CN111939151A

CN111939151A - Composite adriamycin albumin nanoparticle and preparation method and application thereof

Info

Publication number: CN111939151A
Application number: CN202010634393.7A
Authority: CN
Inventors: 唐岚; 张冉; 何峥; 单伟光; 张振海
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2020-07-04
Filing date: 2020-07-04
Publication date: 2020-11-17

Abstract

The invention belongs to the technical field of medicine preparation, and particularly relates to compound adriamycin albumin nanoparticles (VES-DOX-BSANPs), a preparation method thereof and application thereof in anti-tumor aspect. The VES-DOX-BSANPs provided by the invention are prepared by taking Bovine Serum Albumin (BSA) as a carrier material, wrapping drugs such as adriamycin (DOX) and Vitamin E Succinate (VES) and adopting a high-pressure homogenization method. The compound adriamycin albumin nanoparticles have the advantages of uniform size, proper particle size, good dispersibility and stable physicochemical properties. In vivo and in vitro efficacy experiments, the VES-DOX-BSANPs have the effects of enhancing efficacy, reducing toxicity and reversing tumor multidrug resistance.

Description

Composite adriamycin albumin nanoparticle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicine preparation, and particularly relates to a compound adriamycin albumin nanoparticle, a preparation method thereof and application thereof in anti-tumor aspect.

Background

Cancer has long been a disease which troubles human health problems, and the complexity, variability and difficult cure of the cancer are difficult points and hot spots of pharmaceutical research and clinical research. Cancer therapy has made tremendous progress over the past decades. Among the numerous therapeutic approaches, chemotherapy has become the main strategy for the treatment of most cancers due to the high cytotoxicity of the drug against cancer cells, whereas conventional chemotherapeutic drugs such as Doxorubicin (Doxorubicin, DOX) always exhibit inherent limitations such as nonspecific distribution, high toxicity, poor bioavailability, rapid blood clearance, poor solubility in physiological environments, and the like.

DOX belongs to an anthracycline antitumor antibiotic, is derived from streptomyces grey-gray subspecies, and is a chemotherapeutic drug with wide activity. While DOX has been standardized as a first line anticancer drug, its resistance and toxic side effects remain important limiting factors for its clinical use, and its potential cardiotoxicity, including life-threatening myocardial disease, congestive heart failure, and dose-limiting myelosuppression, are side effects that must be considered.

Nanoparticles (NPs) are generally defined as colloidal polymer particles between 1 nm and 1000nm, are good carriers of drugs, and mainly differ from microsphere carriers in that the Nanoparticles have ultramicro volumes, can penetrate through tissue gaps and be absorbed by cells, and can pass through the smallest capillary vessels of a human body, so that the Nanoparticles have unique superiority as a new drug delivery system which is widely researched, particularly in the fields of targeted and positioned drug delivery, mucosal absorption drug delivery, gene therapy, protein polypeptide drug controlled release and the like.

Albumin is a simple protein, in which amino acids are linked by peptide bonds and twisted into a mass, with numerous network voids, creating favorable space conditions for mosaic drug carrying. Animal pathology research shows that bovine serum albumin BSA can be used as a safe and non-toxic carrier, and intravenous injection of albumin microspheres is widely used for diagnosis and has no toxicity related report. The albumin also has biodegradability, and can prevent the drug from releasing from the injection site and make the drug slowly release at the effective site after the drug is combined with the albumin. The nano-particle using albumin as a carrier is non-toxic and has good biocompatibility, and can achieve tumor targeting of a medicament by utilizing a natural albumin approach, such as gp60 and caveolin-mediated transcellular transport and interaction between tumor SPARC proteins. In conclusion, bovine serum albumin is an ideal drug carrier.

The albumin nanoparticle drug delivery system comprises active targeting and passive targeting. The nanoparticles without surface modification are administered intravenously into the body and are mainly taken up by the viscera rich in reticuloendothelial cells. The distribution of the nano drug delivery system after entering the body is mainly influenced by the surface property and the particle size of the nano particles. The nanoparticles with positive charges on the surface generally have lung tissue targeting property, and the nanoparticles with negative charges on the surface have liver tissue targeting property. The active targeting system of albumin nanoparticles comprises nanoparticle surface modification, magnetic albumin nanoparticles and albumin nanoparticles combined by antigen-antibody. The surface modification of the nanoparticles is to change the surface property of the nanoparticles through covalent coupling effect, thereby avoiding phagocytosis by macrophages such as liver, spleen and the like, and having targeting property; magnetic albumin nanoparticles are prepared by coating medicines and magnetic substances (such as ferroferric oxide, ferric oxide and the like) with albumin, and the magnetic albumin nanoparticles are transferred to target tissues or target organs under the control of an external magnetic field after administration.

The preparation method of albumin nanoparticles can be mainly divided into an emulsion solidification method, a desolvation method and Nab^TMTechniques, and other methods.

