilic polymer uniformly dispersed with water for hydration, and carrying out self-assembly on the polymer to obtain a drug-loaded micelle;

the hydrophobic drug comprises a healing promoting drug and an antibacterial drug in a weight ratio of (0.5-2):1, and the weight ratio of the hydrophobic drug to the antibacterial/healing promoting dual-drug delivery system is 1 (5-20);

(II) preparation of temperature-sensitive hydrogel:

mixing Pluronic F127 and Pluronic F68 with water, and stirring at 4-10 ℃ for 36-72h to obtain the temperature-sensitive hydrogel;

the weight ratio of the pluronic F127 to the pluronic F68 is (16-22) to (2-8), and the volume ratio of the total weight of the pluronic F127 to the pluronic F68 to the water is 1mg (2-4.5) mL;

(III) preparation of pharmaceutical composition:

mixing the drug-loaded micelle with the volume ratio of (0.8-1.2):1 and the temperature-sensitive hydrogel at 4-10 ℃ to obtain the drug composition containing the temperature-sensitive hydrogel.

In a fifth aspect, the present invention provides the use of an antibacterial/pro-healing dual drug delivery system according to the first aspect, or a pharmaceutical composition according to the third aspect of the invention, in the manufacture of a medicament for the treatment of chronic difficult-to-heal wounds.

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

(1) the antibacterial/healing promoting dual-drug delivery system can prolong the retention time of the drug at the wound through targeted response, promote the drug to enter cells, kill bacteria in the cells, improve the bioavailability of the drug and further improve the treatment effect of the drug;

(2) the antibacterial/healing promoting dual-drug delivery system can be combined with EGFR, so that the amount of the drug loaded by the nano-carrier entering epithelial cells is increased, and the drug can effectively eliminate bacteria invading the epithelial cells;

(3) in the pharmaceutical composition, the antibacterial/healing-promoting double-effect medicine encapsulated by the nano-carrier can more easily enter epithelial cells of an infected part, so that the accumulation amount of the medicine in the cells is increased, pathogenic bacteria in the epithelial cells are effectively eliminated, the occurrence of repeated infection can be reduced, the bioavailability of the medicine is effectively improved, and the treatment effect of the medicine is improved;

(4) the antibacterial/healing promoting dual-drug delivery system and the preparation method of the pharmaceutical composition are simple in process and convenient to operate.

Drawings

FIG. 1 is a schematic structural diagram of a pharmaceutical composition described in application example 1;

wherein, 1 is a healing promoting drug, 2 is an antibacterial drug, 3 is a hydrophobic chain segment, 4 is MMP-9 response peptide, 5 is a hydrophilic chain segment, and 6 is EGFR targeting peptide.

FIG. 2 is a schematic structural diagram of a pharmaceutical composition according to application example 8;

wherein, 1 is a healing promoting drug, 2 is an antibacterial drug, 3 is a hydrophobic chain segment, 4 is MMP-9 response peptide, and 5 is a hydrophilic chain segment.

Fig. 3 is a particle size distribution diagram of the pharmaceutical composition described in application example 1.

FIG. 4 is an electron microscope image of the pharmaceutical composition of application example 1.

FIG. 5 is a Zeta potential diagram of the pharmaceutical composition described in application example 1.

FIG. 6 is a schematic diagram showing the effect of the pharmaceutical composition of application example 1 on promoting cell migration compared with application examples 1 to 3.

FIG. 7 is a graph showing the mean fluorescence intensity of the pharmaceutical compositions of application example 1 and comparative application examples 1 to 3 against human skin epidermal cells.

FIG. 8 is a graph showing the bactericidal effect of the pharmaceutical compositions of application example 1 and comparative application examples 1 to 3 against human epidermal cell intracellular bacteria.

Fig. 9 is a graph showing wound healing effects of the pharmaceutical compositions according to application example 13, comparative application example 3 and comparative application example 6 on mouse bacterial infection.

Detailed Description

The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The sources of the components in the following examples are as follows:

example 1

The present embodiment provides an antibacterial/healing promoting dual drug delivery system, and the preparation method of the antibacterial/healing promoting dual drug delivery system comprises the following steps:

(1) the reaction formula of the MMP-9 response peptide modified polymer is shown as follows:

the preparation method of the polymer modified by the MMP-9 response peptide specifically comprises the following steps:

(a) dissolving 9.6 mu mol of MMP-9 responsive PVGLIG in 1mL of methanol to obtain solution A; dissolving 12 mu mol of DSPE-Mal in 2mL of chloroform to obtain a solution B;

(b) mixing the solution A and the solution B obtained in the step (a), reacting for 4 hours at 40 ℃, and removing the solvent to obtain an intermediate product;

(c) 13.2 mu mol mPEG2k-NH of the intermediate product obtained in step (b)₂Mixing 24 mu mol of EDC, 1.2 mu mol of DMAP and 3mL of trichloromethane, reacting for 4h at 40 ℃, concentrating to remove the solvent, adding a water redissolved product, filtering by a 0.22 mu m needle filter, transferring the filtrate into a dialysis bag with the molecular weight cutoff of 3500Da for dialysis for 24h, collecting the dialysate, and freeze-drying to obtain DSPE-PVGLIG-mPEG2 k.

