CN110124032B - Anti-tumor implant with local chemotherapy and photothermal therapy functions and preparation method thereof - Google Patents

Abstract

The invention discloses an anti-tumor implant with the functions of local chemotherapy and photothermal therapy and a preparation method thereof, and relates to the technical field of drug preparation. The preparation method of the anti-tumor implant with the functions of local chemotherapy and photothermal therapy includes: mixing the drug-loaded polydopamine particle dispersion with a water-soluble polymer solution to obtain an internal phase spinning solution; dissolving gelatin or chitosan After forming the outer phase spinning solution; the inner phase spinning solution and the outer phase spinning solution are subjected to coaxial electrospinning; wherein, the water-soluble polymer is selected from any one of polyvinyl alcohol, polyvinylpyrrolidone and polyethylene oxide, The drug in the drug-loaded polydopamine particles is an antitumor drug. The anti-tumor implant includes nanofibers with a core-shell structure, and the core-shell structure is loaded with drug-loaded nano-polydopamine particles. This fiber implant has the effects of local chemotherapy and photothermal therapy, and is a biological Compatible and biodegradable implant.

Description

Anti-tumor implant with local chemotherapy and photothermal therapy functions and preparation method thereof

technical field

The invention relates to the technical field of drug preparation, in particular to an anti-tumor implant with local chemotherapy and photothermal therapy functions and a preparation method thereof.

Background technique

Malignant tumors are still a serious threat to human health. In terms of tumor treatment, systemic chemotherapy (accompanied by surgical resection, radiotherapy, etc.) is still the main method at present. . In order to improve this mode of administration, some researchers have begun to use various nanocarriers to encapsulate drugs to reduce the toxic and side effects of chemotherapeutic drugs and improve drug efficacy, but most of the nanocarriers enter the body through systemic administration. Therefore, in the process of reaching the tumor tissue, there are still the following problems, such as: clearance by the reticuloendothelial system, excessive enrichment at non-targeted tissues, excessive dependence on the high permeability and retention effects of solid tumors, clinical experiments Or the cumulative amount at the tumor in animal experiments is still insufficient.

In contrast, topical drug delivery systems can effectively overcome these problems. Because it can maintain the local drug concentration at a high level for a certain period of time, it can improve the therapeutic effect, and at the same time effectively reduce the toxic and side effects of the drug on normal organs and tissues, and it does not require repeated administration, which reduces the pain of the patient. . At present, various non-degradable materials (such as ethylene-vinyl acetate copolymers) and degradable materials (such as polyether-polyanhydride copolymers, polyether-polyester copolymers, degradable hydrogel materials, etc.) It is used to prepare various anti-tumor implants for local delivery to various anti-tumor drugs and factors (such as paclitaxel, doxorubicin and other chemotherapy drugs, as well as anti-angiogenesis factors, endostatin genes, etc. These anti-tumor drugs Implants can be made into various forms by various means, such as spheres, discs, solid films, rods or fiber membranes, etc. Among them, the drug-loaded electrospun micro/nanofiber membrane is simple to prepare, has a wide range of material sources, and is suitable for drugs. It can be effectively encapsulated and controlled release, and is easily degraded in vivo (large specific surface area).

As far as some local drug delivery systems reported so far, there are still some problems, such as some locally injected micro/nano drug carriers, which are easy to escape out of the lesion; some locally injected semi-solid drug delivery systems are difficult to use in practice. The gel is formed in time in the body, which leads to the sudden release of the drug; some locally implanted bulk drug delivery systems have difficult to adjust the degradation performance, and it is difficult to effectively control the release of the drug. Therefore, there is still a need to develop new topical drug delivery systems. Anti-tumor implants, especially fibrous implants, are a very effective local drug delivery system, and will belong to a class of locally implanted drug delivery systems in terms of operation. In addition to being directly used for tumor treatment, it can also be used as a postoperative filler, which can effectively prevent tumor recurrence.

However, traditional anti-tumor implants (including some existing fiber implants) still have some of the problems mentioned above. Moreover, most of the drugs contained in the implants are directly released by the degradation of the carrier, but because the released drugs are small molecules, which are easy to diffuse to other tissues, they may also be cleared, and the targeting performance is poor, so it needs to be improved. . In addition, the existing anti-tumor implants also have problems such as immune rejection after surgery, toxicity, and difficulty in degradation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a preparation method of an anti-tumor implant with the functions of local chemotherapy and photothermal therapy, aiming to prepare an anti-tumor implant with the functions of local chemotherapy and photothermal therapy.

Another object of the present invention is to provide an anti-tumor implant with the functions of local chemotherapy and photothermal therapy, which has the functions of local chemotherapy and photothermal therapy.

The present invention solves its technical problems by adopting the following technical solutions.

The present invention provides a preparation method of an anti-tumor implant with local chemotherapy and photothermal therapy functions, comprising the following steps:

Mixing the drug-loaded polydopamine particle dispersion with the water-soluble polymer solution to obtain an internal phase spinning solution;

Dissolving gelatin or chitosan to form an external spinning solution;

Coaxial electrospinning is performed on the inner phase spinning solution and the outer phase spinning solution;

Wherein, the water-soluble polymer is selected from any one of polyvinyl alcohol, polyvinylpyrrolidone and polyethylene oxide, and the drug in the drug-loaded polydopamine particles is an antitumor drug.