Vitamin E Succinate (VES) has wide antitumor effect, has no toxic or side effect on normal human body cells and tissues, and has good inhibition effect on VES on gastric cancer, breast cancer, leukemia, prostate cancer and the like through numerous researches. VES can induce apoptosis through reactive oxygen species ROS, and because the pH in tumor cells is lower than that of normal cells, VES can selectively inhibit tumor cells without adverse effects on normal tissues.

The mechanism of action of VES against tumors lies in the following two aspects: (1) inhibiting the proliferation of tumor cells, wherein the tumor cells have unlimited proliferation caused by cell cycle disorder, and the VES inhibiting the proliferation of the tumor cells is mainly realized by inhibiting the synthesis of DNA, blocking the cell cycle, regulating cell cycle regulatory protein and other mechanisms; (2) tumor cell apoptosis is induced, and VES induction of tumor cell apoptosis is mainly realized through an endogenous channel and an exogenous channel.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provides composite adriamycin albumin nanoparticles (VES-DOX-BSANPs), a preparation method thereof and application thereof in the aspect of tumor resistance. In order to reduce the toxic and side effects of DOX, enhance the curative effect and reverse the multidrug resistance of tumors, the invention selects VES and adriamycin which have the anti-tumor and anti-drug resistance effects simultaneously to prepare the composite nanoparticle for treating the cancers. By combining DOX and VES, the nanoparticles with proper particle size are prepared, so that a better tumor targeting effect is achieved, the toxic and side effects of adriamycin are reduced, and the anti-tumor effect is enhanced.

In order to achieve the above object, the present invention provides the following technical solutions:

one of the purposes of the invention is to provide composite doxorubicin albumin nanoparticles VES-DOX-BSANPs, wherein the nanoparticles are obtained by dissolving desalted doxorubicin DOX and vitamin E succinate VES in an organic solvent to serve as an organic phase, using an aqueous solution containing bovine serum albumin BSA as an aqueous phase, dropwise adding the organic phase into the aqueous phase under a shearing condition to form colostrum, and removing the organic solvent after high-pressure homogenization.

The invention also aims to provide a preparation method of the composite type adriamycin albumin nano-particles VES-DOX-BSANPs, which comprises the following steps:

(1) weighing a certain amount of desalted adriamycin DOX and vitamin E succinate VES, dissolving in an organic solvent to serve as an organic phase, and taking an aqueous solution containing bovine serum albumin BSA as a water phase;

(2) adding the organic phase into the water phase drop by drop under the shearing condition to form primary emulsion;

(3) and (3) placing the colostrum in a high-pressure homogenizer, performing multiple cycles under certain homogenizing pressure to obtain a DOX and VES-wrapped nanoparticle suspension, and removing the organic solvent to obtain a composite doxorubicin albumin nanoparticle VES-DOX-BSANPs suspension aqueous solution.

Preferably, the method steps are carried out in the absence of light.

Preferably, the preparation method of the desalted adriamycin DOX comprises the steps of precisely weighing a proper amount of DOX & HCl, adding a certain amount of purified water, carrying out ultrasonic treatment for 10min to fully dissolve the DOX & HCl, adding a sodium hydroxide aqueous solution to adjust the pH value to be 8-8.5, extracting with chloroform, taking a chloroform layer, extracting with saturated saline water, removing the undissociated DOX & HCl, adding excessive anhydrous sodium sulfate into a chloroform solution to remove water, and carrying out reduced pressure concentration at 35 ℃ to remove chloroform, thereby obtaining the desalted DOX.

Preferably, the molar ratio of the desalted DOX and VES in the step (1) is 1: 1-3, more preferably the feed molar ratio is 1: 1.

Preferably, in the step (1), the dosage of the desalted adriamycin DOX in the organic phase is 5-10mg/mL of the organic phase, and the more preferred dosage is 10 mg/mL.

Preferably, in the step (1), the content of bovine serum albumin BSA in the aqueous phase is 3-10mg/mL, and more preferably 5 mg/mL.

Preferably, the organic solvent in the step (1) is chloroform; the aqueous solution of the aqueous phase is purified water, a 1% aqueous sodium chloride solution, a 2% aqueous sodium chloride solution or a 3% aqueous sodium chloride solution, and a more preferred aqueous solution is purified water.

Preferably, the volume ratio of the organic phase to the aqueous phase in the step (1) is 1: 40.

Preferably, the organic phase is added to the aqueous phase in step (2) under the shearing conditions of a high shear emulsifier.

Preferably, the organic phase is added into the water phase in the step (2) and then is sheared for 5min to form colostrum.

Preferably, in the step (2), the organic phase and the aqueous phase form a colostrum and then pass through a 100-mesh screen.

Preferably, the homogenization pressure in the step (3) is 900-1300bar, and the cycle times are 7-9 times; more preferably the homogenization pressure is 1100bar and the number of cycles is 7.