(2) The reaction formula of the EGFR targeting peptide modified amphiphilic polymer is shown as follows:

the preparation method of the EGFR targeting peptide modified amphiphilic polymer comprises the following steps: mu.mol of DSPE-PEG2k-NH ₂6 mu mol of polypeptide GE11, 24 mu mol of NHS (20eq.), 24 mu mol of EDC, 0.24 mu mol of DIEA, 5mL of acetonitrile and 5mL of DMF are mixed, the mixture is reacted for 24h at 26 ℃ in a dark place under the nitrogen atmosphere, the solvent is removed by rotary evaporation, the product is dissolved by 5mL of ultrapure water, the solution is filtered by a 0.22 mu m needle filter, then the solution is dialyzed for 24h in a dialysis bag with the molecular weight cutoff of 3500Da, and the dialyzate is frozen and dried to obtain DSPE-PEG2 k-YHWYGYTPQNVI.

(3) Mixing 12.5mg of DSPE-PVGLIG-mPEG2k obtained in the step (1) and 2.5mg of DSPE-PEG2k-YHWYGYTPQNVI obtained in the step (2) to obtain the antibacterial/healing promoting dual-drug delivery system.

Example 2

(a) dissolving 9 mu mol of MMP-9 responsive PVGLIG in 1mL of methanol to obtain a solution A; dissolving 12 mu mol of DSPE-Mal in 2mL of chloroform to obtain a solution B;

(b) mixing the solution A and the solution B obtained in the step (a), reacting for 4.2 hours at 38 ℃, and removing the solvent to obtain an intermediate product;

(c) 13 mu mol mPEG2k-NH of the intermediate product obtained in the step (b)₂Mixing 25 μmol EDC, 1.3 μmol DMAP and 3mL chloroform, reacting at 38 deg.C for 4.2h, concentrating to remove solvent, adding water to redissolve the product, filtering with 0.22 μm syringe needle filterFiltering, transferring the filtrate into a dialysis bag with molecular weight cutoff of 3500Da, dialyzing for 26h, collecting the dialysate, and freeze-drying to obtain DSPE-PVGLIG-mPEG2 k.

the preparation method of the EGFR targeting peptide modified amphiphilic polymer comprises the following steps: weighing 1.2 mu mol of DSPE-PEG2k-NH₂6.2 mu mol of polypeptide GE11, 26 mu mol of NHS, 26 mu mol of EDC, 0.25 mu mol of DIEA, 5mL of acetonitrile and 5mL of DMF, reacting at 28 ℃ in a dark place for 25h under the nitrogen atmosphere, removing the solvent by rotary evaporation, dissolving the product with 5mL of ultrapure water, filtering the solution by using a 0.22 mu m needle filter, dialyzing the solution for 26h in a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying the dialyzate to obtain the DSPE-PEG2 k-YHWYGYTPQNVI.

Example 3

(a) dissolving 10 mu mol of MMP-9 responsive PVGLIG in 1mL of methanol to obtain a solution A; dissolving 12 mu mol of DSPE-Mal in 2mL of chloroform to obtain a solution B;

(b) mixing the solution A and the solution B obtained in the step (a), reacting for 3.8h at 42 ℃, and removing the solvent to obtain an intermediate product;

(c) 13.5 mu mol mPEG2k-NH of the intermediate product obtained in step (b)₂Mixing 23 mu mol of EDC, 1.1 mu mol of DMAP and 3mL of trichloromethane, reacting for 3.8h at 42 ℃, concentrating to remove the solvent, adding a water redissolved product, filtering by a 0.22 mu m needle filter, transferring the filtrate into a dialysis bag with the molecular weight cutoff of 3500Da for dialysis for 22h, collecting the dialysate, and freeze-drying to obtain DSPE-PVGLIG-mPEG2 k.

the preparation method of the EGFR targeting peptide modified amphiphilic polymer comprises the following steps: weighing 1.2 mu mol of DSPE-PEG2k-NH₂5.8 mu mol of polypeptide GE11, 23 mu mol of NHS, 23 mu mol of EDC, 0.23 mu mol of DIEA, 5mL of acetonitrile and 5mL of DMF, reacting for 26h at 25 ℃ in a dark place under the nitrogen atmosphere, removing the solvent by rotary evaporation, dissolving the product with 5mL of ultrapure water, filtering the solution by using a 0.22 mu m needle filter, dialyzing for 26h in a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying the dialyzate to obtain the DSPE-PEG2 k-YHWYGYTPQNVI.