The present invention also provides an anti-tumor implant with the functions of local chemotherapy and photothermal therapy, comprising nanofibers with a core-shell structure, and drug-loaded nano-polydopamine particles are loaded in the core-shell structure;

Among them, the drug loaded in the drug-loaded nano-polydopamine particles is an anti-tumor drug, and the anti-tumor drug is doxorubicin or camptothecin;

Preferably, the particle size of the drug-loaded nano-polydopamine particles is 40-130 nm;

More preferably, the anti-tumor implant is prepared by the above-mentioned preparation method.

The embodiment of the present invention provides a method for preparing an anti-tumor implant with the functions of local chemotherapy and photothermal therapy. The beneficial effect is that an internal phase is formed by mixing a drug-loaded polydopamine particle dispersion with a water-soluble polymer solution. spinning solution, dissolving gelatin or chitosan to form an inner phase spinning solution, mixing the inner phase spinning solution and the outer phase spinning solution, and then performing coaxial electrospinning to prepare nanofibers with a core-shell structure, and Nanoparticles of polydopamine are loaded in the inner core of the nanofibers. The anti-tumor implant prepared by the preparation method provided by the invention has the functions of local chemotherapy and photothermal therapy, and is a novel anti-tumor implant.

The present invention also provides an anti-tumor implant with the functions of local chemotherapy and photothermal therapy, comprising nanofibers with a core-shell structure, and drug-loaded nano-polydopamine particles are loaded in the core-shell structure. The implant has the effect of local chemotherapy and photothermal therapy, and is a non-toxic and easily degradable implant.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the transmission electron microscope picture of the anti-tumor implant provided by the embodiment of the present invention;

Fig. 2 is the heating curve of 44nm particle under different concentrations;

Fig. 3 is the heating curve of 44nm particle at different concentrations;

Fig. 4 is the heating curve of 137nm particles under different concentrations;

Figure 5 is an in vitro photothermal cytotoxicity experiment of electrospun fibers loaded with dopamine particles.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The anti-tumor implants and preparation methods thereof provided in the embodiments of the present invention will be specifically described below.

The embodiment of the present invention provides a method for preparing an anti-tumor implant with local chemotherapy and photothermal therapy functions, comprising the following steps:

S1. Preparation of internal phase spinning solution

The inner phase spinning solution is obtained by mixing the drug-loaded polydopamine particle dispersion with the water-soluble polymer solution; the drug in the drug-loaded polydopamine particles is an antitumor drug. Among them, the water-soluble polymer is selected from any one of polyvinyl alcohol, polyvinylpyrrolidone and polyethylene oxide, preferably polyvinyl alcohol.

It should be noted that the water-soluble polymer is the main part of the internal phase spinning solution. After spinning, the water-soluble polymer is a solid matrix, and the drug-loaded polydopamine particles will be dispersed into it. In the subsequent use process, the water-soluble polymer is used. Will dissolve in water. Preferably, the particle size of the drug-loaded polydopamine particles is 40-130 nm.

Preferably, the mixing process of the drug-loaded polydopamine particle dispersion and the polyvinyl alcohol solution is to drop the drug-loaded polydopamine particle dispersion into the polyvinyl alcohol solution, and mixing by dropwise mixing can make the drug-loaded polydopamine particles It is uniformly dispersed into the polyvinyl alcohol solution to improve the uniformity of the final product.

Preferably, the drug loading amount of the drug-loaded polydopamine particles is 10%-30%. The concentration of polyvinyl alcohol in the inner phase spinning solution is 80-120 g/L, and the mass ratio of drug-loaded polydopamine particles to polyvinyl alcohol is 1:5-20. The uniformity and stability of the final product are controlled by controlling the mass ratio of the drug-loaded polydopamine particles and polyvinyl alcohol. If the amount of polyvinyl alcohol is too small, it will affect the uniformity of the drug-loaded polydopamine particle dispersion and affect the efficacy of the product. .

In some embodiments, the concentration of the polyvinyl alcohol solution is 150-250 g/L, and the concentration of the polyvinyl alcohol is adjusted with deionized water after mixing the drug-loaded polydopamine particle dispersion with the polyvinyl alcohol solution. The concentration of the polyvinyl alcohol solution is slightly higher than the concentration of the polyvinyl alcohol in the inner phase spinning solution, which makes the operation more convenient and feasible. Adjust the concentration of polyvinyl alcohol to the specified range with deionized water.

Specifically, the preparation of the drug-loaded polydopamine particles can be commercially available polydopamine particles and then the drug-loaded dopamine particles are prepared by means of adsorption. Preferably, the drug-loaded polydopamine particle dispersion is concentrated by mixing the drug-loaded polydopamine particles with water, and mixing the drug-loaded polydopamine particle suspension with a higher concentration is conducive to the subsequent regulation of the dosage of each component. If the concentration of polyvinyl alcohol is adjusted to the specified range, it is beneficial to the spinning work.

The preparation process of the drug-loaded polydopamine particles includes: after mixing the polydopamine particles with polyethylene glycol modified with a tumor-targeting molecule at one end, reacting at a temperature of 15-25° C. for 20-30 hours to obtain the targeted polydopamine particles ; Mix the targeted polydopamine particles, antitumor drugs and tris buffer solution in the dark and stir for 20-30h, and then dialyze and spin-evaporate.