Preferably, the nanoparticle suspension aqueous solution obtained in the step (3) is pre-frozen in a low-temperature refrigerator and then taken out, and the mixture is placed in a freeze dryer for freeze drying to obtain the composite type lyophilized powder of the doxorubicin albumin nanoparticles VES-DOX-BSANPs

Preferably, the nanoparticle suspension aqueous solution in the step (3) is pre-frozen in a low-temperature refrigerator at-80 ℃ for 12 hours, and then the pre-frozen sample is taken out and is placed in a freeze dryer for freeze drying for 24 hours to obtain the VES-DOX-BSANPs freeze-dried powder.

The invention also aims to provide the application of the composite adriamycin albumin nanoparticles VES-DOX-BSANPs in the aspect of tumor resistance.

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

(1) the VES-DOX-BSANPs disclosed by the invention take BSA (bovine serum albumin) as a carrier, and the albumin nanoparticles are prepared by adopting a high-pressure homogenization method, compared with the traditional preparation method, the high-pressure homogenization method does not need to add a surfactant or any polymer, and pharmacological active substances exist in an amorphous and amorphous state, so that the nanoparticles which are uniformly dispersed and have proper particle sizes can be obtained.

(2) The VES-DOX-BSANPs disclosed by the invention have stronger cell growth inhibition effect on MCF-7/ADR in an in-vitro toxicity test, and the toxicity of the VES-DOX-BSANPs is stronger than that of DOX bulk drugs. Calculating the drug resistance index and the reversal drug resistance factor, and proving that the VES not only has strong anti-tumor effect, but also has good effect on the anti-adriamycin-resistant drug of MCF-7/ADR. The synergy of DOX and VES realizes the inhibition of drug-resistant strains, and more effectively kills adriamycin-resistant cells (MCF-7/ADR).

(3) The pharmacodynamic test research result of the VES-DOX-BSANPs in the tumor-bearing mice shows that compared with other administration groups, the VES-DOX-BSANPs have the strongest tumor inhibition effect in vivo.

(4) The VES-DOX-BSANPs provided by the invention can better target tumor tissues in a tumor-bearing mouse body, and have the effects of enhancing the curative effect and reducing the toxic and side effects.

Therefore, the composite doxorubicin albumin nanoparticles VES-DOX-BSANPs disclosed by the invention have the effects of avoiding the residue of toxic organic solvents, reversing the multidrug resistance of tumors to DOX and achieving better tumor targeting, so that the curative effect can be enhanced, and the toxic and side effects of DOX are reduced.

Drawings

FIG. 1 TEM image of VES-DOX-BSANPs;

FIG. 2 is a graph of the growth inhibitory effect of DOX, DOX-BSANPs and VES-DOX-BSANPs on MCF-7 cells (n-5);

FIG. 3 is a graph of the growth inhibitory effect of DOX, DOX-BSANPs and VES-DOX-BSANPs on MCF-7/ADR cells;

FIG. 4 is a graph of the growth inhibitory effect of DOX, DOX-BSANPs and VES-DOX-BSANPs on 4T1 cells;

FIG. 5 is a graph showing the change in tumor volume during administration in each group of mice;

FIG. 6 is a tumor profile of various groups of mice;

FIG. 7 is a tumor weight chart of each group of tumor-bearing mice;

FIG. 8 is a graph showing the relative body weight changes of groups of tumor-bearing mice during the administration period.

Detailed Description

The technical solution of the present invention is further specifically described below by way of specific examples and with reference to the accompanying drawings.

Example 1: preparation of VES-DOX-BSANPs

(1) Preparation of desalted DOX

Precisely weighing 77mgDOX & HCl in a small beaker wrapped by tinfoil, adding 10mL of purified water, performing ultrasonic treatment for 10min to fully dissolve the DOX & HCl, adding 0.5% sodium hydroxide aqueous solution, adjusting the pH value to 8-8.5, extracting with chloroform for 5 times, collecting the water layer, adjusting the pH value to 8-8.5 again, extracting with chloroform for 2 times, combining all chloroform layers, extracting the chloroform layer with saturated saline solution, removing the undissociated DOX & HCl, adding excessive anhydrous sodium sulfate into the chloroform solution to remove water, and performing reduced pressure concentration at 35 ℃ to remove chloroform to obtain desalted DOX.

(2) Preparation of VES-DOX-BSANPs

VES-DOX-BSANPs were prepared by high pressure homogenization. 20mg of desalted DOX and 19.53mg of VES (DOX and VES molar ratio 1:1) were weighed out in an amount of 10mg/mL based on the organic phase and dissolved in 2mL of chloroform as an organic phase. 0.4g of BSA was weighed in an amount of 5mg/mL of the aqueous phase and dissolved in 80mL of purified water as an aqueous phase. Under the shearing condition of a high-shear emulsifying machine, slowly and uniformly dropwise adding an organic phase into a water phase, shearing for 5min by the high-shear emulsifying machine to form a primary emulsion, sieving by a 100-mesh sieve, placing the primary emulsion in a high-pressure homogenizer, carrying out 7-time circulation under the homogenizing pressure of 1100bar to obtain a nanoparticle suspension containing DOX and VES, transferring the nanoparticle suspension into a round-bottomed flask coated with tinfoil, carrying out rotary evaporation to remove an organic solvent, thus obtaining a VES-DOX-BSANPs suspension aqueous solution, and carrying out a light-shielding measure in the whole experimental process. The particle size (121.9 +/-0.98) nm and the potential (-21.4 +/-0.44) mv of VES-DOX-BSANPs are measured by a Malvern laser particle sizer.