Example 4

This example provides an antibacterial/healing promoting dual drug delivery system, which differs from example 1 only in that DSPE-Mal is replaced by equimolar amount of PCL3400-Mal, and the other compositions and preparation steps are the same as example 1.

Example 5

This example provides an antibacterial/healing promoting dual drug delivery system, differing from example 1 only in that DSPE-PEG2k-NH was used₂Replacement with equimolar amounts of PCL3400-PEG2000-NH₂The other compositions and preparation procedures were the same as in example 1.

Example 6

This example provides an antibacterial/healing promoting dual drug delivery system, which differs from example 1 only in that DSPE-PVGLIG-mPEG2k is reduced to 9mg in step (3), and the other components and preparation steps are the same as example 1.

Example 7

This example provides an antibacterial/healing promoting dual drug delivery system, which differs from example 1 only in that DSPE-pvgig-mPEG 2k is increased to 20mg in step (3), and the other composition and preparation steps are the same as example 1.

Example 8

This example provides an antibacterial/healing promoting dual drug delivery system, which differs from example 1 only in that it does not contain DSPE-PEG2k-YHWYGYTPQNVI, and the other components and preparation steps are the same as example 1.

Comparative example 1

This comparative example provides an antibacterial/healing promoting dual drug delivery system, which differs from example 1 only in that it does not contain DSPE-PVGLIG-mPEG2k, and the other components and preparation steps are the same as example 1.

Comparative example 2

This comparative example provides an antibacterial/healing promoting dual drug delivery system of 15mg of commercially available DSPE-PEG2 k.

Application example 1

The application example provides a pharmaceutical composition, and a preparation method of the pharmaceutical composition comprises the following steps:

weighing 1mg of curcumin, 0.5mg of rifampicin and the antibacterial/healing promoting dual-drug delivery system provided in example 1 (12.5mg of DSPE-PVGLIG-mPEG2k and 2.5mg of DSPE-PEG2k-YHWYGYTPQNVI), dissolving in a mixed solution of 2.5mL of methanol and 2.5mL of chloroform, transferring the mixture into a round-bottomed flask, performing rotary evaporation for 40min at a set rotation speed of 80r/min under the condition of 36 ℃ water bath, adding 2mL of deionized water after the organic solvent is evaporated to dryness and the rest components form a film on the wall, then placing into a 60 ℃ water bath kettle for hydration for 40min, performing ultrasonic treatment for 10min at a power of 300W, and filtering with a 0.22 mu m water-based filter membrane to obtain the pharmaceutical composition.

The pharmaceutical composition comprises curcumin, rifampicin and the antibacterial/healing promoting dual drug delivery system prepared in example 1; wherein the curcumin and rifampicin are located in the hydrophobic core of the antibacterial/healing promoting dual drug delivery system.

As shown in fig. 1, the pharmaceutical composition according to application example 1 includes a hydrophobic drug and an antibacterial/healing promoting dual drug delivery system;

wherein the outer layer of the antibacterial/healing promoting dual drug delivery system comprises a hydrophilic segment 5 and the inner core comprises a hydrophobic segment 3; an MMP-9 responsive peptide 4 is embedded between the hydrophilic segment 5 and the hydrophobic segment 3; the EGFR targeting peptide 6 is connected with the hydrophilic chain segment 5 of the amphiphilic polymer through a covalent bond;

wherein the hydrophobic drug is located in the inner core of the antibacterial/healing promoting dual drug delivery system, and the hydrophobic drug comprises healing promoting drug 1 and antibacterial drug 2.

Application examples 2 to 7

Application examples 2 to 7 provide 6 kinds of pharmaceutical compositions, which are different from application example 1 only in that the antibacterial/healing promoting dual drug delivery system prepared in example 1 was replaced with the antibacterial/healing promoting dual drug delivery system prepared in examples 2 to 7, respectively, having the same weight, and the other components and preparation methods were the same as application example 1.

Application example 8

Application example 8 provides a pharmaceutical composition, which is different from application example 1 only in that the antibacterial/healing promoting dual drug delivery system prepared in example 1 is replaced with the antibacterial/healing promoting dual drug delivery system prepared in example 8 with the same weight, and other components and preparation methods are the same as application example 1.

