CN111110659A

CN111110659A - Anti-tumor nano preparation and application thereof in targeted therapy of malignant tumor

Info

Publication number: CN111110659A
Application number: CN202010127408.0A
Authority: CN
Inventors: 李翔; 张婧; 邹茜
Original assignee: Jiangxi University of Traditional Chinese Medicine; Jiangxi Bencao Tiangong Technology Co Ltd
Current assignee: Jiangxi University of Traditional Chinese Medicine; Jiangxi Bencao Tiangong Technology Co Ltd
Priority date: 2020-02-28
Filing date: 2020-02-28
Publication date: 2020-05-08
Anticipated expiration: 2040-02-28
Also published as: CN111110659B

Abstract

The invention discloses an anti-tumor nano preparation and application thereof in targeted therapy of malignant tumors, wherein poly-dopamine nanoparticles are used as a photothermal conversion material to prepare the anti-tumor nano preparation with high photothermal conversion efficiency, good degradability in vivo, good biocompatibility and almost no cytotoxicity; meanwhile, the treatment effect of killing tumor cells is achieved by combining photothermal treatment. After the preparation is injected into female mice inoculated with 4T1-luc cell BALB/C in tail vein, the photothermal therapy has obviously better therapeutic effect than the conventional therapy, and the therapeutic mode has small wound area, almost no toxic or side effect, reduces the administration dosage and reduces the pain compared with the surgical therapy.

Description

Anti-tumor nano preparation and application thereof in targeted therapy of malignant tumor

Technical Field

The invention relates to research and application of chemotherapy in combination with photothermal therapy on malignant tumors, in particular to an anti-tumor nano preparation and application thereof in targeted therapy on malignant tumors.

Background

In recent years, photothermal therapy has been widely focused and advanced by academia, and the therapeutic principle is that photothermal material absorbs laser to convert light energy into heat energy to rapidly raise the temperature of tumor part, so that the temperature exceeds the tolerance temperature of cancer cells by 50 ℃, thereby killing cancer cells. The effect of the combination therapy of malignant tumor by various means is obviously increased to a certain extent, the photothermal therapy and the chemical therapy of the tumor are combined to obtain good results to a great extent, and the photothermal therapy and the chemical therapy have good application prospects due to small toxic and side effects and low wound, so that the photothermal therapy and the chemical therapy become a great hotspot of research. The precious metal nanoparticles Au, Ag and the like have high photothermal conversion efficiency, but are expensive, have poor degradability in vivo and the like, so that the application of the precious metal nanoparticles is limited, and the polydopamine nanoparticles not only have high photothermal conversion efficiency which is comparable to that of the precious metal nanoparticles, but also have good degradability in vivo, good biocompatibility and almost no cytotoxicity, so that the polydopamine nanoparticles can be used as a preferred photothermal material. And how to highly concentrate the photothermal material at the tumor part so as to reduce the damage to other tissues is a great problem. The long-circulation targeting high molecular material phospholipid modified poly-dopamine nanoparticle nucleus loaded epirubicin is enriched at a tumor part, the tumor part contains a large amount of phospholipase, so that the anti-cancer drug is exposed to achieve the anti-cancer effect, and the long-circulation targeting high molecular material phospholipid modified poly-dopamine nanoparticle nucleus loaded epirubicin is combined with photothermal therapy to thoroughly kill cancer cells.

Epirubicin is an isomer of adriamycin, and belongs to antibiotic antineoplastic drugs. Epirubicin prevents the formation of mRNA by interfering with the transcription process of tumor cells; thereby inhibiting the synthesis of DNA and RNA. In addition, epirubicin also has an inhibitory effect on topoisomerase II, is a cell cycle nonspecific drug, is effective on various transplantable tumors, has a good treatment effect on various tumors, and has less myocardial toxicity compared with doxorubicin.

