CN112569206A

CN112569206A - Albumin nano drug delivery system with anti-tumor metastasis and targeting functions and preparation method thereof

Info

Publication number: CN112569206A
Application number: CN201910937259.1A
Authority: CN
Inventors: 陈钧; 王家豪
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-09-29
Filing date: 2019-09-29
Publication date: 2021-03-30

Abstract

The invention belongs to the field of medicinal preparations, and relates to an albumin nano drug delivery system with anti-tumor metastasis and targeting functions and a preparation method thereof. The invention combines a biguanide group on albumin, utilizes albumin to coat biodegradable high molecular material polylactic acid-glycolic acid copolymer (PLGA), and coats chemotherapeutic drug paclitaxel by an emulsification-solvent evaporation method, thereby constructing a multifunctional targeting drug-carrying nano system. The delivery system is accumulated at a tumor part in vivo and is preferentially taken by tumor cells, the paclitaxel and the biguanide albumin are combined to kill the tumor cells and inhibit tumor metastasis, the life cycle is prolonged, and the delivery system has good biological safety and broad prospects in the aspect of treating triple negative breast cancer and metastasis thereof.

Description

Albumin nano drug delivery system with anti-tumor metastasis and targeting functions and preparation method thereof

Technical Field

The invention belongs to the field of medicinal preparations, and relates to an albumin nano drug delivery system with anti-tumor metastasis and targeting functions and a preparation method thereof.

Background

The prior art discloses that cancer is one of the biggest killers of human health at present, Triple Negative Breast Cancer (TNBC) accounts for about 10% -20% of the present breast cancer, and surface estrogen receptor, progesterone receptor and HER2 expression are all negative, and are the only breast cancer types without targeted therapy approved at present; in addition, it has the problems of high interstitial pressure, high invasion, easy recurrence and the like, and the prognosis is poor. Traditional treatments, including surgery, radiation and chemotherapy, can treat tumors well before cancer cells spread, but there is no good way to target the metastatic and recurrent processes of tumors, resulting in high patient mortality.

The traditional chemotherapeutic drugs generally have the problems of poor solubility, short in-vivo circulation half-life, easy removal and difficult accumulation at tumor sites, and the existing coping methods mostly adopt nano-therapy, so that not only can hydrophobic drugs be effectively encapsulated, but also the permeation and retention effects (EPR effect) of the tumor sites can be utilized to achieve high accumulation effects. However, nano-therapies also have certain limitations, such as low encapsulation efficiency and drug loading for chemotherapeutic drugs, easy clearance by the monocyte phagocytic system during in vivo circulation, and the like. Meanwhile, the pure delivery of chemotherapeutic drugs by using a nano system can inhibit the growth of in situ tumors, but is difficult to inhibit metastasis and even promotes metastasis. Therefore, when the nano system is used for delivering the chemotherapeutic drug, the nano system overcomes the drug delivery difficulty, improves the treatment and metastasis inhibition effects and has higher clinical application prospect and conversion value.

Biguanide drugs have recently been newly found in cancer therapy as a conventional therapeutic agent for type II diabetes. Research shows that the biguanide medicine can enhance the treatment effect of chemotherapeutic medicine and has the functions of inhibiting tumor metastasis and improving tumor microenvironment.

The albumin is a serum protein with extremely low immunogenicity, can be used as a good carrier of hydrophobic or hydrophilic drugs, has the ingestion tendency of tumor cells to the albumin, and improves the targeting property of the albumin carrier.

Based on the basis and the current situation of the prior art, the inventor of the present application intends to provide an albumin nano drug delivery system with anti-tumor metastasis and targeting functions and a preparation method thereof, wherein the albumin nano drug delivery system comprises a chemotherapeutic drug and a nano drug delivery system which targets tumors, improves the chemotherapeutic effect and inhibits metastasis.

Disclosure of Invention

The invention aims to provide an albumin nano drug delivery system with anti-tumor metastasis and targeting functions and a preparation method thereof based on the basis and the current situation of the prior art.