As shown in fig. 2, the pharmaceutical composition of application example 8 includes a hydrophobic drug and an antibacterial/healing promoting dual drug delivery system;

wherein the outer shell of the antibacterial/pro-healing dual drug delivery system comprises a hydrophilic segment 5, the inner core comprises a hydrophobic segment 3, and the MMP-9 responsive peptide 4 is embedded between the hydrophilic segment 5 and the hydrophobic segment 3.

Application example 9

The application example provides a pharmaceutical composition, which is different from the application example 1 only in that no curcumin is contained, the rifampicin is increased to 1.5mg, and other components and a preparation method are the same as the application example 1.

Application example 10

The application example provides a pharmaceutical composition, which is different from the application example 1 only in that rifampicin is not contained, curcumin is increased to 1.5mg, and other components and a preparation method are the same as the application example 1.

Application example 11

The application example provides a pharmaceutical composition, which is different from the application example 1 only in that curcumin is replaced by quercetin with the same weight, and other components and a preparation method are the same as the application example 1.

Application example 12

The application example provides a pharmaceutical composition, which is different from the application example 1 only in that rifampicin is replaced by doxycycline of the same weight, and other components and a preparation method are the same as the application example 1.

Application example 13

(1) preparation of hydrogel: mixing 0.8g of pluronic F127, 0.16g of pluronic F68 and 2mL of water, and stirring for 48 hours at 4 ℃ until solid particles are completely dissolved to obtain a temperature-sensitive hydrogel solution;

(2) preparation of a pharmaceutical composition containing a temperature-sensitive hydrogel: and (2) uniformly mixing the medicinal composition prepared in the application example 1 and the temperature-sensitive hydrogel solution at 4 ℃ according to the volume ratio of 1:1 to prepare the medicinal composition containing the temperature-sensitive hydrogel.

Comparative application example 1

The comparative application example provides a pharmaceutical composition, and the preparation method of the pharmaceutical composition comprises the following steps: 1.5mg of curcumin was dissolved in 5mL of dimethyl sulfoxide solvent to prepare a free curcumin solution.

Comparative application example 2

The comparative application example provides a pharmaceutical composition, and the preparation method of the pharmaceutical composition comprises the following steps: 1.5mg rifampicin was dissolved in 5mL dimethylsulfoxide to prepare a free rifampicin solution.

Comparative application example 3

The comparative application example provides a pharmaceutical composition, and the preparation method of the pharmaceutical composition comprises the following steps: 1mg curcumin and 0.5mg rifampicin were dissolved in 5mL dimethylsulfoxide as a mixed solution of free curcumin and rifampicin.

Comparative application examples 4 to 5

Comparative application examples 4 to 5 provide 2 pharmaceutical compositions, which are different from application example 1 only in that the antibacterial/healing promoting dual drug delivery system prepared in example 1 was replaced with the antibacterial/healing promoting dual drug delivery system prepared in comparative examples 1 to 2, respectively, having the same weight, and the other components and preparation methods were the same as in application example 1.

Comparative application example 6

This comparative application example provides a pharmaceutical composition, which is different from application example 13 only in that the pharmaceutical composition prepared in application example 1 was replaced with the pharmaceutical composition prepared in comparative application example 3 in the same weight, and the other components and the preparation method were the same as in application example 13.

Test example 1

Performance testing

Testing a sample: pharmaceutical compositions provided in application examples 1-12 and comparative application examples 4-5

The test method comprises the following steps: the freeze dryer and the microplate reader are used for testing and calculating the drug loading rate and the encapsulation rate, the dynamic light scattering laser particle size analyzer is used for testing the particle size distribution and the zeta potential, and the specific test results are shown in table 1.

TABLE 1

As can be seen from the above test results, the drug-loading rate of the pharmaceutical composition provided by the present invention (application examples 1 to 12) is between 6.94 and 7.69 wt%; the encapsulation rate is between 97.60 and 99.99 weight percent; the average grain diameter is between 15 and 30nm, and the zeta potential is between 22.7 and 10.2mv below zero; the pharmaceutical composition provided by the invention has good physical properties.

As shown in fig. 3, the distribution diagram of the particle size of the pharmaceutical composition provided in application example 1 shows that the average particle size of the pharmaceutical composition provided in application example 1 is 20 nm; as shown in an electron microscope image of the pharmaceutical composition of application example 1 shown in fig. 4, the particle size of the pharmaceutical composition provided in application example 1 is 18-25 nm; as shown in FIG. 5, the Zeta potential of the pharmaceutical composition provided in application example 1 was-12.1 mv. Application example 1 provides a pharmaceutical composition having a smaller particle size, a larger drug loading, a higher encapsulation efficiency, and a negative zeta potential value. The negative charge nano-particles have higher biocompatibility to wound tissues than the positive charge nano-particles, and the more the positive charge is, the more toxicity to the wound tissues is easily generated.