In some embodiments, the preparation process of the polydopamine particles includes: mixing ammonia water, an organic solvent and water to obtain a first mixed solution, adding the dopamine hydrochloride solution dropwise to the first mixed solution, and at a temperature of 15-25° C. The reaction is carried out for 20-30h under temperature conditions, and then the polydopamine particles are separated;

In another embodiment, the preparation process of polydopamine particles includes: adding sodium hydroxide aqueous solution dropwise to the dopamine hydrochloride aqueous solution, reacting at a temperature of 30-70° C. for 3-10 hours, and then separating the polydopamine particles . The particle size can be adjusted by adjusting the concentration of dopamine hydrochloride, the amount of sodium hydroxide and the temperature. The decrease of the concentration of dopamine hydrochloride, the increase of the amount of sodium hydroxide or the increase of the reaction temperature will make the particle size smaller.

Specifically, the organic solvent used in the process of preparing the polydopamine particles can be a solvent that is miscible with water, such as ethanol, methanol, dimethyl sulfoxide, acetone, tetrahydrofuran, and the like. When the particle size of the polydopamine particles is greater than 100 nm, the separation of the polydopamine particles is by centrifugal separation; preferably, the particles obtained after centrifugal separation are re-dispersed with water and then centrifuged; when the particle size of the polydopamine particles is less than 100 nm, the polydopamine particles are separated. The separation method of dialysis and then rotary evaporation is adopted; preferably, during the dialysis process, a dialysis bag with a molecular weight of 13500-14500 is used for dialysis for 30-40 hours, and the rotary evaporation temperature is 25-35 °C. The particle size of the final product is controlled by the amount of ammonia water. The more ammonia water is used, the smaller the particle size of the product.

In some embodiments, the polyethylene glycol modified with the tumor targeting molecule at one end can be a commercially available product, such as a Sigma-Aldrich manufacturer.

In another embodiment, the preparation process of the polyethylene glycol modified with the tumor targeting molecule at one end includes: after mixing and dissolving the double-ended amino-modified polyethylene glycol and the tumor targeting molecule, under the action of a catalyst amide reaction, then isolated, purified and lyophilized. Specifically, the tumor targeting molecule is selected from any one of folic acid, phenylboronic acid, aptamer and RGD polypeptide, and the catalyst is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) ) carbodiimide hydrochloride.

Specifically, the anti-tumor drug is selected from doxorubicin or camptothecin, and the two anti-tumor drugs can be adsorbed on the targeted polydopamine particles, thereby loading the anti-tumor drug on the targeted polydopamine particles.

S2. Preparation of external phase spinning solution

The outer phase spinning solution is formed after dissolving gelatin or chitosan, and the shell structure can be formed in electrospinning by using gelatin or chitosan.

In some embodiments, the external phase spinning solution is a mixed solution formed by dissolving gelatin and genipin (ie, the first spinning solution); adding genipin to the raw materials of the external phase spinning solution can make genipin and gelatin Cross-linking occurs, improving the mechanical properties of the product and making the product easy to degrade.

In another embodiment, the external phase spinning solution is a mixed solution (ie, the second spinning solution) formed by dissolving chitosan and polyethylene oxide. Preferably, the mass ratio of chitosan: polyethylene oxide in the second spinning solution is 1:0.8-1.2, and the total concentration of chitosan and polyethylene oxide is 50-70 g/L. More preferably, when using the second spinning solution as the external phase spinning solution, after spinning, the filaments are washed 5-15 times with the ethanol solution of genipin, and then the filaments are placed in the ethanol solution of genipin. Soak in ethanol for 10-24 hours, and then soak in absolute ethanol for 2 hours; further preferably, the concentration of genipin in ethanol solution is 0.2-1% by mass/volume.

Preferably, the solvents of the external phase spinning solution are acetic acid and water, and the volume ratio of acetic acid and water is 7-11:1; preferably, the external phase spinning solution is stirred for 8-15 hours before electrospinning. Using the mixed solvent formed by acetic acid and water as the raw material can enhance the spinning effect and make the stability of the spinning process and the uniformity of spinning better.

Further, the total concentration of gelatin and genipin in the external phase spinning solution is 200-250 g/L, and the mass ratio of gelatin and genipin is 25-35:3. The dosage of gelatin and genipin needs to be controlled. Gelatin is the main raw material for spinning. Controlling the dosage of the two within the above range can effectively improve the uniformity and strength of the spinning product, while giving the product good degradability.

S3, coaxial electrospinning

The inner phase spinning solution and the outer phase spinning solution are coaxially electrospun. The specific operation of coaxial spinning can refer to the existing technology. Generally speaking, the coaxial electrospinning process includes: placing the inner phase spinning solution and the outer phase spinning solution in a syringe respectively, and connecting the syringe to the coaxial spinning solution. The inner and outer inlets of the needle are then fixed horizontally on the microfluidic syringe pump; the aluminum foil-wrapped drum is fixed at 10-20 cm from the needle tip as a fiber receiver, and the rotation speed is about 80-120 rpm. In the process of coaxial electrospinning, the operating voltage is 12-20kv, the pushing speed is 0.1-0.2mL/h, the receiving distance is 10-20cm, the operating temperature is 15-25℃, and the operating humidity should be controlled within 40%.