Based on the fact that Triple Negative Breast Cancer (TNBC) is a highly aggressive and easily relapsed malignant tumor, three targets which are common in common breast cancer do not exist on the surface of the TNBC, and the TNBC is the only breast cancer type without targeted therapy at present. Paclitaxel (Paclitaxel) is an antimicrotubule drug and is also a common chemotherapeutic drug for treating breast cancer, but has poor solubility, short in-vivo half-life and poor targeting property. Guanidyl is used as a cationic group, and has a great deal of application in the aspect of drug delivery, and biguanide drugs have an anti-tumor effect and also have an effect in the aspect of inhibiting tumor metastasis.

The invention combines a biguanide group on albumin, utilizes albumin to coat biodegradable high molecular material polylactic acid-glycolic acid copolymer (PLGA), and coats chemotherapeutic drug paclitaxel by an emulsification-solvent evaporation method, thereby constructing a multifunctional targeting drug-carrying nano system.

More specifically, the biguanide group is modified on the surface of albumin through chemical reaction, the PLGA polymer core carrying paclitaxel is prepared by an emulsification-solvent evaporation method, and the biguanide albumin is coated outside the PLGA polymer core, so that an albumin-based nano system is constructed. Can target tumor parts in vivo, co-deliver chemotherapeutic drugs and active biguanide groups, improve tumor microenvironment and inhibit cancer metastasis while jointly inhibiting in-situ tumor growth. Meanwhile, the medicine has good biological safety and has wide prospect in the aspect of treating triple negative breast cancer and metastasis thereof.

The biguanide-based modified albumin and the biodegradable polymer PLGA are used as carriers, and the multifunctional nanoparticles are prepared by encapsulating the chemotherapeutic drug paclitaxel, so that the common drug delivery of the paclitaxel and the biguanide is realized, the accumulation of the drug is promoted, the synergistic anti-tumor effect is further exerted, the sensitivity of tumor cells to chemotherapy is increased by utilizing the pharmacological activity of the biguanide, the metastasis of in-situ tumors is inhibited, and the good anti-tumor effect is achieved.

According to the invention, the adopted materials are PLGA and albumin, a biguanide group is modified on the surface of the albumin through a chemical reaction, a high-molecular auxiliary material polylactic acid-glycolic acid copolymer PLGA is used as an inner core of the nanoparticle, and human serum albumin is wrapped outside the inner core, wherein the amino acid residue on the surface of the albumin is used for modifying the biguanide group, so that the targeting property of the nanoparticle is improved, and the anti-tumor transfer effect of the nanoparticle is enhanced.

In the invention, the molecular weight of the polylactic acid-glycolic acid copolymer PLGA is 10-30 kDa.

In the invention, the chemotherapeutic drug paclitaxel is coated on the hydrophobic core of PLGA by an emulsification-solvent evaporation method; the model drug adopted by the invention is paclitaxel, which is encapsulated in biodegradable polylactic acid-glycolic acid material in a physical encapsulation way

In the invention, the surface of the albumin is conjugated with biguanidino (HSA-NH) through nucleophilic reaction, and the biguanidino albumin replaces natural albumin to construct the nanoparticles.

In the invention, the amphiphilic albumin is distributed on a two-phase interface in the oil-water emulsification process, and finally coats a PLGA core to form nanoparticles.

In the invention, after being taken by tumor cells, the nanoparticles are dissociated in the cells and release albumin and chemotherapeutic drugs, and the biguanide group can inhibit the metastasis of in-situ tumors, reduce the polarization of macrophages to M2 type and improve the tumor microenvironment.

In the invention, the paclitaxel and the biguanidino compound exist in the same drug delivery system, exert the synergistic effect and inhibit the transfer, and have the effect remarkably better than that of single drug.

In the invention, in an in-situ breast cancer animal model, the growth of in-situ tumors and the occurrence of pulmonary metastasis can be obviously inhibited through long-term administration, the life cycle of animals is prolonged, and meanwhile, the in-situ breast cancer animal model has good safety in vivo.

The cells adopted by the invention are three-negative breast cancer cells 4T1 of mice, luciferase-labeled 4T1 cells (4T1-Luc) and mouse macrophage RAW264.7 which are recognized in the field and are commercially available.

The mice employed in the present invention are female Balb/c mice, which are recognized in the art and commercially available.

The invention describes a preparation method of a drug co-delivery nano system, and related preparation characterization, in vivo targeting evaluation and pharmacodynamic evaluation.

The invention proves the killing effect of the preparation on cancer cells through cytotoxicity and TUNEL experiments, and the guanidyl and the taxol have combined curative effect.