Test example 2

Cell scratch test for pharmaceutical compositions

Testing a sample: pharmaceutical compositions provided in application examples 1-12, comparative application examples 1-5

The test method comprises the following steps: the scarification test of epidermal cells of human skin was carried out by the method reported by encapsidation of cups nanoparticules with MMP 9-reactive and thermo-Sensitive Hydrogel improvings diagnostic round health. PBS without any medicine is taken as a control group, and the curcumin concentration in the pharmaceutical compositions provided by application examples 1-8, application example 10, application example 12, comparative application example 1 and comparative application examples 3-5 is 0.5 mu g/mL; the concentration of rifampicin in application example 9, application example 11, and comparative application example 2 was 0.5 μ g/mL; the scratch healing rate after 24h of administration was counted, and the test results are shown in table 2:

TABLE 2

As can be seen from the data in Table 2, the healing rate of the pharmaceutical composition provided by the invention (application examples 1-12) to scratches after being administrated for 24 hours reaches 30-92%; the healing rate of the scratch of the pharmaceutical composition provided by the preferred technical scheme (application examples 1-3) after 24 hours of administration reaches 90-92%.

As can be seen from comparison of application example 1 and application examples 4 to 5, the replacement of DSPE-PVGLIG-mPEG2000 with PCL3400-PVGLIG-mPEG2000 or the replacement of DSPE-PEG2000-YHWYGYTPQNVI with PCL3400-PEG2000-YHWYGYTPQNVI results in a slightly poor effect of the pharmaceutical composition in promoting the healing of the scratch.

According to the comparative examples of the application example 1, the application example 8 and the comparative application examples 4 to 5, the MMP-9 response peptide does not affect the endocytosis of the nano-carrier-encapsulated drug by cells, and the EGFR targeting peptide can promote the uptake of the nano-carrier-encapsulated drug by cells, so that the healing of scratches is promoted.

Through comparison among application example 1, application examples 9 to 10 and application example 12, curcumin encapsulated in the nano-carrier can promote healing of scratches, and the encapsulated antibacterial drug does not affect healing of scratches.

As can be seen from a comparison between application example 1 and application example 11, curcumin has an effect of promoting healing of scratches better than quercetin.

It can be seen from the comparison of application example 1 and comparative application examples 1 to 3 that only the drug encapsulated in the nanocarrier can better promote healing of the scratch.

As shown in fig. 6, the pharmaceutical composition provided in application example 1 achieved substantial healing of the wound at 24h compared to the comparative application examples 1-3 and the control group. The medicine composition provided by the application 1 can ensure that the scratch part of the medicine is specifically enriched and quickly reaches the concentration required by treatment.

Test example 3

Flow cytometry experiments

Testing a sample: pharmaceutical compositions provided in application example 1 and comparative application examples 1-3

The test method comprises the following steps: flow cytometry experiments were performed using the method reported by bioinsered Polymerization of query to product a serum with content cytoxicity and Cancer-Targeting Potential in Vivo, using PBS without any drug as a control. Curcumin has fluorescence at 488nm, so that the accumulation amount of curcumin in the cell in the pharmaceutical composition can be judged by the fluorescence intensity. When P is greater than 0.05, the difference is not significant; significant variability was indicated when 0.01< P < 0.05; when P <0.01, the difference was very significant.

As shown in fig. 7, for human skin epidermal cells, the fluorescence intensity value of the pharmaceutical composition provided in application example 1 after being taken up by epithelial cells is the strongest, which indicates that the drug entrapped in the nanocarrier is easier to enter into the cells; and P <0.0001 was calculated, indicating that application example 1 is very different than comparative example application 3. The pharmaceutical composition provided by application example 1 can be combined with EGFR, so that the drug encapsulated by the nano-carrier can enter epithelial cells of an infected part more easily, the bioavailability of the drug is effectively improved, and the treatment effect of the drug is improved.

Test example 4

Intracellular sterilization experiment

The test method comprises the following steps: the intracellular sterilization experiment is carried out by adopting the method reported by Antibiotic Therapy, namely Sublethal Levels of Antibiotics and Bacterial Persitus in epistalial cells, and MRSA clinical isolates are taken as sterilization targets, and PBS without any medicine is taken as a control group.