An embodiment of the present invention also provides an anti-tumor implant, comprising nanofibers with a core-shell structure, and drug-loaded nano-polydopamine particles are loaded in the inner core of the core-shell structure; wherein, the drug-loaded nano-polydopamine particles are The loaded drug is an anti-tumor drug, and the anti-tumor drug is doxorubicin or camptothecin; preferably, the particle size of the drug-loaded nano-polydopamine particles is 40-130 nm.

It should be noted that the anti-tumor implant is a nanofiber with a core-shell structure, and drug-loaded nano-polydopamine particles are loaded in the inner core, and the implant has tumor targeting and photothermal therapy effects.

Preferably, the anti-tumor implant can be prepared by the above-mentioned preparation method.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

The present embodiment provides a method for preparing an anti-tumor implant, comprising the following steps:

First, the preparation of drug-loaded polydopamine particles:

(1) Preparation of polydopamine particles. (a) Mix together 5 mL of ammonia water, 40 mL of absolute ethanol and 90 mL of deionized water, and stir for about 30 minutes to make the mixture uniform. (b) Weigh out 0.5 g of dopamine hydrochloride and completely dissolve it in 30 mL of deionized water. (c) The dopamine hydrochloride solution in (b) was added dropwise to the mixed solution in (a), and stirring was continued for 24 hours. It can be found that the color of the mixed solution gradually changed to dark brown during the dropwise addition. (d) After 20 hours of reaction at 15°C, the generated polydopamine particles were collected by centrifugation, and the particles were washed three times by means of redispersion and centrifugation with distilled water to obtain dopamine particles with better monodispersity.

(2) Preparation of polyethylene glycol molecules with single-end modified tumor targeting molecules. Dissolve 10 g of double-ended amino-modified polyethylene glycol (NH ₂ -PEG-NH ₂ ) and 1 g of folic acid in 80 mL of dimethyl sulfoxide solvent, in 0.5 g of N-hydroxysuccinimide and 1 g of 1- Under the catalysis of ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, the folic acid molecule was linked to NH ₂ -PEG-NH ₂ through an amide reaction (reaction at 15 °C for 20 h). on the single-ended amino group. Then, it is separated and purified, and after freeze-drying, a polyethylene glycol molecule modified with single-end folic acid is obtained.

(3) Weigh 20 mg of polydopamine particles and 10 mg of doxorubicin, add them to 20 mL of TIRS buffer (pH 8.5), stir in the dark for 20 hours, dialyze with a dialysis bag of 1500 molecular weight for 30 hours, and then rotate at 30°C to obtain Concentrate.

Secondly, the preparation of internal phase spinning solution:

The dispersion liquid formed after mixing the above-mentioned drug-loaded polydopamine particles with water is concentrated by centrifugal collection and redispersion, and then the high-concentration dispersion is added dropwise to the polyvinyl alcohol aqueous solution with a concentration of 150 g/L dissolved in advance, And add deionized water to the mixed solution so that the concentration of polyvinyl alcohol is 80g/L, and stir evenly. The mass ratio of drug-loaded polydopamine particles to polyvinyl alcohol was controlled to be 1:5.

Then, the preparation of the external phase spinning solution:

The external phase spinning solution is a mixed solvent formed by dissolving gelatin and genipin in acetic acid and water. The total concentration of the solute in the external phase spinning solution is 200 g/L, the mass ratio of gelatin and genipin is 25:3, and the acetic acid is 25:3. The volume ratio to water is 7:1. Before electrospinning, the external phase spinning solution was stirred for about 8 hours, so that the gelatin was cross-linked by genipin, and the whole was dark blue.

Finally, coaxial electrospinning

Put equal volumes of the inner phase spinning solution and the outer phase spinning solution into a 5 mL syringe respectively, and connect them to the inner and outer inlets of the coaxial spinning needle, and then fix the syringe horizontally on the microfluidic syringe pump. A clean and grounded foil-wrapped drum was fixed about 10 cm from the needle tip as a fiber receiver at about 80 rpm (low speed). During spinning, an electrostatic high voltage of about 12 kV was applied between the needle and the receiving plate, and at the same time, the driving speed of the inner and outer phases of the spinning solution was controlled at 0.1 mL/h. All operations were completed at about 15°C. After the fibers were collected, they were placed in a vacuum drying oven for vacuum drying to remove the solvent that was not completely volatilized.

Example 2

The present embodiment provides a method for preparing an anti-tumor implant, comprising the following steps:

First, the preparation of drug-loaded polydopamine particles:

(1) Preparation of polydopamine particles. (a) Mix together 9 mL of ammonia water, 40 mL of absolute ethanol and 90 mL of deionized water, and stir for about 30 minutes to make the mixture uniform. (b) Weigh out 0.5 g of dopamine hydrochloride and completely dissolve it in 30 mL of deionized water. (c) The dopamine hydrochloride solution in (b) was added dropwise to the mixed solution in (a), and stirring was continued for 24 hours. It can be found that the color of the mixed solution gradually changed to dark brown during the dropwise addition. (d) After 30 hours of reaction at 25°C, the generated polydopamine particles were dialyzed with a dialysis bag with a molecular weight of 13500 for 30 hours, and then obtained by rotary evaporation at 25°C. The dopamine particle concentrate with better monodispersity can be obtained.