The invention proves that the drug delivery system can inhibit epithelial-mesenchymal transition (EMT) effect of breast cancer cells and inhibit tumor metastasis through immunoblotting (Western Blot) experiments and flow cytometry experiments, and can reduce M2 type macrophages to regulate tumor microenvironment compared with a commercially available taxol preparation. The in vivo and in vitro targeting property and the inhibition effect on in-situ tumor of the preparation are proved through high content experiments and in vivo pharmacodynamic experiments, and meanwhile, the preparation can reduce the generation of breast cancer pulmonary metastasis, prolong the life cycle of mice and has good application prospect.

Drawings

FIG. 1: characterization of materials, nano-formulations and cytotoxicity, wherein,

a: the rate and progress of the reaction of monoguanidinoalbumin (HSA-AH),

b: the rate and rate of the reaction of biguanide-based albumin (HSA-NH),

c: particle size distribution plots of PLGANP, HSANP, HSA-AH NP, and HSA-NH NP,

d: zeta potential quantitation maps for PLGANP, HSANP, HSA-AH NP, and HSA-NH NP,

e: MTT curve of free paclitaxel to 4T1 cell for 24h,

f: IC of various preparation groups for 4T1 cells for 24h₅₀。

FIG. 2: the uptake and uptake mechanism of tumor cells and macrophages to the nanoparticles, wherein,

a: the results of quantitative uptake of free coumarin-6, PLGANP, HSANP, HSA-AH NP, and HSA-NH NP by Raw264.7 cells,

b: the results of quantitative determination of uptake of free coumarin-6, PLGANP, HSANP, HSA-AH NP and HSA-NH NP into 4T1 cells,

c: results of the effect of different endocytosis inhibitors on the uptake of coumarin, PLGANP, HSANP, HSA-AH NP, HSA-NH NP by 4T1 cells.

FIG. 3: the effect of several kinds of nanometer preparation on the expression of 4T1 cell and tumor tissue protein, and the expression of several kinds of nanometer preparation,

a: effects of Taxol (Taxol), PLGANP, HSANP, HSA-AH NP, HSA-NH NP on E-cadherin, Vimentin (Vimentin) in 4T1 cells,

b: effects of paclitaxel albumin injection (Abraxane), PLGANP, HSANP, HSA-AH NP, and HSA-NHNP on E-cadherin and Vimentin of 4T1 tumor tissue,

c: quantification results of the effects of Taxol (Taxol), PLGANP, HSANP, HSA-AH NP, HSA-NH NP on E-cadherin (cadherin E) expression of 4T1 cells,

d: quantification results of the influence of paclitaxel albumin injection (Abraxane), PLGANP, HSANP, HSA-AH NP, and HSA-NHNP on the expression of E-cadherin (cadherin E),

e: quantitative results of the effect of Taxol (Taxol), PLGANP, HSANP, HSA-AH NP, HSA-NH NP on the expression of Vimentin (Vimentin) in 4T1 cells,

f: quantification of the effect of paclitaxel albumin injection (Abraxane), PLGANP, HSANP, HSA-AH NP, and HSA-NHNP on Vimentin expression.

FIG. 4: flow cytometry was used to examine the effect of the nanopreparations on the polarization of macrophages M1, M2, wherein,

a: flow results of the effect of each formulation group on macrophage M1 typing,

b: results quantifying the effect of each formulation group on macrophage M1 typing,

c: flow results of the effect of each formulation group on macrophage M2 typing,

d: quantification of the effect of each formulation group on macrophage M2 typing.

FIG. 5: the in vivo targeting results of the nanosystems, wherein,

a: as a result of the change of DiR fluorescence with time in tumor-bearing mice in each preparation group,

b: the DiR fluorescence qualitative result of each preparation group in the main organs of the mice,

c: DiR fluorescence quantification in mouse major organs was performed for each formulation group.

FIG. 6: in vivo pharmacodynamic evaluation of nanosystems, wherein,

a: the qualitative result of the isolated lung transfer bioluminescence of each preparation group of mice,

b: the mouse in vitro lung transfer bioluminescence quantitative result of each preparation group,

c: the qualitative result of mouse tumor tissue cell proliferation and apoptosis of each preparation group,

d: the tumor volume of the tumor-bearing mice after the multiple administration of each preparation group,

e: the life cycle of tumor-bearing mice after multiple administration of each preparation group.