The nano-drug preparation refers to a class of innovative drug preparations researched and developed by applying a nano-carrier technology, and compared with the traditional preparation, the nano-drug preparation shows many new pharmacodynamic and metabolic dynamics characteristics, such as long circulation, targeting, sustained and controlled release, high biological adhesion, a special cell-entering mechanism, multi-component co-delivery and the like. The research and development of nano-drug preparations or drug nano-carriers have become the leading edge and hot spot of the current international medical and pharmaceutical community. Traditional chemotherapeutic drugs have strong toxic and side effects, which greatly affect the therapeutic effect and the quality of life of patients. The important reason for the toxic side effects of chemotherapeutic drugs is that they are highly toxic and non-selective, killing tumor cells while also damaging normal cells and tissues. Although epirubicin is less toxic than doxorubicin, its cumulative toxicity over prolonged use can still cause tissue damage. The nano drug delivery system can improve the curative effect of the antitumor drug and reduce the toxic and side effects thereof, and a plurality of different types of nano materials are used for constructing the nano drug delivery system, thereby obtaining good effect. The epirubicin is prepared into the antitumor drug nano preparation, so that the drug is slowly released and acts on tumor cells more intensively, the curative effect of the antitumor drug can be improved, and the toxic and side effects of the antitumor drug can be reduced.

The photothermal therapy is a new method for treating tumors, and is expected to become an important method for treating tumors due to short treatment time, small toxic and side effects and great development potential. Photothermal therapy is to use a material with higher photothermal conversion efficiency to convert light energy into heat energy to make the temperature of the heat energy exceed the tolerance temperature of tumor cells so as to ablate the tumor cells, such as: the polydopamine nanoparticles are widely applied due to good degradability, high photo-thermal conversion efficiency and good biocompatibility. The defects that the optimal effect cannot be achieved only by means of photothermal therapy, and the problem that the high targeting of the antitumor drug to the tumor part is solved. The invention relates to a treatment method for thoroughly killing cancer cells by combining the light-heat treatment by adopting phospholipid to coat an anticancer drug epirubicin to highly target tumor parts. At present, the research of targeting a tumor part by using phospholipid is few, particularly the research application of targeting the tumor part and combining photothermal therapy is less, and the research of highly targeting and conveying a medicament to the tumor part by combining chemotherapy is less.

Disclosure of Invention

The invention provides an anti-tumor nano preparation and application thereof in targeted therapy of malignant tumors, wherein the nano preparation is prepared by coating epirubicin with a photo-thermal material polydopamine nanoparticle, and has long circulation and high targeted anti-tumor effect, and the treatment effect of killing tumor cells is achieved by combining photo-thermal treatment.

The technical scheme of the invention is as follows:

an anti-tumor nano preparation and an application thereof in targeted therapy of malignant tumors, wherein the anti-tumor nano preparation is a nano preparation of epirubicin coated by polydopamine nanoparticles; the preparation method comprises the following specific steps:

s1: uniformly stirring and mixing 25% ammonia water, 30% ethanol solution and L-cysteine, carrying out dark water bath at 0-40 ℃ for 5-10 min, adding 0.26 mol/L dopamine hydrochloride into the mixed solution, and continuously stirring and reacting for 0.5-72 h to obtain polydopamine nanoparticles (PDA-NPS);

s2: dialyzing the polydopamine nanoparticles obtained in the step S1 in a dialysis tube, and after dialysis is finished, continuously diluting the polydopamine nanoparticles by 2-10 times with water to obtain a solution A;

s3: sequentially adding auxiliary material drug carrier, aqueous binder and Epirubicin (EPI) into methanol for full dissolution to obtain solution B;

s4: slowly dropwise adding the solution B obtained in the step S3 into the solution A obtained in the step S2, shaking while dropwise adding, and placing the mixed solution in a water bath at 30-45 ℃ for 25-35 min after fully and uniformly mixing; and (3) dialyzing the mixed solution after the water bath is finished, and obtaining the epirubicin nano preparation (EPCM-NPS) coated with the polydopamine nano particles, namely the anti-tumor nano preparation.

Furthermore, the particle size of the anti-tumor nano preparation is 80-250 nm.

Further, the anti-tumor nano preparation comprises the following components in parts by weight: 6-60 parts of a drug loading agent, 2-20 parts of an aqueous binder, 1-10 parts of epirubicin and 2-6 parts of polydopamine nanoparticles.