And pre-freezing the prepared suspension aqueous solution of VES-DOX-BSANPs in a low-temperature refrigerator at the temperature of-80 ℃ for 12 hours, taking out a pre-frozen sample, and freeze-drying the pre-frozen sample in a freeze dryer for 24 hours to obtain the lyophilized powder of VES-DOX-BSANPs. Dissolving a proper amount of VES-DOX-BSANPs freeze-dried powder in purified water, dripping the solution on a copper net by using a capillary after uniform dissolution, standing the copper net for a few minutes under an infrared lamp to dry the copper net, observing the form of the copper net by using a TEM and taking a picture. The morphology of the nanoparticles observed by TEM is shown in figure 1, and VES-DOX-BSANPs have spherical appearance, smooth surface, good dispersibility and particle size similar to that measured by a Malvern particle sizer.

Example 2: when preparing VES-DOX-BSANPs, the feed amount of the desalted adriamycin DOX is 5mg/mL of organic phase; the molar ratio of desalted DOX to VES is 1: 3; the homogenization pressure is 900 bar. The rest of the procedure was the same as in example 1. The particle size (102.2 +/-1.06) nm and the potential (-18.1 +/-0.36) mv of VES-DOX-BSANPs are measured by a Malvern laser particle sizer.

Example 3: when preparing VES-DOX-BSANPs, the feeding amount of the desalted adriamycin DOX is 7.5mg/mL of organic phase, and the homogenizing pressure is 1300 bar; the cycle number was 9 times, and the rest of the operation was the same as in example 1. The particle size (106.7 +/-0.62) nm and the potential (-17.4 +/-0.36) mv of VES-DOX-BSANPs are measured by a Malvern laser particle sizer.

Example 4: when preparing VES-DOX-BSANPs, the bovine serum albumin BSA content of the water phase is 3 mg/mL; the water phase solvent is 2% sodium chloride aqueous solution; the organic phase was 1.5mL of chloroform. The rest of the procedure was the same as in example 1. The particle size (131.9 +/-0.77) nm and the potential (-25.4 +/-0.51) mv of VES-DOX-BSANPs are measured by a Malvern laser particle sizer.

Example 5: when preparing VES-DOX-BSANPs, the bovine serum albumin BSA content of the water phase is 10mg/mL water phase; the water phase solvent is 3% sodium chloride water solution; the organic phase was 3mL of chloroform. The rest of the procedure was the same as in example 1. The particle size (98.6 +/-0.39) nm and the potential (-19.5 +/-0.58) mv of VES-DOX-BSANPs are measured by a Malvern laser particle sizer.

Comparative example 1: preparation of DOX-BSANPs

DOX-BSANPs are prepared by a high-pressure homogenization method. 20mg of desalted DOX was weighed out and dissolved in 2mL of chloroform as an organic phase, and 0.4mg of BSA was weighed out and dissolved in 80mL of purified water as an aqueous phase. Under the shearing condition of a high-shear emulsifying machine, slowly and uniformly dropwise adding an organic phase into a water phase, shearing for 5min to form a primary emulsion, sieving the primary emulsion through a 100-mesh sieve, placing the primary emulsion in a high-pressure homogenizer, carrying out 7-time circulation under the homogenizing pressure of 1100bar to obtain a DOX-coated nanoparticle suspension, transferring the nanoparticle suspension into a tin foil-coated round-bottom flask, carrying out rotary evaporation to remove an organic solvent, and thus obtaining a DOX-BSANPs suspension aqueous solution. And pre-freezing the prepared DOX-BSANPs suspension water solution in a low-temperature refrigerator at the temperature of-80 ℃ for 12 hours, taking out a pre-frozen sample, and freeze-drying the pre-frozen sample in a freeze dryer for 24 hours to obtain the DOX-BSANPs freeze-dried powder.

Test example 1: research on in vitro anti-tumor activity of VES-DOX-BSANPs

In order to examine the in vitro anti-tumor effect of VES-DOX-BSANPs, the application takes human breast cancer cells (MCF-7), DOX-resistant breast cancer cells (MCF-7/ADR) and mouse breast cancer cells (4T1) as cell models, and examines the effects of DOX, example 1VES-DOX-BSANPs and comparative example 1DOX-BSANPs on the proliferation of the three cells and the anti-drug resistance of example 1VES-DOX-BSANPs through an MTT method.

And (3) culturing the cells: 4T1, MCF-7/ADR dispersed in 25cm²The culture flask of (1) was incubated with 10% fetal bovine serum-containing RPMI-1640 medium at 37 ℃ under 5% CO₂Culturing in a cell culture box, and replacing fresh culture solution every other day for culturing in the culture process.