The specific implementation mode is as follows:

example 1: preparation, characterization and cytotoxicity of materials and Nanodimulants

Connecting a biguanide group to albumin through a chemical reaction, self-condensing 1H-pyrazole-1-formamidine hydrochloride (AH) to form biguanide hydrochloride (NH), dissolving 133mg of HSA and 35mg of AH and NH in 2mL of triple distilled water respectively, adding 30 mu LN, N-Diisopropylethylamine (DIEA), stirring for 24H at room temperature, dialyzing for two days by using the triple distilled water, freeze-drying to obtain HSA-AH and HSA-NH, and detecting the reaction progress by using an ultraviolet spectrophotometer;

the nano drug delivery system is prepared by an emulsification-solvent evaporation method. 20mg of PLGA was weighed out, dissolved in 1ml of 0.5mg/ml PTX in dichloromethane and vortexed to dissolve. Adding 2ml of 1% sodium cholate solution, and carrying out ultrasonic treatment for 2.5min in an ice-water bath state, wherein the working time and the interval time are both 2s, and the ultrasonic power is 200-220W. Pouring the emulsified system into 8ml of 0.5% sodium cholate solution, stirring and dispersing, and removing dichloro-chloride by rotary evaporation after 0.5hMethane. And centrifuging the rest system by a high-speed centrifuge at 14000rpm for 45 min. The supernatant was discarded and the nanoparticle pellet was washed 2 times with PBS. And (3) carrying out heavy suspension by using 1ml of PBS or normal saline to obtain a PLGA nanoparticle solution. HSA, HSA-AH, and HSA-NH nanoparticle solutions were prepared in the same manner as described above, except that 2ml of 1% sodium cholate solution was replaced with 2% albumin aqueous solution. The particle size and the potential of the nanoparticles are measured by a Malvern particle size/Zeta potential measuring instrument. After 4T1 cells adhere to the wall for 24h, free paclitaxel, HSA-AH and HSA-NH nanoparticles with different concentration gradients are added, and after incubation for 24h, the cytotoxicity of each preparation group to 4T1 is detected by using an MTT colorimetric method, and IC is calculated₅₀A value;

the results show that: FIGS. 1A and 1B show that the HSA-AH reaction rate is greater than that of HSA-NH, since the reactivity of 1H-pyrazole-1-carboxamidine hydrochloride (AH) itself is about 30% higher than that of the biguanide hydrochloride (NH) in both final reaction schedules. FIGS. 1C and 1D show that the PLGANP has a particle size of about 80nm and a potential of about-25 mV; HSANP, HSA-AHNP, HSA-NH have a particle size of about 180nm and a potential of about-12 mV, since there is a significant increase in particle size after coating with albumin, and the surface potential shows the potential of albumin itself (about-12 mV). FIG. 1E shows IC of paclitaxel free drug₅₀The value was about 350ng/ml, while FIG. 1F shows that PLGA, HSA-AH group nanoparticles have IC lower than free drug by passive diffusion into cells due to lower efficiency of cell entry₅₀The value is larger than that of free drugs, and the albumin has certain nutritive value on tumor cells and can influence the cell viability. And IC of HSA-NH formulation group₅₀The value is obviously lower than that of a control albumin preparation group, and the results prove that the biguanide also generates a synergistic effect on chemotherapy and increases the cytotoxicity of the chemotherapeutic drug paclitaxel while the chemotherapeutic drug paclitaxel plays a role.

Example 2: cellular uptake and pathway investigation of NanoPreprants

In order to prove the importance of albumin and biguanide groups on the outer layer of the nanoparticles to the cellular uptake, 4T1 breast cancer cells and Raw264.7 macrophages are selected to investigate the uptake conditions of the nanoparticles to various nano preparations. Each nanoparticle carries coumarin-6 to carry out fluorescence labeling by an emulsification-solvent evaporation method. After the cells are planted in the 96-well plate of 4T1 and Raw264.7 for 24 hours, sucking out a culture medium, adding nanoparticles according to a preset concentration gradient table, sucking out the nanoparticles after the cells take up for 2 hours, washing by Phosphate Buffer Solution (PBS), fixing by 4% paraformaldehyde for 15 minutes and staining nuclei by nuclear dye (Hoechst reagent) in a dark place for 8 minutes, and then inspecting qualitative and quantitative results of cell uptake by a high content analysis system;