Further, the ratio of dopamine hydrochloride to L-cysteine in the step S1 is (1-4): 1.

further, the step S3 is to include one or more of dilauroyl phosphatidyl glycerol (DLPG), dipalmitoyl phosphatidyl glycerol (DPPG), distearoyl phosphatidyl glycerol (DSPG), and dimyristoyl phosphatidyl glycerol (DMPG).

Further, the aqueous binder in step S3 is composed of one or more components selected from distearoyl phosphatidyl acetamide-methoxy polyethylene glycol 5000 (DSPE-MPEG 5000), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000), distearoyl phosphatidyl acetamide-N-hydroxysuccinimide-polyethylene glycol 2000 (DSPE-PEG 2000-NHS), distearoyl phosphatidyl ethanolamine-polyethylene glycol-cyclopeptide polypeptide (DSPE-PEG 2000-cRGD), distearoyl phosphatidyl ethanolamine-polyethylene glycol-methotrexate (DSPE-PEG 2000-MTX).

Further, the molecular weight of the dialysis device in the steps S1 and S4 is 1-300 kd, and the dialysis time is 0.5-100 h.

Furthermore, the anti-tumor nano preparation is applied to targeted therapy of malignant tumors.

Furthermore, the anti-tumor nano preparation can be used for targeted treatment of breast cancer and malignant tumor of superficial growth of skin cancer.

Further, the application of the anti-tumor nano preparation in targeted therapy of 4T1-luc cell BALB/C female mice is as follows: injecting the anti-tumor nanometer preparation into mouse via tail vein for 0.1-24 h, and irradiating tumor part with 808 nm laser at irradiation power of 1-15 w/cm for photothermal treatment²The irradiation time is 10-120 min.

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

1. the invention adopts the polydopamine nanoparticles as the photothermal conversion material to prepare the anti-tumor nano preparation, and realizes the characteristics of high photothermal conversion efficiency, good in-vivo degradability, good biocompatibility and almost no cytotoxicity;

2. the adjuvant drug loading agent for preparing the nano preparation is composed of phosphatidyl glycerol components, the adjuvant water-based binder is composed of polyethylene glycol phospholipid compound components, and the core loading can be effectively carried out on the epirubicin inner core coated by the polydopamine nano particles under the synergistic effect of the phospholipid components and the polyethylene glycol phospholipid compound components; wherein the phosphatidyl glycerol provides negative charge and is combined with the positively charged drug to play a role in loading the drug; because the polyethylene glycol phospholipid can improve the hydration degree of the nanoparticles and the steric hindrance in vivo, the nano preparation is highly enriched at the tumor part, thereby achieving the effect of long-circulating targeting tumor;

3. the invention adopts the photothermal material polydopamine nanoparticles to coat epirubicin to prepare an anti-tumor nano preparation, and simultaneously combines the method of photothermal treatment to kill tumor cells, so that the double inactivation effect on the tumor cells can be achieved, and the nano preparation can be effectively used for targeted treatment of breast cancer and malignant tumor of superficial growth of skin cancer;

4. after the preparation is injected into female mice inoculated with 4T1-luc cell BALB/C in tail vein, the photothermal therapy has obviously better therapeutic effect than the conventional therapy, and the therapeutic mode has small wound area, almost no toxic or side effect, reduces the administration dosage and reduces the pain compared with the surgical therapy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort;

the drawings shown in the present specification are only for the purpose of matching with the disclosure of the specification, and are not intended to limit the practical and practical conditions of the present invention, so that the present invention has no technical significance, and the present invention should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.