(1) Cell seeding

Cells in the logarithmic growth phase were taken and digested with trypsin until the cells became round, and the digestion was stopped by adding a culture medium containing 10% fetal bovine serum. Blowing and beating the cells by a gun head to completely drop the cells, transferring the cells into a centrifuge tube, centrifuging the centrifuge tube for 5min at 1000rpm, removing supernatant, adding a proper amount of fresh complete culture solution, and blowing and beating the cells to form uniformly dispersed cell suspension. Sucking a small amount of cell suspension, counting in a blood cell counting plate, diluting with RPMI 1640 complete culture medium containing fetal calf serum to density of 5 × 10⁴And (3) uniformly dispersing the cell suspension per mL, inoculating the cell suspension into a 96-well plate by using a pipette, adding 100 mu L of cell suspension into each well, adding 100 mu L of PBS into the periphery of the 96-well plate in a circle, and placing the 96-well plate in an incubator for overnight culture so as to adhere to the wall. For each concentration gradient, 5 replicate wells were set, 5 blank wells and 5 control wells were set, and the 96-well plate was placed in an incubator and incubated for 24h to allow cells to adhere.

(2) Preparation of different medicinal liquids

Precisely weighing DOX bulk drug, DOX-BSANPs and VES-DOX-BSANPs freeze-dried powder, dissolving the DOX bulk drug, DOX-BSANPs and VES-DOX-BSANPs freeze-dried powder with RPMI-1640 culture solution to prepare 0.5mg/mL (calculated by DOX content) mother solution, filtering the mother solution with a 0.22 mu m microporous filter membrane, and diluting the mother solution with RPMI-1640 culture solution until DOX concentration is 50, 25, 5, 1, 0.2 and 0.04 mu g/mL, wherein the mother solution is used for toxicity tests of MCF-7 cells and 4T1 cells. The DOX preparation is diluted by RPMI-1640 culture solution to make the DOX concentration of 100, 50, 10, 2, 0.4 and 0.08 mu g/mL for toxicity test of MCF-7/ADR cells.

(3) MTT assay for detecting cytotoxic effects

After the cells are attached to the wall, 100 mu L of the culture solution containing the medicine is added into each well according to the set concentration in the step (2), 100 mu L of the culture solution is added into a control group, and only 200 mu L of the culture solution without the cells is added into a blank group. After 48h incubation, the plates were removed, 10. mu.L of MTT solution (5mg/mL) was added to each well, placed in the incubator for an additional 4h, the supernatant was aspirated off, and the plates were inverted over filter paper and carefully blotted to remove residual solvent. Add 100. mu.L DMSO/well and shake for ten minutes in a horizontal shaker to completely dissolve the purple crystals. And (4) measuring the absorbance at the wavelength of 570nm by using a microplate reader, calculating the inhibition rate of the cells, and calculating the survival rate of the cells.

(4) Cell drug resistance test

The IC of each material was calculated by GraphPad prism5.0 software₅₀The prepared formulations and the multidrug resistance of the drugs were evaluated by the Resistance Index (RI). The extent to which the agent reverses multidrug resistance in tumor cells was assessed by reversing Resistance Factors (RF). The RI and RF are calculated as follows:

RI＝IC₅₀(MCF-7/ADR)/IC₅₀(MCF-7)

RF＝IC₅₀(DOX)/IC₅₀(VES-DOX-BSANPs)

the growth inhibition effect of DOX, DOX-BSANPs and VES-DOX-BSANPs on MCF-7, MCF-7/ADR and 4T1 cells after incubation for 48h was examined. The results of the MTT assay show that all three drugs have certain cytotoxicity to the three cells. From the overall graph, it can be seen that as the concentration of the drug increases, the survival rate of the cells decreases, and the inhibitory effect of the drug on the cells has concentration dependence. Cell viability after incubation of DOX, DOX-BSANPs and VES-DOX-BSANPs with MCF-7, MCF-7/ADR and 4T1 cells at different concentrations for 48h is shown in FIG. 2, FIG. 3, FIG. 4, IC for each group₅₀The values are shown in Table 1.

TABLE 1 IC of DOX, DOX-BSANPs and VES-DOX-BSANPs on MCF-7, MCF-7/ADR and 4T1 cells₅₀

As can be seen in FIGS. 2 and 4, in the concentration range of 0.04-50 μ g/mL (calculated as DOX content), DOX has higher toxicity to MCF-7 than the two nano preparations, for 4T1 cells, DOX still has higher toxicity to MCF-7 than the two nano preparations at the concentration of 0.04-5 μ g/mL, and VES-DOX-BSANPs show higher cytotoxicity than the bulk drug at the concentration of 25-50 μ g/mL, which indicates that the toxicity to 4T1 cells is increased by the combined administration of VES and DOX at higher concentration, and the higher antitumor effect is achieved.