The experimental result shows that aiming at the epidermal cells of the human skin, the pharmaceutical composition provided by the application example 1 has the strongest killing capability on intracellular bacteria and the minimum total number of the intracellular bacteria. As shown in fig. 8, the difference in significance p between application example 1 and comparative application example 3 was very significant, and the difference in significance p between the control group and comparative application example 3 was very significant, namely 0.0008, with respect to human skin epidermal cells. The pharmaceutical composition provided by application example 1 can be combined with EGFR, so that the healing promoting drugs and/or antibacterial drugs encapsulated by the nano-carrier can more easily enter epithelial cells of infected parts, the accumulation amount of the drugs in the cells is increased, pathogenic bacteria in the epithelial cells are effectively eliminated, the occurrence of repeated infection can be reduced, the bioavailability of the drugs is effectively improved, and the treatment effect of the drugs is improved.

Experimental example 5

Animal experiments: mouse skin wound healing curve

Testing a sample: application example 13, comparative application example 3, and comparative application example 6

The test method comprises the following steps: animal experiments were performed using the method reported by Multifunctional Chinese inventor optics for round health, with PBS without any drug added as control 1 and PBS hydrogel solution as control 2; the amount of the pharmaceutical composition administered was 1mg/kg relative to rifampicin.

The test result is shown in fig. 9, after the pharmaceutical composition is adopted for 15 days, the wound healing rate of the mouse is as high as 96.4% by using the pharmaceutical composition provided in application example 13; by using the pharmaceutical composition provided in comparative application example 6, the wound healing rate of the mice reaches 79.9%; using the pharmaceutical composition provided in comparative application example 3, the wound healing rate of the mice was 68.7%; the wound healing rate of the mouse is 58.2% by using the hydrogel solution of PBS; the wound healing rate of the mice was 48.2% using PBS solution. The above data indicate that hydrogel alone has no efficacy in promoting wound healing, and that both the free drug and its hydrogel solution of free drug have a weak ability to promote wound healing.

The applicant states that the present invention is illustrated by the above examples to provide an antibacterial/healing promoting dual drug delivery system, a pharmaceutical composition, a method of preparation and use thereof, but the present invention is not limited to the above examples, i.e. it is not meant to imply that the present invention must be practiced in the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. An antibacterial/pro-healing dual drug delivery system, wherein the outer layer of the antibacterial/pro-healing dual drug delivery system comprises a hydrophilic segment, the inner core comprises a hydrophobic segment, and an MMP-9 responsive peptide is embedded between the hydrophilic segment and the hydrophobic segment.

2. The anti-bacterial/pro-healing dual drug delivery system according to claim 1, wherein the MMP-9 responsive peptide is embedded between the hydrophilic segment and the hydrophobic segment by forming covalent bonds with hydrophilic polymers, hydrophobic compounds, respectively;

preferably, an amide bond is formed between the MMP-9 responsive peptide and a hydrophilic polymer, and a carbon-nitrogen bond is formed between the MMP-9 responsive peptide and a hydrophobic compound;

preferably, the molar ratio of the MMP-9 responsive peptide, hydrophilic polymer, and hydrophobic compound is (0.7-0.9): (1-1.2):1, preferably 0.8:1.1: 1;

preferably, the amino acid sequence of the MMP-9 response peptide includes any one of PVGLIG, PLGLAG or GPLGVR, preferably PVGLIG;

preferably, the hydrophilic polymer comprises any one or a combination of at least two of methoxy polyethylene glycol amine, succinamide polyethylene glycol amine or methoxy polyethylene glycol maleimide, preferably methoxy polyethylene glycol amine;

preferably, the number average molecular weight of the hydrophilic polymer is 1000-4000, preferably 2000;

preferably, the hydrophobic compound comprises any one or a combination of at least two of polycaprolactone-maleimide, aminocaprolactam, distearoylphosphatidylethanolamine-maleimide or aminodistearoylphosphatidylethanolamine, preferably distearoylphosphatidylethanolamine-maleimide.

3. The anti-bacterial/healing-promoting dual drug delivery system according to claim 1 or 2, further comprising an amphiphilic polymer modified with an EGFR-targeting peptide located at the end of the hydrophilic segment of the amphiphilic polymer;

preferably, the loading amount of the EGFR-targeting peptide modified amphiphilic polymer accounts for 11.3-22.7% of the total mass of the antibacterial/healing promoting double-drug delivery system;

preferably, the amino acid sequence of the EGFR targeting peptide comprises KYFPPLALYNPTEYFY or YHWYGYTPQNVI, preferably YHWYGYTPQNVI;

preferably, the hydrophilic segment of the amphiphilic polymer has a number average molecular weight of 1000-4000, preferably 2000.