FIG. 1 is a graph showing a particle size distribution of PDA-NPS;

FIG. 2 is a graph showing a particle size distribution of EPCM-NPS;

FIG. 3 is an SEM image of a PDA-NPS;

FIG. 4 is an SEM image of EPCM-NPS;

FIG. 5 is a graph showing temperature rise curves of different concentrations of PDA-NPS under 808 nm laser irradiation;

FIG. 6 shows the in vitro release rates of EPCM-NPS at pH 5.0, 6.5, 7.4 for 72 h;

FIG. 7 is a graph showing the survival rate of 4T1-luc cells with different concentrations of PDA-NPS;

FIG. 8 is a graph of the viability of EPI, PDA-NPS and EPCM-NPS and photothermal therapy on 4T1-luc cells, respectively;

FIG. 9 is a graph of the time-concentration profile of EPI and EPCM-NPS, respectively, in rats;

FIG. 10 is a graph showing the drug content profiles of the EPI and EPCM-NPS distribution in various organs (heart, liver, spleen, lung, kidney, tumor) after 1 h in BALB/C mice, respectively;

FIG. 11 is a graph showing the drug content distribution of EPI and EPCM-NPS in various organs (heart, liver, spleen, lung, kidney, tumor) after 12 h in BALB/C mice, respectively;

FIG. 12 is a graph showing the drug content profiles of the EPI and EPCM-NPS distribution in various organs (heart, liver, spleen, lung, kidney, tumor) 24 h after BALB/C mice;

FIG. 13 is a graph of weight gain of control, EPI and EPCM-NPS, respectively, on BALB/C mice;

FIG. 14 is a graph of tumor volume growth inhibition in BALB/C mice for control group, EPI and EPCM-NPS, respectively;

FIG. 15 is a histogram of tumor suppression weights of control, EPI and EPCM-NPS, respectively, in BALB/C mice.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

Example 1

The preparation steps of the poly-dopamine nanoparticle and poly-dopamine nanoparticle core-loaded epirubicin nano preparation are as follows:

s1: adding 1g of L-cysteine and 6 ml of 25% ammonia water into 260 ml of 30% ethanol solution, stirring and mixing uniformly, carrying out water bath in a dark place at 35 ℃ for 10 min, adding 0.26 mol/1 dopamine hydrochloride 40 m1 into the mixed solution, and continuously stirring and reacting for 24 h to obtain PDA-NPS;

s2: continuously diluting the PDA-NPS dialysis tube obtained in the step S1 by 2 times with water to obtain a solution A after dialysis in the PDA-NPS dialysis tube;

s3: mixing the auxiliary materials of a drug loading agent, an aqueous binder and Epirubicin (EPI) in a ratio of 12: 4: 1, respectively and sequentially adding the mixture into 2 ml of methanol for full dissolution to obtain a solution B;

s4: slowly dropwise adding the solution B obtained in the step S3 into the solution A obtained in the step S2, shaking while dropwise adding, and placing the mixed solution in a water bath at 37 ℃ for 30 min after fully and uniformly mixing; dialyzing the mixed solution at 100 kd for 6 h after the water bath is finished to obtain EPCM-NPS, namely the anti-tumor nano preparation;

the average particle size of EPCM-NPS obtained by detection by a particle size analyzer is 80 nm.

Example 2

s1: adding 1g of L-cysteine and 6 ml of 25% ammonia water into 260 ml of 30% ethanol solution, stirring and mixing uniformly, carrying out 0 ℃ dark water bath for 5 min, adding 60 ml of 0.26 mol/L dopamine hydrochloride into the mixed solution, and continuously stirring and reacting for 72 h to obtain PDA-NPS;

s2: continuously diluting the PDA-NPS dialysis tube obtained in the step S1 by 6 times with water to obtain a solution A after dialysis in the PDA-NPS dialysis tube;

s3: mixing the auxiliary materials of a drug loading agent, an aqueous binder and Epirubicin (EPI) in a ratio of 6: 10: 1, respectively and sequentially adding the mixture into 2 ml of methanol for full dissolution to obtain a solution B;

s4: slowly dropwise adding the solution B obtained in the step S3 into the solution A obtained in the step S2, shaking while dropwise adding, and placing the mixed solution in a water bath at 45 ℃ for 25 min after fully and uniformly mixing; dialyzing the mixed solution at 100 kd for 40 h after the water bath is finished to obtain EPCM-NPS, namely the anti-tumor nano preparation;

the average particle size of EPCM-NPS obtained by detection by a particle size analyzer is 200 nm.