For the drug-resistant strain MCF-7/ADR, the tolerance degree to DOX is obvious, the cell survival rate is close to 20% when the concentration of DOX raw drug is as high as 100 mu g/mL, the survival rate of MCF-7/ADR is 25.2% when the concentration of DOX is 50%, and the survival rate of MCF-7 is only 6.8%. In the concentration range of 0.4-100 mu g/mL, the compound shows better cytotoxicity to MCF-7/ADR and VES-DOX-BSANPs than to raw drugs and DOX-BSANPs. The drug resistance of VES makes MCF-7/ADR sensitive, enhances the toxicity of DOX single administration, and the anti-tumor effect and the drug resistance of VES make the growth inhibition effect on MCF-7/ADR cells obvious, and the drug effect is further enhanced.

The single-drug DOX has higher cytotoxicity to MCF-7 and 4T1 cells, IC50 is 0.06 +/-0.006 mu g/mL and 0.16 +/-0.003 mu g/mL respectively, the cell survival rate of VES-DOX-BSANPs is lower than that of DOX-BSANPs in MCF-7 cells or 4T1 cells, and the IC50 ratios of the DOX-BSANPs to the VES-DOX-BSANPs are 1.6 and 4 in MCF-7 and 4T1 cells respectively, which shows that the addition of the VES in the nanoparticle preparation obviously improves the toxicity of the DOX preparation with the same concentration to breast cancer cells.

TABLE 2 drug resistance index and Reversal drug resistance factors of VES-DOX-BSANPs against MCF-7/ADR cells

The results of the reversal of multidrug resistance of VES-DOX-BSANPs to the drug-resistant strain MCF-7/ADR on tumor cells are shown in Table 2. MCF-7/ADR has obvious DOX tolerance degree, and the drug resistance index is far greater than that of a VES-DOX-BSANPs preparation. Compared with DOX, the reversal drug resistance factor of VES-DOX-BSANPs to MCF-7/ADR is 1.71, which shows that the nano preparation has better effect of reversing tumor multidrug resistance.

Test example 2: in vivo efficacy study of VES-DOX-BSANPs

This test example two nanoformulations prepared according to example 1 and comparative example 1 were used for the following pharmacodynamic tests:

(1) establishment of tumor model of tumor-bearing mouse

Collecting breast cancer cell 4T1 in logarithmic growth phase, washing with PBS twice, digesting with pancreatin, adding culture solution, blowing to make cell fall off, centrifuging at low speed (1000r/min, 5min), collecting lower layer cell, adding PBS, blowing with liquid transfer gun to make cell suspension, and making into cell suspension with concentration of 5 × 10⁷one/mL. The cell suspension is subcutaneously injected to the left forelimb armpit of a healthy mouse, the amount of the cell suspension injected into each mouse is 0.15mL, and the cells are resuspended before each injection, so that the uneven cell concentration caused by sedimentation is avoided. The growth of the tumor was observed every other day after injection, and the major and minor diameters of the tumor were measured with a vernier caliper to calculate the volume of the tumor. When the tumor volume is 150-200 mm³In time, the molding is considered to be successful.

(2) Dosing regimens

When the tumor volume grows to 150-200 mm³The tumor-bearing mice were dosed periodically. The tumor-bearing mice were randomly divided into 3 groups, namely, a normal saline group, a DOX group, DOX-BSANPs and a VES-DOX-BSANPs, and each group contained 5 mice. The medicine is administered once every three days at the medicine concentration DOX of 5mg/kg for four times continuously, the administration mode is tail vein injection, and the administration volume is 0.2 mL/unit.

(3) In vivo pharmacodynamic investigation and toxic and side effect evaluation

The in vivo pharmacodynamics inspects the growth inhibition effect of the nanoparticle preparation on tumors. After dosing, body weights were weighed daily and the longest (a) and shortest (B) diameters of the tumors were measured using a vernier caliper. And (3) drawing a mouse tumor volume-time and body weight-time change curve. The formula for tumor volume is as follows:

v: tumor volume

A: maximum diameter of tumor

B: minimum tumor diameter

And stopping administration when the tumor volume between the tumor-bearing mouse model group and the administration group is obviously different. The mice are sacrificed by adopting a cervical dislocation method, subcutaneous complete tumors are stripped, the tumor weight is weighed, and the tumor inhibition rate is calculated by the following formula:

within 14 days of administration, the body types and body weights of the mice of the control group and the administration group were observed, the mice were periodically weighed, and the survival state, hair color and activity behavior of the mice were observed, and the excreta thereof were observed.