in order to prove the mechanism of taking up the nano preparation by the 4T1 breast cancer cells, several taking-up inhibitors, namely methyl beta-cyclodextrin (M beta-CD), Amiloride (Amilolide), necolazole (Nocodazole) and Genistein (Genistein), are selected, the 4T1 cells are planted in a 96-well plate for 24 hours, the culture medium is sucked out, and the taking-up inhibitors with certain concentration are respectively added for incubation for 0.5 hour. Absorbing a culture medium, adding nanoparticles with a certain concentration, incubating for 2h, then removing the culture medium, washing by PBS, fixing by 4% paraformaldehyde for 15min, and staining nuclei by Hoechst reagent in a dark place for 8min, and analyzing the quantitative result of cell uptake with high content;

the results show that: fig. 2A is a high content quantitative result of the uptake of several agents by raw264.7 cells, macrophages are the main phagocytic cells in the circulatory system that can recognize foreign substances in the body and phagocytose, resulting in the off-target of nanoparticles. And the HSA, HSA-AH and HSA-NH three groups of nano preparations are less taken up by macrophages compared with the pure PLGA nano preparation, so that the fact that the albumin on the surface brings low immunogenicity for the nano preparations is proved, and loss in vivo circulation is reduced. Figure 2B is a high content quantification of the uptake of several formulations by 4T1 cells, with significantly higher uptake of biguanide-based modified albumin formulations compared to the other groups, in addition to the tumor targeting of surface albumin, due to the basic cationic high transmembrane effect of biguanide. Fig. 2C shows that methyl β -cyclodextrin group has significant inhibitory effect on the formulation invasion through the action of different uptake inhibitors, demonstrating that the invasion pathway of several nano-formulations is the cell membrane cave-like invagination pathway, which is the same as the previous albumin nano-formulation invasion pathway.

Example 3: effect of Nanodichest on tumor EMT Effect

According to the results of previous experiments in the literature and laboratories, metformin has the ability to inhibit epithelial-to-mesenchymal transition (EMT) of cancer cells, and thus can inhibit metastasis of cancer. In order to examine the influence of several nano preparations on the EMT effect of 4T1 tumors, after a 6-well plate is used for 4T1 cells for 24 hours, a free drug, PLGANP, HSANP, HSA-AH and HSA-NHNP are respectively added with a paclitaxel equivalent of 150ng/mL serving as a dosing concentration, after the cells are incubated in an incubator for 24 hours, supernatant is sucked off, the 6-well plate is placed on ice after being washed for 3 times by PBS, 250 mu L of RIPA lysate is added into each well for cracking for 30 minutes, cells are scraped by a cell scraper, cell fragments and lysate are added into a 1.5mL centrifuge tube, and the centrifuge tube is centrifuged for 10 minutes at 4 ℃ and 12000 rpm/min. Transferring the supernatant to a new precooled EP tube, discarding the precipitate, quantifying by using a BCA kit, adding a proper amount of 5x sample loading buffer solution, uniformly mixing by vortex, heating on a 95 ℃ dry heater for 5min, and loading. For the study of EMT protein expression at the tissue level, several formulations were administered to tumor-bearing mice, each mouse administered an equivalent of 5mg/kg paclitaxel and one dose every two days, four times, with each group of n-6. Perfusing the heart of the mouse, taking out tumor tissue, and cracking, quantifying and sampling the tumor tissue according to the method;

the results show that: fig. 3A, 3C, and 3E show that, after incubation with several kinds of nano-preparations, the nano-preparation of biguanide albumin can significantly up-regulate the expression of E-Cadherin (E-Cadherin) in cancer cells, and simultaneously reduce the expression of Vimentin (Vimentin) in cancer cells, thereby reducing the EMT effect of 4T1 breast cancer cells, inhibiting the invasion and metastasis of cancer cells, and proving that the biguanide group on albumin can play a role in regulating the EMT effect at the in vitro cell level. Fig. 3B, 3D, and 3F show that, in animal in vivo experiments, the biguanide-based nano-formulation can also up-regulate E-cadherin, down-regulate vimentin expression, and reduce the occurrence of EMT effect. In summary, the biguanide group albumin can exert the anti-transfer effect of biguanide drugs and is effectively proved in vivo and in vitro, while other preparation groups including monoguanidino albumin have no obvious regulation effect on the aspect of EMT effect, and the necessity of adopting biguanide group modification is proved.