(2) Preparation of polyethylene glycol molecules with single-end modified tumor targeting molecules. Dissolve 4g of double-ended amino-modified polyethylene glycol (NH ₂ -PEG-NH ₂ ) and 0.3g of phenylboronic acid monohydrate in 50mL of dimethyl sulfoxide solvent, in 0.2g of N-hydroxysuccinimide and 4g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyzed by amide reaction (after 48h of reaction at 25°C, 5mL of deionized water was added to terminate the reaction) A folic acid molecule is attached to the single-terminal amino group of _NH2 -PEG- _NH2 . The solid was filtered off, and then the filtrate was dialyzed with a dialysis bag (MWCO 1000) for 3 days, and the residue was freeze-dried to obtain a polyethylene glycol molecule modified with single-end folic acid.

(3) Weigh 20 mg of polydopamine particles and 10 mg of doxorubicin, add them to 20 mL of TIRS buffer (pH 8.5), stir in the dark for 30 hours, dialyze them with a dialysis bag of 1500 molecular weight for 40 hours, and then rotate at 30°C to obtain Concentrate.

Secondly, the preparation of internal phase spinning solution:

The dispersion liquid formed after mixing the above-mentioned drug-loaded polydopamine particles with water is concentrated by centrifugal collection and re-dispersion, and then the high-concentration dispersion is added dropwise to the polyvinyl alcohol aqueous solution with a concentration of 250 g/L dissolved in advance, And add deionized water to the mixed solution to make the polyvinyl alcohol concentration 120g/L, and stir evenly. The mass ratio of drug-loaded polydopamine particles to polyvinyl alcohol was controlled to be 1:20.

Then, the preparation of the external phase spinning solution:

The external phase spinning solution is a mixed solvent formed by dissolving gelatin and genipin in acetic acid and water. The total concentration of the solute in the external phase spinning solution is 250 g/L, the mass ratio of gelatin and genipin is 35:3, and the acetic acid is 35:3. The volume ratio to water is 11:1. Before electrospinning, the external phase spinning solution was stirred for about 15 hours, so that the gelatin was cross-linked by genipin, and the whole was dark blue.

Finally, coaxial electrospinning

Put equal volumes of the inner phase spinning solution and the outer phase spinning solution into a 5 mL syringe respectively, and connect them to the inner and outer inlets of the coaxial spinning needle, and then fix the syringe horizontally on the microfluidic syringe pump. A clean and grounded foil-wrapped drum was fixed about 20 cm from the needle tip as a fiber receiver at about 120 rpm (low speed). During spinning, an electrostatic high voltage of about 20 kV was applied between the needle and the receiving plate, and the pushing speed of the inner and outer phases of the spinning solution was controlled at 0.2 mL/h. All operations were completed at about 30°C. After the fibers were collected, they were placed in a vacuum drying oven for vacuum drying to remove the solvent that was not completely volatilized.

Example 3

The present embodiment provides a method for preparing an anti-tumor implant, comprising the following steps:

First, the preparation of drug-loaded polydopamine particles:

(1) Preparation of polydopamine particles. (a) Mix together 9 mL of ammonia water, 40 mL of absolute ethanol and 90 mL of deionized water, and stir for about 30 minutes to make the mixture uniform. (b) Weigh out 0.5 g of dopamine hydrochloride and completely dissolve it in 30 mL of deionized water. (c) The dopamine hydrochloride solution in (b) was added dropwise to the mixed solution in (a), and stirring was continued for 24 hours. It can be found that the color of the mixed solution gradually changed to dark brown during the dropwise addition. (d) After reacting at 25°C for 30 hours, the generated polydopamine particles were dialyzed with a dialysis bag with a molecular weight of 14500 for 40 hours, and then obtained by rotary evaporation at 35°C. The dopamine particle concentrate with better monodispersity can be obtained.

(2) Preparation of polyethylene glycol molecules with single-end modified tumor targeting molecules. Dissolve 4g of double-ended amino-modified polyethylene glycol (NH ₂ -PEG-NH ₂ ) and 0.3g of phenylboronic acid monohydrate in 50mL of dimethyl sulfoxide solvent, in 0.2g of N-hydroxysuccinimide and 0.4g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride catalyzed by amide reaction (reaction at 20°C for 24h) to connect the folic acid molecule to NH ₂ - on the single-terminal amino group of PEG- _NH2 . Then, it is separated and purified, and after freeze-drying, a polyethylene glycol molecule modified with single-end folic acid is obtained.

(3) Weigh 20 mg of polydopamine particles and 10 mg of doxorubicin into 20 mL of TIRS buffer (pH 8.5), stir in the dark for 30 hours, dialyze with a dialysis bag with a molecular weight of 1500 for 36 hours, and then rotate at 30°C to obtain Concentrate.

Secondly, the preparation of internal phase spinning solution:

The dispersion liquid formed after mixing the above-mentioned drug-loaded polydopamine particles and water is concentrated by centrifugal collection and re-dispersion, and then the high-concentration dispersion is added dropwise to the polyvinyl alcohol aqueous solution with a concentration of 200 g/L dissolved in advance, And add deionized water to the mixed solution to make the polyvinyl alcohol concentration 100g/L, and stir evenly. The mass ratio of drug-loaded polydopamine particles to polyvinyl alcohol was controlled to be 1:10.