The maximum diameter and the minimum diameter of the tumor are measured every day, and the volume of the tumor is calculated to be used as one index for observing the tumor growth of the VES-DOX-BSANPs inhibiting mice. As can be seen from FIG. 5, the tumor volume changes were most significant in mice in Saline group, and the tumor growth rate was not affected. The growth speed of the tumor of the mice in the DOX-BSANPs group is relatively slow, the change of the tumor volume of the VES-DOX-BSANPs group is minimum, and the tumor growth inhibition effect is strongest. After the last administration of the experiment, each group of mice is sacrificed, tumor tissues are taken out and weighed, and the tumor inhibition rates of the DOX group, the DOX-BSANPs group and the VES-DOX-BSANPs group are calculated, wherein the tumor inhibition rates of the groups are 40.3%, 49.1% and 71.9% respectively. It can be seen from FIG. 6 that the tumor volume of the saline group is the largest, the tumor volume of the VES-DOX-BSANPs group is the smallest, and the inhibition effect is the most obvious. After sacrifice, tumors of the VES-DOX-BSANPs group were classified as 27.5% (p <0.0001) of the saline group, 47.1% (p <0.05) of the DOX group, and 55.2% (p <0.05) of the DOX-BSANPs group, respectively. The tumor growth of the DOX group, the DOX-BSANPs group and the VES-DOX-BSANPs group is inhibited in different degrees, namely 59.7% (P <0.01), 50.9% (P <0.001) and 27.6% (P <0.0001) of the normal saline group respectively, which indicates that the growth inhibition effect of the VES-DOX-BSANPs group on the mouse tumor is most obvious, because the albumin nanoparticle formed by compounding the VES and the DOX is used as a P-gp inhibitor, the drug concentration of the DOX at the tumor part is increased, the VES has good anti-tumor effect and better anti-drug resistance, and the two drugs are accumulated more at the tumor part due to double effects, so that the stronger anti-tumor effect of the VES-DOX-BSANPs is ensured.

The body weight change of the mice in the blank control group and the respective administration groups during the administration period is shown in FIG. 8. The body weight of mice in the saline group changes most obviously and tends to rise, while the body weight of the mice in the VES-DOX-BSANPs group increases first and then slowly decreases within the first 7 days, and the change of the total body weight is not obvious. Similarly, the DOX-BSANPs group showed a body weight fluctuation during the administration period and a general slow-rising trend. The tumor-bearing mice in the DOX group had a significant weight loss, and the mice had dull hair color and died during the administration. The results show that the raw material drug adriamycin has great toxic and side effects, and the prepared VES-DOX-BSANPs can reduce the toxic and side effects of the adriamycin.

The health state of the mice is observed every day during the administration period, the mice in the normal saline group have normal body shape, are rich and have plump and glossy skin, and the mice in the VES-DOX-BSANPs have good state. In contrast, mice in the DOX group were lean in size and slightly blackened in skin color, and during dosing, the DOX group developed diarrhea to a different extent.

Test example 3: study of tissue distribution in vivo of VES-DOX-BSANPs

This test example two kinds of nano-formulations prepared in example 1 and comparative example 1 were used for the following small animal in vivo tests:

(1) establishment of tumor model of tumor-bearing mouse

Refer to test example 2.

(2) Small animal in vivo imaging

The experiment utilizes the characteristic of DOX fluorescence and can be used for observing the migration and distribution of the medicament in a nude mouse body in real time. The targeting of VES-DOX-BSANPs is proved by adopting a small animal living body imaging technology, namely, MCF-7 tumor-bearing nude mice successfully molded are divided into 3 groups, namely a DOX group, a DOX-BSANPs group and a VES-DOX-BSANPs group. Each group was injected via tail vein with 200. mu.L of a solution containing DOX at 5mg/kg, and was imaged in vivo at 5 time points of 1, 3, 6, 9, and 24h, respectively. 3 minutes prior to imaging, mice were rapidly anesthetized with isoflurane and then tested on the machine.

In vivo imaging results show that, in the DOX group, after the administration for 3 hours, the DOX group has the systemic distribution, when the administration is carried out for 9 hours, the fluorescence of the DOX can be observed at a tumor part, the DOX distribution becomes narrow in 24 hours, the fluorescence can also be observed at the tumor part, but the fluorescence intensity is not enhanced, and the fluorescence intensity shows random change along with the administration time, which indicates that the action time of the drug at the tumor part is not long and the amount of the drug is not large, and the drug disperses in other tissues or is metabolized and discharged out of the body along with the blood circulation, thereby confirming that the tissue distribution of the DOX solution is poor.

For the DOX-BSANPs and VES-DOX-BSANPs groups, after administration for 1h, the drugs are distributed systemically, however, fluorescence of DOX can be observed at tumor parts, the fluorescence intensity of the tumor parts of the VES-DOX-BSANPs group is continuously enhanced along with the time extension, the fluorescence intensity of the tumor parts of nude mice of the DOX-BSANPs group is firstly enhanced and then weakened, and the fluorescence intensity of the DOX at the tumor parts of the VES-DOX-BSANPs group reaches the highest when administration is carried out for 9 h. The results show that VES-DOX-BSANPs have stronger targeting property in a nude mouse, the medicament can be more effectively gathered at a tumor part, VES inhibits the efflux of DOX at the tumor part, and the concentration of the medicament at the tumor part is increased.