Example 3

s1: adding 1g of L-cysteine and 6 ml of 25% ammonia water into 260 ml of 30% ethanol solution, stirring and mixing uniformly, carrying out water bath in a dark place at 40 ℃ for 7 min, adding 80 ml of 0.26 mol/L dopamine hydrochloride into the mixed solution, and continuously stirring and reacting for 0.5 h to obtain PDA-NPS;

s2: continuously diluting the PDA-NPS dialysis tube obtained in the step S1 by 8 times with water to obtain a solution A after dialysis in the PDA-NPS dialysis tube;

s3: mixing auxiliary material drug loading agent, aqueous binder, Epirubicin (EPI) in a ratio of 1: 2: 1, respectively and sequentially adding the mixture into 2 ml of methanol for full dissolution to obtain a solution B;

s4: slowly dropwise adding the solution B obtained in the step S3 into the solution A obtained in the step S2, shaking while dropwise adding, and placing the mixed solution in a water bath at 30 ℃ for 30 min after fully and uniformly mixing; dialyzing the mixed solution at 100 kd for 0.5 h after the water bath is finished to obtain EPCM-NPS, namely the anti-tumor nano preparation;

the average particle size of EPCM-NPS obtained by detection by a particle size analyzer is 100 nm.

Example 4

s1: adding 1g of L-cysteine and 6 ml of 25% ammonia water into 260 ml of 30% ethanol solution, stirring and mixing uniformly, carrying out dark water bath at 20 ℃ for 6 min, adding 100 ml of 0.26 mol/L dopamine hydrochloride into the mixed solution, and continuously stirring and reacting for 12 h to obtain PDA-NPS;

s3: mixing auxiliary material drug loading agent, aqueous binder, Epirubicin (EPI) in a ratio of 3: 2: 1, respectively and sequentially adding the mixture into 2 ml of methanol for full dissolution to obtain a solution B;

s4: slowly dropwise adding the solution B obtained in the step S3 into the solution A obtained in the step S2, shaking while dropwise adding, and placing the mixed solution into a water bath at 25 ℃ for 32 min after fully and uniformly mixing; dialyzing the mixed solution at 100 kd for 100h after the water bath is finished to obtain EPCM-NPS, namely the anti-tumor nano preparation;

the average particle size of EPCM-NPS obtained by particle size analyzer is 160 nm.

Example 5

s1: adding 1g of L-cysteine and 6 ml of 25% ammonia water into 260 ml of 30% ethanol solution, stirring and mixing uniformly, carrying out water bath in a dark place at 30 ℃ for 8 min, adding 120 ml of 0.26 mol/L dopamine hydrochloride into the mixed solution, and continuously stirring and reacting for 48 h to obtain PDA-NPS;

s3: mixing auxiliary material drug loading agent, aqueous binder, Epirubicin (EPI) in a ratio of 3: 3: 5, respectively and sequentially adding the mixture into 2 ml of methanol for full dissolution to obtain a solution B;

s4: slowly dropwise adding the solution B obtained in the step S3 into the solution A obtained in the step S2, shaking while dropwise adding, and placing the mixed solution into a water bath at 30 ℃ for 28 min after fully and uniformly mixing; dialyzing the mixed solution at 100 kd for 20 h after the water bath is finished to obtain EPCM-NPS, namely the anti-tumor nano preparation;

the average particle size of EPCM-NPS obtained by detection by a particle size analyzer is 240 nm.

As is clear from examples 1 to 5 above, the EPCM-NPS prepared in example 1 had an average particle diameter of 80 nm; the particle size is the smallest for example 1 compared to examples 2-5. In the following experiments, the PDA-NPS and EPCM-NPS prepared under the experimental conditions of example 1 were used as the experimental samples.