4. A method of preparing an antibacterial/healing promoting dual drug delivery system according to any one of claims 1 to 3, comprising the steps of: the MMP-9 response peptide is firstly reacted with hydrophobic compounds and then reacted with hydrophilic polymers to prepare the antibacterial/healing promoting dual-drug delivery system.

5. The method of making an anti-microbial/healing promoting dual drug delivery system according to claim 4, wherein the method of making the anti-microbial/healing promoting dual drug delivery system comprises the steps of:

(c) mixing the intermediate product obtained in the step (b), a hydrophilic polymer and an organic solvent C, and reacting to obtain an amphiphilic polymer modified by MMP-9 response peptide;

preferably, in step (a), the organic solvent a comprises any one of methanol, ethanol or acetone or a combination of at least two thereof, preferably methanol;

preferably, in step (a), the organic solvent B comprises any one or a combination of at least two of chloroform, methanol or ethyl acetate, preferably chloroform;

preferably, in the step (b), the reaction temperature is 30-50 ℃, and the reaction time is 3-5 h;

preferably, in step (C), the organic solvent C comprises any one or a combination of at least two of chloroform, methanol or ethyl acetate, preferably chloroform;

preferably, in the step (c), the reaction temperature is 30-50 ℃, and the reaction time is 3-5 h;

preferably, in step (c), the reaction is carried out by adding a catalyst and/or a coupling agent;

preferably, the catalyst comprises any one or a combination of at least two of 4-dimethylaminopyridine, 4- (dimethylamino) pyridine-N-oxide or polypyrrole, preferably 4-dimethylaminopyridine;

preferably, the molar ratio of the catalyst to the hydrophobic compound is (0.05-0.2): 1;

preferably, the coupling agent comprises any one of or a combination of at least two of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, dicyclohexylcarbodiimide or dimethyl carbonate, preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide;

preferably, the molar ratio of the coupling agent to the hydrophobic compound is (1-3): 1;

preferably, in step (c), after the reaction, post-treatment is further performed, and the post-treatment sequentially comprises concentration, redissolution, filtration, dialysis and drying;

preferably, the redissolution is water;

preferably, the filtration is performed by using a 0.22 μm needle filter;

preferably, the molecular weight cut-off of the dialysis bag used for dialysis is 3000-4000Da, and the dialysis time is 20-40 h;

preferably, the drying is vacuum freeze drying.

6. The method of preparing an anti-bacterial/healing promoting dual drug delivery system according to claim 4 or 5, further comprising an EGFR-targeting peptide modified amphiphilic polymer, the method of preparing the anti-bacterial/healing promoting dual drug delivery system comprising the steps of: mixing the MMP-9 response peptide modified polymer with the EGFR targeting peptide modified amphiphilic polymer to obtain the antibacterial/healing promoting double-drug delivery system;

preferably, the weight ratio of the MMP-9 response peptide modified polymer to the EGFR targeting peptide modified amphiphilic polymer is (3-7): 1;

preferably, the preparation method of the EGFR targeting peptide modified amphiphilic polymer comprises the following steps: mixing an EGFR (epidermal growth factor receptor) targeting peptide, an amphiphilic polymer and a solvent, and reacting to obtain the amphiphilic polymer modified by the EGFR targeting peptide;

preferably, the molar ratio of the EGFR-targeting peptide to the amphiphilic polymer is 1 (4-8);

preferably, the solvent comprises any one of acetonitrile, N-dimethylformamide or methanol or a combination of at least two thereof, preferably a combination of acetonitrile and N, N-dimethylformamide;

preferably, the volume ratio of acetonitrile to N, N-dimethylformamide is (0.8-1.2) to 1, preferably 1: 1;

preferably, the reaction time is 20-28h, and the reaction temperature is 25-35 ℃;

preferably, the reaction is carried out under dark conditions;

preferably, the reaction is carried out under a nitrogen atmosphere;

preferably, the reaction also requires the addition of a coupling agent comprising N-hydroxysuccinimide and/or 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine, preferably N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine;

preferably, the molar ratio of the EGFR-targeting peptide, N-hydroxysuccinimide, and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine is 1 (15-25) to (15-25);

preferably, the reaction also requires the addition of N, N-diisopropylethylamine;

preferably, the molar ratio of the EGFR-targeting peptide to N, N-diisopropylethylamine is 1 (0.15-0.25);

preferably, after the reaction, post-treatment is required, and the post-treatment sequentially comprises concentration, re-dissolution, filtration, dialysis and drying;

preferably, the redissolution is water;

preferably, the filtration is performed by using a 0.22 μm needle filter;

preferably, the drying is vacuum freeze drying.