Then, the preparation of the external phase spinning solution:

The external phase spinning solution is a mixed solvent formed by dissolving gelatin and genipin in acetic acid and water. The total concentration of the solute in the external phase spinning solution is 220 g/L, the mass ratio of gelatin and genipin is 30:3, and the acetic acid is 30:3. The volume ratio to water is 9:1. Before electrospinning, the external phase spinning solution was stirred for about 10 hours, so that the gelatin was cross-linked by genipin, and the whole was dark blue.

Finally, coaxial electrospinning

Put equal volumes of the inner phase spinning solution and the outer phase spinning solution into a 5 mL syringe respectively, and connect them to the inner and outer inlets of the coaxial spinning needle, and then fix the syringe horizontally on the microfluidic syringe pump. A clean and grounded foil-wrapped drum was fixed about 14 cm from the needle tip as a fiber receiver at about 100 rpm (low speed). During spinning, an electrostatic high voltage of about 18 kV was applied between the needle and the receiving plate, and at the same time, the driving speed of the inner and outer phases of the spinning solution was controlled at 0.15 mL/h. All operations were completed at about 27°C. After the fibers were collected, they were placed in a vacuum drying oven for vacuum drying to remove the solvent that was not completely volatilized.

Example 4

The present embodiment provides a method for preparing an anti-tumor implant. The specific steps are roughly the same as those in Example 3, except that the solute in the external phase spinning solution only includes gelatin, and the dosage is equivalent to that of gelatin and genipin. Sum.

Example 5

This example provides a method for preparing an anti-tumor implant, the specific steps are roughly the same as those in Example 3, the differences are: (1) the solutes in the external phase spinning solution are chitosan and polyethylene oxide, and the shell The total concentration of polysaccharide and polyethylene oxide was 50 g/L, and the mass ratio of the two was 1:1. After successful spinning, the filaments are washed 5-10 times with an ethanol solution of genipin, and then the filaments are soaked in the solution for 24 hours to achieve the purpose of cross-linked chitosan. Soak in absolute ethanol for 2 hours. The concentration of genipin in ethanol solution is 0.5% by mass/volume. (2) The polydopamine particles in the inner phase are pure polydopamine particles without drug (doxorubicin).

Example 6

The present embodiment provides a method for preparing an anti-tumor implant. The specific steps are roughly the same as those in Example 3, except that:

The preparation of polydopamine particles includes the following steps: (a) 180 mg of dopamine hydrochloride is added to 90 mL of deionized water and mixed together, and stirred for about 30 minutes to make it evenly mixed. (b) Dopamine hydrochloride:hydroxide = 1:0.8 in molar ratio, the corresponding aqueous sodium hydroxide solution (37.6 mg sodium hydroxide dissolved in 1 mL water) was added dropwise to the mixed solution in (a), in Continuous stirring at 50°C for 5 hours, the generated polydopamine particles were collected by centrifugation, and the particles were washed three times by means of redispersion and centrifugation with distilled water to obtain dopamine particles with better monodispersity.

Example 7

The present embodiment provides a method for preparing an anti-tumor implant. The specific steps are roughly the same as those in Example 3, except that:

The preparation of polydopamine particles includes the following steps: (a) 120 mg of dopamine hydrochloride is added to 90 mL of deionized water and mixed together, and stirred for about 30 minutes to make it uniformly mixed. (b) Dopamine hydrochloride:hydroxide=1:1 in molar ratio, the corresponding aqueous sodium hydroxide solution (31.34mg sodium hydroxide dissolved in 1mL water) was added dropwise to the mixed solution in (a), in The resulting polydopamine particles were dialyzed with a dialysis bag with a molecular weight of 13,500 for 30 hours under continuous stirring at 70°C for 5 hours, and then obtained by rotary evaporation at 25°C. The dopamine particle concentrate with better monodispersity can be obtained.

Comparative Example 1

This comparative example provides a preparation method of an anti-tumor implant, and the specific steps are roughly the same as those in Example 3, except that the pushing speed in the coaxial electrospinning is 0.05 mL/h.

Comparative Example 2

This comparative example provides a preparation method of an anti-tumor implant, and the specific steps are roughly the same as those in Example 3, except that the pushing speed in the coaxial electrospinning is 0.3 mL/h.

Comparative Example 3

This comparative example provides a preparation method of an anti-tumor implant. The specific steps are roughly the same as those in Example 3, except that the size of the polydopamine nanoparticles contained in the inner phase is 400 nm.

Test Example 1

The transmission electron microscope image of the anti-tumor implant prepared in Test Example 5, and the results are shown in FIG. 1 . It can be seen from Figure 1 that the fiber as a whole is indeed in a core-shell structure through transmission electron microscopy, and the interior is darker, which may be due to the presence of polydopamine, so the contrast under the lens is more obvious than that of the shell.

In Comparative Example 1, it is easy to cause blockage at the outlet of the needle because the pushing speed is too slow. In Comparative Example 2, because the pushing speed is too fast, it will cause a large loss of raw materials and uneven fibers. In Comparative Example 3, because the particle size is too large, it cannot be completely removed. Fiber encapsulation, while not conducive to the spread of deep tumor tissue. Therefore, although the method of coaxial spinning is roughly the same as the existing method, the parameter control during the spinning process has a significant impact on the stability of the spinning process and the uniformity of the product.