Different concentrations of PDA-NPS solution photothermal conversion experiment:

respectively taking 1 ml of PDA-NPS (10 mug/ml, 25 mug/ml, 50 mug/ml and 100 mug/ml) with different concentrations in a quartz pool, and continuously irradiating by a laser (808 nm; 4.9 w/cm)²(ii) a 10 min), recording the temperature every 30 s, and drawing a temperature rise curve, wherein the specific image is shown in the attached figure 5;

with the increase of the concentration of the PDA-NPS, the number of the PDA-NPS nanoparticles contained in the PDA-NPS is increased, the efficiency of converting light energy into heat energy is increased, the temperature of the solution is continuously increased, when the concentration is 100 mug/ml, the temperature of the solution is increased by more than 60 ℃ after 10 min of irradiation, the temperature of the solution is higher than the heat-resistant temperature of a cancer cell by 50 ℃, and the temperature of a control group is only increased to 37 ℃, and the temperature of the control group is within the normal cell bearable range. Therefore, the poly-dopamine nanoparticles can be safely and efficiently applied to photothermal therapy.

In vitro dissolution release experiments within 72 h:

respectively putting 1 ml of 3 groups of same EPCM-NPS in dialysis bags of 100 kd for dialysis, respectively putting three dialysis bags in media with pH of 5.0 (pH environment of lysosome), 6.5 (pH microenvironment of tumor) and 7.4 (pH environment in blood) at 37 ℃, stirring in a 100r magnetic stirrer, respectively sampling at different time points, filtering, and performing liquid phase analysis, wherein the specific image is as shown in figure 6;

the results show that the EPCF-NPS release accumulation amount gradually increases along with the prolonging of the time, and the slow release behavior is shown. The EPCF-NPS has higher release in an acid environment with the pH value of 5.0, the accumulative release amount at the pH value of 7.4 is 10 times, the EPI release speed of the EPCF-NPS is accelerated along with the reduction of the pH value, and the accumulative release amount is obviously increased. The poly-dopamine nanoparticle loaded with epirubicin has good drug slow-release capacity, can respond to the intracellular environment of cancer cells, and has a selective treatment effect.

MTT method cytotoxicity assay:

1) the MTT method is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value of the formazan is measured by an enzyme-labeling instrument, so that the quantity of living cells can be indirectly reflected. At a rate of 1X 10 per hole⁴4T1-LUC cells were seeded in 96-well plates at 37 ℃ in CO₂After incubation for 24 hours in an incubator with the concentration of 5%, removing the culture medium, respectively diluting PDA-NPS with different concentrations by using the culture medium, adding 100 mu l of the PDA-NPS into a 96-well plate per well, continuously incubating for 24 hours, removing liquid medicine containing polydopamine nanoparticles, washing for 3 times by using PBS, adding an MTT solution, incubating for 4 hours, removing the culture medium, adding 150 mu l of a DMSO solution per well, shaking for 10 minutes, and measuring the absorbance at 490nm of an enzyme labeling instrument, wherein a specific image is shown in the attached figure 7;

the result shows that the cell survival rate of the PDA-NPS is still maintained at about 87% even at a high concentration of 960 mug/ml, which indicates that the toxicity of the polydopamine nanoparticle concentration on 4T1-luc cells is low when the concentration of the polydopamine nanoparticle is not higher than 960 mug/ml, and the concentration of the polydopamine nanoparticle is selected to be 150 mug/ml in the subsequent photo-thermal treatment so as to eliminate the influence of the toxicity.

2) Cell photothermal therapy:

dividing 4T1-LUC cells into 5 groups, respectively adding PBS as a control group, EPI of 25 mug/ml, EPI of 200 mug/ml, PDA-NPS of 150 mug/ml, EPCM-NPS of 150 mug/ml (EPI 25 mug/ml) at 37 ℃, CO₂After incubation for 12 h in an incubator with the concentration of 5%, performing photo-thermal treatment on each group, removing liquid medicine containing polydopamine nanoparticles, cleaning for 3 times by using PBS, adding MTT solution, incubating for 4 h, removing a culture medium, adding 150 mu l of DMSO solution into each hole, shaking for 10 min, and measuring the absorbance of an enzyme-labeling instrument at 490nm, wherein the specific image is shown in the attached figure 8;

the result shows that after photothermal treatment, the cell activity of the PDA-NPS group is reduced to 30%, the cell activity of the EPCF-NPS preparation group is only 15%, the cell killing effect of the EPCF-NPS preparation is more obvious, and the cell activity of the control group, the EPI 25 mug/ml solution group and the EPI200 mug/ml solution group are almost not obviously different from those of the non-illuminated group. The poly-dopamine nanoparticle nuclear-loaded epirubicin nano preparation is proved to be capable of well combining chemotherapy and photothermal therapy and performing more effective synergistic treatment on cancer cells.