After the living body imaging is finished for 24h, two groups of nude mice are killed by taking off cervical vertebrae, are quickly dissected on a super clean bench, take out heart, liver, spleen, lung and kidney tissues and tumors, are washed by physiological saline, are placed on filter paper to absorb moisture, are sequentially placed on a black plastic plate, and are subjected to machine imaging and photographing recording. The tissue fluorescence imaging result shows that the DOX group has stronger fluorescence in the liver part, and weak fluorescence is detected in the tumor part. The fluorescence of the liver part of the DOX-BSANPs group is weaker than that of the DOX group, the fluorescence of the liver part of the DOX-BSANPs group becomes stronger, and the fluorescence signals of other organs are not seen, so that the DOX-BSANPs group also has weak toxic and side effects on the liver. In the VES-DOX-BSANPs group, fluorescence is gathered at a tumor part in a large amount, and the fluorescence intensity is obviously stronger than that of the DOX-BSANPs group. Tissue distribution experiments prove that VES-DOX-BSANPs can be targeted to tumor parts efficiently, and the targeting effect is stronger than that of DOX-BSANPs without VES, so that the tumor resisting effect is stronger.

The above-described embodiments are merely preferred embodiments of the present invention, which is not intended to be limiting in any way, and other variations and modifications are possible without departing from the scope of the invention as set forth in the appended claims.

Claims

1. The composite adriamycin albumin nanoparticle is characterized in that desalted adriamycin and vitamin E succinate are dissolved in an organic solvent to serve as an organic phase, an aqueous solution containing bovine serum albumin BSA serves as a water phase, the organic phase is gradually dripped into the water phase under the shearing condition to form colostrum, and the organic solvent is removed after high-pressure homogenization to obtain the composite adriamycin albumin nanoparticle.

2. A preparation method of the compound adriamycin albumin nanoparticle of claim 1, which is characterized by comprising the following steps:

(1) weighing a certain amount of desalted adriamycin and vitamin E succinate, dissolving the desalted adriamycin and the vitamin E succinate in an organic solvent to serve as an organic phase, and taking an aqueous solution containing bovine serum albumin BSA as a water phase;

(3) and (3) placing the colostrum in a high-pressure homogenizer, circulating for multiple times under certain homogenizing pressure to obtain a nanoparticle suspension wrapping the adriamycin and the vitamin E succinate, and removing the organic solvent to obtain the compound adriamycin albumin nanoparticle suspension aqueous solution.

3. The preparation method of the compound adriamycin albumin nanoparticles according to claim 2, wherein the desalted adriamycin is prepared by adding adriamycin hydrochloride into purified water for ultrasonic dissolution, adjusting the pH value to 8-8.5 by using a sodium hydroxide aqueous solution, extracting by using chloroform, and then performing reduced pressure concentration to remove the chloroform.

4. The method for preparing the compound adriamycin albumin nanoparticles as claimed in claim 2, wherein the dosage of the desalted adriamycin in the step (1) is 5-10mg/mL of the organic phase, and the feeding molar ratio of the desalted adriamycin to the vitamin E succinate is 1: 1-3, and the organic solvent is chloroform.

5. The method for preparing the compound adriamycin albumin nanoparticles as claimed in claim 4, wherein the content of bovine serum albumin BSA in the aqueous phase in the step (1) is 3-10mg/mL of the aqueous phase; the aqueous solution of the water phase in the step (1) is purified water or sodium chloride aqueous solution; the volume ratio of the aqueous phase to the organic phase was 40: 1.

6. The method for preparing the compound adriamycin albumin nanoparticles as claimed in claim 2, wherein in the step (2), after the organic phase is added into the aqueous phase under the shearing condition of the high-shear emulsifying machine, the mixture is continuously sheared for 5min to form colostrum and is sieved by a 100-mesh screen.

7. The method for preparing nanoparticles of doxorubicin albumin as claimed in claim 6, wherein the homogenization pressure in step (3) is 900-1300bar, and the number of cycles is 7-9.

8. The preparation method of the compound adriamycin albumin nanoparticles as claimed in claim 2, wherein the feeding amount of desalted adriamycin DOX in the step (1) is 10mg/mL of organic phase, the feeding molar ratio of desalted adriamycin DOX to vitamin E succinate VES is 1:1, the organic solvent is chloroform, the aqueous solution of the aqueous phase is purified water, the volume ratio of the aqueous phase to the organic phase is 40:1, and the content of bovine serum albumin BSA in the aqueous phase is 5mg/mL of the aqueous phase; the homogenizing pressure in the step (3) is 1100bar, and the cycle times are 7 times.

9. The preparation method of the compound adriamycin albumin nanoparticles as claimed in claim 2, wherein the nanoparticle suspension aqueous solution obtained in the step (3) is pre-frozen in a low-temperature refrigerator, taken out, and placed in a freeze dryer for freeze drying to obtain the compound adriamycin albumin nanoparticle VES-DOX-BSANPs freeze-dried powder.

10. An application of the compound adriamycin albumin nanoparticle of claim 1 in the aspect of tumor resistance.