Test Example 2

The polydopamine particle diameter prepared in Example 3 is roughly 44nm, and it is mixed with water to form suspensions of different concentrations, and the photothermal heating effect of the polydopamine particle suspensions of different concentrations is tested, and the test results are shown in Figure 2- image 3. The particle diameter of the polydopamine particles prepared in Example 1 was approximately 137 nm, and it was mixed with water to form suspensions of different concentrations, and the photothermal heating effects of the polydopamine particle suspensions of different concentrations were tested. The test results are shown in Figure 4.

Test method: Take 44nm polydopamine particles as an example, put 3mL of deionized water in a centrifuge tube with a capacity of 5mL as a control group, and put 3mL of polydopamine particles at concentrations of 50μg/mL, 100μg/mL, 200μg/mL and 400μg The polydopamine/water dispersion of /mL was placed in a 5mL centrifuge tube as the experimental group, irradiated with an 808nm (1W/cm ² or 2W/cm ² ) near-infrared laser for 600s, and an infrared thermometer was used to record the temperature every 30s. . The photothermal heating effect test steps of 137nm polydopamine particles are roughly the same.

It can be seen from Figure 2-Figure 4 that the temperature rise of particles with different concentrations increases with the prolongation of illumination time; and under the same illumination conditions, the higher the concentration, the greater the temperature rise of the nanoparticles, that is, the stronger the photothermal conversion ability. Under the same size conditions (Figures 2 and 3), the greater the light power, the greater the temperature rise. Under the same laser (808nm, 1W/cm ² ) irradiation conditions (Figures 2 and 4), the larger the particle size, the larger the temperature rise and the stronger the photothermal conversion ability.

Test Example 3

The mouse breast cancer cells were co-cultured with the anti-tumor implant prepared in Example 5, and the anti-tumor implant was irradiated with a near-infrared laser (1.5W/cm ² ) under the condition of near-infrared laser (1.5W/cm 2 ) in vitro. The anti-tumor effect, the results are shown in Figure 5.

Test method: The mouse breast cancer cells were inoculated in a 48-well plate and cultured for 24 hours (5.0×10 ⁴ cells per well, 800 μL of medium), and then the anti-tumor implants (fibrous membranes) prepared in Example 5 were added. ) gently transfer to the well plate. The samples were exposed to 808 nm laser at 1.5 W/cm ² for 5, 10, 15 or 20 minutes, respectively, and then cell viability was determined by Alamar-Blue method. For details of Alamar-Blue method, please refer to the instruction manual of Alamar-Blue reagent.

It can be seen from Fig. 5 that the survival rate of tumor cells in the non-irradiated group is about 100%, which does not affect the normal activity of the cells, indicating that the anti-tumor embedding agent has no effect on tumor cells under the condition of no drug loading and no light. It has no toxicity; and the tumor cell survival rate after applying light is only about 20%, indicating that the anti-tumor implant can achieve good anti-tumor effect without drug loading and after a short period of light irradiation.

To sum up, the present invention provides a method for preparing an anti-tumor implant with the functions of local chemotherapy and photothermal therapy, which forms an internal phase by mixing a drug-loaded polydopamine particle dispersion with a water-soluble polymer solution. spinning solution, dissolving gelatin or chitosan to form an inner phase spinning solution, mixing the inner phase spinning solution and the outer phase spinning solution, and then performing coaxial electrospinning to prepare nanofibers with a core-shell structure, and Nanoparticles of polydopamine are loaded in the inner core of the nanofibers. The anti-tumor implant prepared by the preparation method provided by the invention has the functions of local chemotherapy and photothermal therapy.

The present invention also provides an anti-tumor implant, comprising nanofibers with a core-shell structure and loaded with drug-loaded nano-polydopamine particles in the core-shell structure, and the fiber implant has local chemotherapy and photothermal therapy. Effect.

The above-described embodiments are some, but not all, embodiments of the present invention. The detailed descriptions of the embodiments of the invention are not intended to limit the scope of the invention as claimed, but are merely representative of selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims (14)

1. A preparation method of an anti-tumor implant with local chemotherapy and photothermal therapy functions is characterized by comprising the following steps:

mixing the drug-loaded polydopamine particle dispersion liquid with a water-soluble polymer solution to obtain an internal phase spinning solution;

dissolving gelatin or chitosan to form external phase spinning solution;

carrying out coaxial electrostatic spinning on the internal phase spinning solution and the external phase spinning solution;

wherein the water-soluble polymer is selected from any one of polyvinyl alcohol, polyvinylpyrrolidone and polyethylene oxide, and the drug in the drug-loaded polydopamine particles is an anti-tumor drug;

the particle size of the drug-loaded polydopamine particles is 40-130 nm;

the water-soluble polymer is polyvinyl alcohol, the concentration of the polyvinyl alcohol in the internal phase spinning solution is 80-120g/L, and the mass ratio of the drug-loaded polydopamine particles to the polyvinyl alcohol is 1: 5-20;

the external-phase spinning solution is a first spinning solution or a second spinning solution, the first spinning solution is a mixed solution formed by dissolving gelatin and genipin, and the second spinning solution is a mixed solution formed by dissolving chitosan and polyethylene oxide;

in the first spinning solution, the total concentration of gelatin and genipin is 200-250g/L, and the mass ratio of gelatin to genipin is 25-35: 3;

and (3) chitosan in the second spinning solution: the mass ratio of the polyoxyethylene is 1:0.8-1.2, and the total concentration of the chitosan and the polyoxyethylene is 50-70 g/L;

in the coaxial electrostatic spinning process, the operating voltage is 12-20kv, the pushing speed is 0.1-0.2mL/h, the receiving distance is 10-20cm, and the operating temperature is 15-30 ℃.