In vivo drug time-concentration curve distribution experiment of rats:

randomly dividing 18 SD rats (each half of male and female) with 180-200 g into 3 groups, injecting physiological saline into the tail vein of the blank group, respectively injecting EPCM-NPS and EPI aqueous solutions into the other 2 groups, wherein the administration dose is 10 mg/kg, taking 0.3 ml of plasma from the orbital venous plexus at different time points, centrifuging for 10 min at 5000r-10000r, taking supernatant, performing liquid mass analysis after treatment, and drawing a pharmaceutical time-concentration curve chart, wherein the specific image is shown in figure 9;

the result shows that the maximum blood concentration of the EPCF-NPS is obviously higher than that of an EPI aqueous solution group in half-life period, and the circulation time of the drug in blood is effectively prolonged. The nano preparation modified by the long-circulating phospholipid can slowly release the medicament, improve the curative effect of the anti-tumor medicament and reduce the toxic and side effects of the anti-tumor medicament.

Drug distribution experiments in mice in vivo organs:

54 female mice inoculated with BALB/C4T 1-LUC cells until tumors grow to 100 mm²Randomly dividing into 3 groups, each group comprises 18 groups, each time point comprises 6 groups, injecting physiological saline into tail vein of blank group, injecting EPI aqueous solution and EPCM-NPS nanometer preparation into the other 2 groups, respectively, with administration dosage of 10 mg/kg, then taking out organs (heart, liver, spleen, lung, kidney, tumor) at different time interval points (1 h, 12 h, 24 h), and detecting content distribution of EPI in organism by LC-MS, wherein the specific images are shown in figures 10-12;

the result shows that compared with an EPI group aqueous solution, the EPCF-NPS nano preparation has the most distributed EPI content in a mouse tumor part, and because CRGD can specifically identify α v β 3 integrin receptors overexpressed on the surface of a tumor cell, under the action of the receptors, the nano preparation penetrates through a cell membrane and plays a role in resisting tumors, so that the EPCF-NPS nano preparation modified by CRGD can successfully enrich the medicine in the tumor part and play a role in targeting the tumor part so as to kill the cancer cell.

Mouse tumor chemotherapy combined photothermal therapy experiment:

36 BALB/C female mice with tumor volume of about 100 mm²Randomly dividing the raw materials into 6 groups, each group comprises 6 mice, a blank group and a light group (NIR), an EPI and EPI light group, an EPCM-NPS group and an EPCM-NPS light group, injecting 0.2 ml into tail vein, administering the dose of 5mg/kg, performing photothermal treatment on tumors for 5 min after 4 h, observing and measuring the tumor volume and the body weight every other day, administering the doses for 3 times, taking the tumors after the experiment is finished, analyzing and weighing the tumors, and finally drawing a mouse tumor inhibition curve and a mouse body weight change curve, wherein specific images are shown in figures 13-15;

in fig. 13, the body weight of mice in each group did not fluctuate significantly and the side effects were small compared to the blank control group, and in fig. 14 and 15, the EPI group had a certain therapeutic effect compared to the blank control group, but the EPCF-NPS group without the light group had a better therapeutic effect, since CRGD could recognize α v β 3 integrin receptor on the surface of cancer cells, activate specific proteolytic enzyme on the surface of cancer cells, thereby cleaving CRGD cyclic peptide to form activated CendR peptide, specifically recognize and activate specific receptor on the surface of cancer cells, and under the action of receptor, the nano-formulation penetrated cell membrane and acted as an anti-tumor effect, indicating that the EPCF-NPS nano-formulation modified by CRGD could deliver drugs to the tumor site, increasing the local drug concentration of the tumor, thereby achieving the purpose of selective killing.