7. A pharmaceutical composition comprising a hydrophobic drug and the antibacterial/pro-healing dual drug delivery system of any one of claims 1-3; wherein the hydrophobic drug is located in the hydrophobic inner core of the antibacterial/pro-healing dual drug delivery system.

8. The pharmaceutical composition according to claim 7, wherein the hydrophobic drug comprises a healing-promoting drug and/or an antibacterial drug, preferably a combination of a healing-promoting drug and an antibacterial drug;

preferably, the weight ratio of the healing promoting drug to the antibacterial drug is (0.5-2) to 1;

preferably, the healing promoting drug comprises any one or combination of at least two of curcumin, quercetin, cinnamaldehyde or soybean isoflavone, preferably curcumin;

preferably, the antibacterial drug comprises any one of rifampicin, vancomycin, linezolid or doxycycline or a combination of at least two thereof;

preferably, the pharmaceutical composition is in the form of spherical micelles, and the average particle size of the spherical micelles is 15-30 nm;

preferably, the drug loading of the pharmaceutical composition is 4-10 wt%;

preferably, the encapsulation efficiency of the pharmaceutical composition is 90-99.9 wt%;

preferably, the pharmaceutical composition further comprises a temperature-sensitive hydrogel, and the drug-loaded micelle is uniformly dispersed in the temperature-sensitive hydrogel;

preferably, the temperature-sensitive hydrogel comprises a polyoxyethylene polyoxypropylene ether block copolymer, preferably a combination of pluronic F127 and pluronic F68;

preferably, the weight ratio of the pluronic F127 and the pluronic F68 is (16-22): (2-8);

preferably, the temperature-sensitive hydrogel also comprises water;

preferably, the volume ratio of the total weight of pluronic F127 and pluronic F68 to water is 1mg (2-4.5) mL;

preferably, the temperature-sensitive hydrogel is prepared by the following preparation method: mixing Pluronic F127 and Pluronic F68 with water, and stirring to obtain the temperature-sensitive hydrogel;

preferably, the stirring temperature is 4-10 ℃, and the stirring time is 36-72 h.

9. A process for the preparation of a pharmaceutical composition according to claim 7 or 8, characterized in that it comprises the following steps: loading the hydrophobic drug into a hydrophobic core of the antibacterial/healing promoting dual-drug delivery system by a film dispersion method to prepare a drug-loaded micelle;

preferably, the thin film dispersion method comprises the following specific steps: mixing the hydrophobic drug, the antibacterial/healing promoting double-drug delivery system and an organic solvent D, and performing rotary evaporation to form a film in which the hydrophobic drug and the amphiphilic polymer are uniformly dispersed; mixing the hydrophobic drug and the film with the amphiphilic polymer uniformly dispersed with water for hydration, and carrying out self-assembly on the polymer to obtain a drug-loaded micelle;

preferably, the organic solvent D comprises any one of tetrahydrofuran, chloroform, methanol or acetone or a combination of at least two thereof, preferably a combination of chloroform and methanol;

preferably, the volume ratio of trichloromethane to methanol is (0.5-2) to 1, preferably 1: 1;

preferably, the weight ratio of the hydrophobic drug to the antibacterial/healing promoting dual drug delivery system is 1 (5-20), preferably 1: 10;

preferably, the temperature of the rotary evaporation is 30-40 ℃, the rotating speed of the rotary evaporation is 60-100r/min, and the time of the rotary evaporation is 0.5-1.5 h;

preferably, the volume of water needed for hydrating the uniformly dispersed film of the hydrophobic drug and the amphiphilic polymer is 1-4 mL;

preferably, the temperature of the hydration is 55-70 ℃, and the time of the hydration is 0.5-1 h;

preferably, the hydration is followed by ultrasound and/or filtration;

preferably, the power of the ultrasound is 250-350W, and the time of the ultrasound is 5-30 min;

preferably, the filtration is performed by using a 0.22 μm water system filter membrane;

preferably, the pharmaceutical composition further comprises a temperature-sensitive hydrogel, and the preparation method of the pharmaceutical composition comprises the following steps: mixing the drug-loaded micelle and the temperature-sensitive hydrogel to obtain a preparation in which the drug composition is uniformly dispersed in the temperature-sensitive hydrogel;

preferably, the volume ratio of the drug-carrying micelle to the temperature-sensitive hydrogel is (0.8-1.2): 1;

preferably, the temperature of the mixing is 4-10 ℃.

10. Use of the dual antibacterial/pro-healing drug delivery system according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 7 or 8, for the preparation of a medicament for the treatment of chronic difficult-to-heal wounds.