Example 4: effect of nano preparation on macrophage M1 and M2 typing

To verify whether the biguanide-based albumin nano-preparation affects tumor-associated macrophages in the tumor microenvironment, the change of the proportion of M1 and M2 types of macrophages after administration was examined. RAW264.7 cells activated with 50ng/ml IL-13 served as M2 type positive control group, and RAW264.7 cells activated with 100ng/ml LPS served as M1 type positive control group. Specifically, a 6-pore plate of RAW264.7 cell species is provided, each pore is respectively added with a stimulating factor IL-1350ng/ml or LPS 100ng/ml, paclitaxel with the equivalent of 150ng/ml and a plurality of nano preparations are respectively given after the cells are placed in an incubator to grow for 24h, the cells are collected after 12h, IL-13 group is added with M2 type macrophage marker (CD206) antibody labeled by fluorescent dye PE for staining for 30min, LPS group is added with M1 type macrophage marker (CD86) antibody labeled by fluorescent dye APC for staining for 30min, and after PBS cleaning, the proportion of positive cells is scanned by flow cytometry;

the results show that: fig. 4A, 4B show that when RAW264.7 cells were incubated with LPS for 24h, a large number of CD86 positive cells were produced, the ratio of macrophage M1 phenotype was slightly increased by the free drug, PLGA, HSA groups, while the M1 phenotype was suppressed by the guanidino agent due to the anti-inflammatory properties of the guanidino group, but there was no significant difference, demonstrating that several agents had little effect on macrophage M1 phenotype in the tumor microenvironment; FIGS. 4C and 4D show that after Raw264.7 cells are incubated with IL-13 for 24h, a large number of CD206 positive cells are generated, and the biguanide-based preparation group has obvious inhibition capability of M2 phenotype compared with the free drug, PLGA, HSA and HSA-AH groups, so that the biguanide active group has M2 macrophage regulating effect and can improve the tumor microenvironment.

Example 5: in vivo targeting effect of the NanoPreparation

Establishing an in-situ breast cancer mouse model: the 4T1 cells in logarithmic growth phase were taken and resuspended in serum-free medium, the cell suspension concentration was adjusted to 1X 10⁸cells/mL, and standing at 4 ℃ for later use. Carrying out intraperitoneal injection of 120 mu L of 5% chloral hydrate for anesthesia on female Balb/c mice, using depilatory cream to remove body hair near the third and fourth milk fat pads on the left side of the mice, carefully inoculating cell suspension into the milk fat pads on the fourth left side of the mice, slowly moving out a needle after the milk fat pads of the mice are obviously filled, dripping a proper amount of antibiotic on wounds, and continuing to raise the mice after the mice are recovered;

in a small animal living body imaging experiment, the nano-particles use a cell membrane deep red fluorescent probe (DiR) as a fluorescent label, and PLGA, HSA-AH and HSA-NH nano-particles loaded with the DiR are respectively prepared by an emulsification-solvent evaporation method. Tumor-bearing mice were randomly divided into 6 groups of 6 mice each, and administered at a dose of 1mg/kg DiR. Anesthetizing and fixing a mouse by using 5% chloral hydrate after the preparation is administered for 1h,2h,4h, 12h, 24h, 36h and 48h, perfusing by using normal saline, taking tumor tissues and heart, liver, spleen, lung and kidney, flushing by using the normal saline, and then placing in a small animal living body imaging instrument to acquire images;

the results show that: as can be seen in fig. 5, the fluorescence intensity of each nano-preparation group at the tumor site after administration is higher than that of the free DiR group, and the fluorescence intensity can be maintained for a longer time, which proves the EPR effect and long-circulating effect of the nano-preparation. The PLGA formulation is strongly accumulated at the beginning because it has a small particle size and can be accumulated in a large amount by the EPR effect in a short time. However, the albumin preparation group has better tumor site retention effect over time, and the monoguanidino and biguanide albumin groups have strongest fluorescence, because albumin has certain tumor targeting capacity, and guanidino also increases the uptake of tumor cells to the preparation, so that the albumin obtains better tumor retention effect.