While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive, and any modifications, equivalents, improvements and the like that come within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. An anti-tumor nano preparation and application thereof in targeted therapy of malignant tumor are characterized in that: the anti-tumor nano preparation is a nano preparation of poly-dopamine nano particles coated with epirubicin; the preparation method comprises the following specific steps:

s1: uniformly stirring and mixing 25% ammonia water, 30% ethanol solution and L-cysteine, carrying out dark water bath at 0-40 ℃ for 5-10 min, adding 0.26 mol/L dopamine hydrochloride into the mixed solution, and continuously stirring and reacting for 0.5-72 h to obtain polydopamine nanoparticles;

s3: respectively and sequentially adding the auxiliary material drug loading agent, the water-based binder and the epirubicin into methanol for full dissolution to obtain a solution B;

s4: slowly dropwise adding the solution B obtained in the step S3 into the solution A obtained in the step S2, shaking while dropwise adding, and placing the mixed solution in a water bath at 30-45 ℃ for 25-35 min after fully and uniformly mixing; and (4) dialyzing the mixed solution after the water bath is finished, and obtaining the epirubicin nano preparation coated with the polydopamine nano particles, namely the anti-tumor nano preparation.

2. The anti-tumor nano preparation and the application thereof in targeted therapy of malignant tumors according to claim 1, wherein the nano preparation comprises the following components in percentage by weight: the particle size of the anti-tumor nano preparation is 80-250 nm.

3. The anti-tumor nano preparation and the application thereof in targeted therapy of malignant tumors according to claim 1, wherein the nano preparation comprises the following components in percentage by weight: the anti-tumor nano preparation comprises the following components in parts by weight: 6-60 parts of a drug loading agent, 2-20 parts of an aqueous binder, 1-10 parts of epirubicin and 2-6 parts of polydopamine nanoparticles.

4. The anti-tumor nano preparation and the application thereof in targeted therapy of malignant tumors according to claim 1, wherein the nano preparation comprises the following components in percentage by weight: the ratio of the dopamine hydrochloride to the L-cysteine in the step S1 is (1-4): 1.

5. the anti-tumor nano preparation and the application thereof in targeted therapy of malignant tumors according to claim 1, wherein the nano preparation comprises the following components in percentage by weight: the step S3 pharmaceutical carrier is composed of one or more components of dilauroyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, distearoyl phosphatidyl glycerol, and dimyristoyl phosphatidyl glycerol.

6. The anti-tumor nano preparation and the application thereof in targeted therapy of malignant tumors according to claim 1, wherein the nano preparation comprises the following components in percentage by weight: the aqueous binder in the step S3 is composed of one or more components of distearoyl phosphatidyl acetamide-methoxy polyethylene glycol 5000, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, distearoyl phosphatidyl acetamide-N-hydroxysuccinimide-polyethylene glycol 2000, distearoyl phosphatidyl ethanolamine-polyethylene glycol-cyclopeptide polypeptide, and distearoyl phosphatidyl ethanolamine-polyethylene glycol-methotrexate.

7. The anti-tumor nano preparation and the application thereof in targeted therapy of malignant tumors according to claim 1, wherein the nano preparation comprises the following components in percentage by weight: the molecular weight of the dialysis device in the steps S1 and S4 is 1-300 kd, and the dialysis time is 0.5-100 h.

8. The use of the anti-tumor nano-preparation according to claim 1 for the targeted treatment of malignant tumors.

9. The use of the nano-formulation for the targeted therapy of malignant tumors according to claim 8, wherein: the anti-tumor nano preparation can be used for targeted treatment of breast cancer and malignant tumor of superficial growth of skin cancer.

10. The use of the nano-formulation for the targeted therapy of malignant tumors according to claim 9, wherein: the application of the anti-tumor nano preparation in the targeted therapy of 4T1-LUC cell BALB/C female mice is as follows: injecting the anti-tumor nanometer preparation into mouse via tail vein for 0.1-24 h, and irradiating tumor part with 808 nm laser at irradiation power of 1-15 w/cm for photothermal treatment²The irradiation time is 10-120 min.