2. The method for preparing an anti-tumor implant with local chemotherapy and photothermal therapy functions as claimed in claim 1, wherein the process of mixing the drug-loaded polydopamine particle dispersion with the polyvinyl alcohol solution is dropping the drug-loaded polydopamine particle dispersion into the polyvinyl alcohol solution.

3. The preparation method of the anti-tumor implant with local chemotherapy and photothermal therapy functions as claimed in claim 1, wherein the drug-loaded polydopamine particles have a drug loading of 10% -30%.

4. The method for preparing the anti-tumor implant with local chemotherapy and photothermal therapy functions according to claim 1, wherein the process for preparing the drug-loaded polydopamine particles comprises the following steps:

mixing polydopamine particles and polyethylene glycol with a tumor targeting molecule modified at one end, and reacting at the temperature of 15-25 ℃ for 20-30h to obtain targeted polydopamine particles with a tumor targeting function;

mixing the targeted polydopamine particles, the antitumor drugs and the tris buffer solution, stirring in the dark for 20-30h, and dialyzing and steaming to obtain drug-loaded nanoparticles with a tumor targeting function;

wherein the antitumor drug is selected from adriamycin or camptothecin.

5. The method for preparing the antitumor implant with local chemotherapy and photothermal therapy functions as claimed in claim 4, wherein the polydopamine particle is prepared by a process comprising: mixing ammonia water, an organic solvent and water to obtain a first mixed solution, dropwise adding a dopamine hydrochloride solution into the first mixed solution, reacting for 20-30h at the temperature of 15-25 ℃, and then separating out polydopamine particles.

6. The method for preparing the antitumor implant with local chemotherapy and photothermal therapy functions as claimed in claim 4, wherein the polydopamine particle is prepared by a process comprising: dropping the aqueous solution of sodium hydroxide into the aqueous solution of dopamine hydrochloride, reacting for 3-10h at the temperature of 30-70 ℃, and then separating out the polydopamine particles.

7. The method for preparing an anti-tumor implant with local chemotherapy and photothermal therapy functions as claimed in claim 6, wherein when the particle size of the polydopamine particles is greater than 100nm, the separation of the polydopamine particles is performed by centrifugation; re-dispersing the particles obtained after centrifugal separation with water and re-centrifuging to remove free drug;

when the particle size of the polydopamine particles is less than 100nm, the polydopamine particles are separated and concentrated in a dialysis and rotary evaporation mode; during dialysis, dialyzing with a dialysis bag with molecular weight of 13500-14500 at rotary evaporation temperature of 25-35 deg.C for 30-40 h.

8. The method for preparing the antitumor implant with local chemotherapy and photothermal therapy functions as claimed in claim 4, wherein the preparation process of the polyethylene glycol with the tumor targeting molecule modified at the single end comprises: mixing and dissolving the polyethylene glycol modified by ammonia at the two ends and the tumor targeting molecules, carrying out an amide reaction under the action of a catalyst, and then separating, purifying, freezing and drying.

9. The method for preparing an antitumor implant with local chemotherapy and photothermal therapy functions according to claim 8, wherein the tumor targeting molecule is selected from any one of folic acid, phenylboronic acid, aptamers and RGD polypeptide, and the catalyst is a mixture of N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.

10. The method for preparing an antitumor implant having local chemotherapy and photothermal therapy effects according to claim 1, wherein the external phase spinning solution is stirred for 8-15 hours before electrospinning.

11. The method for preparing an anti-tumor implant having local chemotherapy and photothermal therapy functions according to claim 1, wherein the solvent of the external phase spinning solution is acetic acid and water, and the volume ratio of acetic acid to water is 7-11: 1.

12. The method for preparing the antitumor implant with local chemotherapy and photothermal therapy functions according to claim 1, wherein when the second spinning solution is used as the external phase spinning solution, after spinning, the fiber filaments are washed with genipin ethanol solution for 5-15 times, then soaked in genipin ethanol solution for 10-24 hours, and then soaked in absolute ethanol for 2 hours; the concentration of the ethanol solution of genipin is 0.2-1% of mass/volume percent.

13. The method for preparing the antitumor implant with local chemotherapy and photothermal therapy functions as claimed in claim 1, wherein the coaxial electrospinning process comprises: respectively placing the internal phase spinning solution and the external phase spinning solution into an injector, connecting the injector to an internal inlet and an external inlet of a coaxial spinning needle, and horizontally fixing the injector on a micro-flow injection pump; and fixing a roller wrapped by the aluminum foil paper at a position 10-20cm away from the needle point as a fiber receiver, wherein the rotating speed is 80-120 rpm.

14. An anti-tumor implant with local chemotherapy and photothermal therapy functions, which is prepared by the preparation method of any one of claims 1 to 13, and comprises nanofibers with a core-shell structure, wherein drug-loaded nano poly dopamine particles are loaded in the inner core of the core-shell structure.

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