Example 6: in vivo tumor inhibiting effect and anti-metastasis effect of nano preparation

The mouse model of in situ breast cancer was established as in example 5, using 4T1-Luc cells for in situ tumor inoculation. When the tumor volume reaches 100mm³On the left and right, experimental mice were randomly divided into 6 groups (n ═ 15), the control group was injected with physiological saline, and the experimental groups were administered with Taxol, Abraxane and several nano-formulations, respectively, once every three days from day 0 for 5 times (paclitaxel dose 5mg/kg, other formulations were administered in the same equivalent), while the body weight of the mice was recorded every two days. On day 20 (third day after the last administration), 5 mice in each group were sacrificed, the heart was perfused with physiological saline and the lungs were taken out, and the lung tissues were immediately immersed in 0.5mg/mL D-luciferin solution for 5min and then placed in a live imager to detect bioluminescence while performing semi-quantitative analysis. After the administration period, the normal saline and each preparation group were anesthetized with 150 μ L of 5% chloral hydrate 5 per group, fixed with 4% paraformaldehyde after cardiac perfusion with normal saline, and paraffin-embedded forSections were stained with Ki67 and TUNEL, respectively, to show cell proliferation and apoptosis within tumor tissue. Another 5 per group were used to investigate the life cycle status;

the results show that: FIGS. 6A and 6B show that the saline, Taxol and Abraxane groups produced severe lung tumor metastasis; PLGA, HSA-AH groups all transferred but to a lesser extent; the HSA-NH group produced the least transferred luminescence intensity. Quantitative analysis shows that the nanometer preparation group can inhibit in-situ tumor and reduce metastasis due to high drug delivery effect. The biguanide albumin group can inhibit lung metastasis of breast cancer to a greater extent by regulating microenvironment and EMT effect due to the existence of biguanide active groups. Meanwhile, as shown in fig. 6C-E, commercially available preparations Taxol and Abraxane have similar anti-tumor effects, and both cannot inhibit lung metastasis of tumors, while the nano preparation has a slightly better anti-tumor effect due to better drug delivery and targeting capabilities, but according to the experimental results, the biguanide-based albumin nano preparation has the best in-situ tumor inhibition and anti-proliferation and pro-apoptosis effects, so that the life cycle of mice is obviously prolonged, and the lung metastasis of tumors can be obviously inhibited by inhibiting the EMT effect. In summary, the biguanide albumin has the effect of inhibiting tumor metastasis while having a good in-situ tumor treatment effect.

Claims

1. An albumin nano drug delivery system with anti-tumor metastasis and targeting functions is characterized in that a polymer adjuvant polylactic acid-glycolic acid copolymer (PLGA) is used as an inner core of a nanoparticle, and human serum albumin is wrapped outside the inner core of the nanoparticle, wherein amino acid residues on the surface of the albumin are used for modifying biguanidino, so that the targeting property of the nanoparticle is improved, and the anti-tumor metastasis effect of the nanoparticle is enhanced.

2. The albumin nano drug delivery system with anti-tumor metastasis and targeting functions as claimed in claim 1, wherein the molecular weight of the poly (lactic-co-glycolic acid) (PLGA) is 10-30 kDa.

3. The albumin nano drug delivery system with anti-tumor metastasis and targeting functions as claimed in claim 1, wherein the chemotherapeutic drug paclitaxel is coated on the hydrophobic core of PLGA by emulsion-solvent evaporation.

4. The albumin nano drug delivery system with anti-tumor metastasis and targeting functions as claimed in claim 1, wherein the albumin surface is conjugated with biguanidino (HSA-NH) through nucleophilic reaction, and the biguanidino albumin replaces natural albumin to construct nano particles.

5. The albumin nano drug delivery system with anti-tumor metastasis and targeting functions as claimed in claim 1, wherein the amphiphilic albumin is distributed at two phase interfaces in the oil-water emulsification process, and finally coats the PLGA core to form nanoparticles.

6. The albumin nano drug delivery system with anti-tumor metastasis and targeting functions as claimed in claim 1, wherein the tumor is triple negative breast cancer.