CN112999363A - Multi-drug mixed micelle based on metformin and preparation method and application thereof - Google Patents

Multi-drug mixed micelle based on metformin and preparation method and application thereof Download PDF

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CN112999363A
CN112999363A CN202010592797.4A CN202010592797A CN112999363A CN 112999363 A CN112999363 A CN 112999363A CN 202010592797 A CN202010592797 A CN 202010592797A CN 112999363 A CN112999363 A CN 112999363A
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metformin
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陈钧
蒋天泽
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Fudan University
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Abstract

The invention belongs to the technical field of medicinal preparations, and relates to a nano drug delivery system based on metformin and a preparation method thereof. The mixed micelle delivers OA-Met (or metformin), pro-DHA and Trip to in-situ tumor tissues in a targeted manner at the same time through the high affinity of HA and over-expressed CD44 on the surface of tumor cells, the three medicines play a synergistic effect to prevent EMT and tumor metastasis through different action mechanisms, and simultaneously effectively inhibit the growth of in-situ tumors, prolong the life cycle, improve the treatment effect of metastatic tumors and have good in-vivo safety.

Description

Multi-drug mixed micelle based on metformin and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicinal preparations, and relates to a multi-medicament mixed micelle based on metformin and a preparation method thereof. The multi-drug mixed micelle delivers three drugs to in-situ tumor tissues in a targeted mode, prevents EMT and tumor metastasis through different action mechanisms in a synergistic mode, and achieves the purpose of improving the treatment effect of metastatic tumors by inhibiting the growth of in-situ tumors in a synergistic mode.
Background
The prior art discloses that tumor metastasis is the most important cause of high mortality of tumor patients, and taking the strong metastatic tumor triple negative breast cancer as an example, clinical practice shows that more than 50% of patients have tumor metastasis, and nearly 90% of patients die due to tumor metastasis. In the course of tumor treatment, conventional therapeutic methods such as chemotherapy can be used for in situ tumor treatment and achieve therapeutic effects, however, these therapeutic methods lack treatment for the process of metastasis, resulting in difficulty in complete cure of the tumor; the mechanism of metastasis formation is becoming an important target for tumor therapy of interest to the skilled artisan.
Studies have shown that tumor metastasis generally goes through the following processes: (1) the in-situ tumor cells acquire invasive ability, migrate in the stroma and enter the circulatory system of the body to become Circulating Tumor Cells (CTCs); (2) CTCs spread throughout the body through the circulatory system and migrate to distant organs; (3) CTCs colonize specific distal organs by extravasation angiogenesis and subsequently form tumor metastases. In the first step of metastasis, the EMT process in the in situ tumor tissue is an important mechanism of tumor metastasis, and in the tumor tissue, the epithelioid tumor cells generate EMT, lose intercellular tight connection, remodel cytoskeleton, change cell morphology and gene expression, acquire invasiveness, migrate and leave the in situ tumor tissue, and finally enter the body circulation system to diffuse to the distant organs to generate metastasis. An over-expressed cytokine, such as transforming growth factor-beta (TGF-beta), in tumor tissue can down-regulate the expression of the epithelial-like intercellular adhesion molecule E-cadherin and reduce intercellular adhesion, thus causing the tumor cells to EMT and providing guarantee for self-migration, and the down-regulation of E-cadherin is a main mark for the EMT process. In addition, the dissemination of aggressive tumor cells to distant organs requires the self-renewal and survival differentiation capacity of stem cells, so the generation of tumor stem cells (CSCs) is closely related to the EMT process and is also one of the important features of EMT. In the traditional tumor treatment, cytotoxic drugs can promote tumor metastasis by inducing the EMT process of tumor cells, so that the treatment needs to be carried out by selecting proper low-toxicity drugs. For example, metformin is a first-line therapeutic drug for type II diabetes, and the toxicity of metformin itself is low, and studies show that metformin can reverse the abnormal expression of E-cadherin and specifically inhibit the formation of CSCs by activating AMPK besides the therapeutic action of diabetes, thereby inhibiting the EMT process of tumor cells and preventing tumor metastasis, and simultaneously inhibit the growth of in-situ tumor by the activation of AMPK, metformin can improve the comprehensive therapeutic effect of metastatic tumor by simultaneously inhibiting the generation of EMT and the growth of tumor, while metformin has short half-life and lacks targeting property in vivo, and the administration dosage of metformin required for treatment can cause lactic acidosis and other side effects, so that the therapeutic effect can be increased and the side effects can be solved by increasing targeting property or combined administration and other modes.
The research discloses that the HDAC inhibitor is a compound with a plurality of unsaturated carbon chain structures, and can be mixed with OA-Met to form a stable self-assembled micelle to realize combined treatment. The HDAC inhibitor can promote the expression of certain genes related to cell cycle locking and apoptosis through the inhibition of HDAC to inhibit tumor growth, and can also restore the normal expression of E-cadherin by regulating the expression of the genes, promote the differentiation of CSCs and other ways to reverse the EMT process so as to prevent the formation of metastasis. Pro-DHA has HDAC inhibitory activity in vitro, and Pro-DHA is suitable to be mixed with OA-Met to obtain a more stable self-assembled micelle due to the long fatty carbon chain with high unsaturation degree, so that the synergistic treatment of HDAC inhibitory action and metformin is realized, and the problem of weak in-vivo administration effect (drug resistance) of the HDAC inhibitor is solved. In addition, the cytotoxic drug has a very obvious effect on the treatment of in-situ tumors, and the encapsulation of the toxic drug in the hydrophobic core of the OA-Met and pro-DHA mixed micelle can enhance the treatment effect of in-situ tumors.
Based on the current situation of the prior art, the application aims to provide a multi-drug mixed micelle based on metformin and a preparation method thereof, and the aim of inhibiting the generation of tumor metastasis is achieved by taking the EMT process occurring in-situ tumor tissues as a target point for drug administration intervention.
Disclosure of Invention
The invention aims to provide a multi-drug mixed micelle based on metformin, a preparation method thereof and application thereof in metastatic tumor treatment based on the current situation of the prior art.
The invention provides a nano drug delivery system based on metformin, which is characterized in that a metformin amphiphilic derivative (OA-Met) and a Histone Deacetylase (HDAC) inhibitor pro-DHA are mixed, self-assembled in water and simultaneously entrap triptolide (Trip) to form cationic micelle OPTs, and finally Hyaluronic Acid (HA) of anions is adsorbed on the surface of the cationic micelle OPTs through electrostatic adsorption to construct novel multi-drug mixed micelles (HOPTs). The multi-drug mixed micelle delivers OA-Met (or metformin), pro-DHA and Trip to in-situ tumor tissues in a targeted mode simultaneously through the high affinity of HA and CD44 overexpressed on the surface of tumor cells. The three medicines play a synergistic role through different action mechanisms to prevent the occurrence of EMT and tumor metastasis, and simultaneously effectively inhibit the growth of in-situ tumor to prolong the life cycle, thereby achieving the effect of improving the treatment effect of metastatic tumor.
Based on the tumor EMT process, the pharmacological action of the metformin and the advantages of nano-drugs, the aim of effectively treating metastatic tumors is fulfilled by constructing the nano-drugs based on the metformin by using the nanotechnology, however, the metformin is a water-soluble drug, and the targeted drug delivery of the metformin is difficult to realize by using a conventional nano-carrier form, so that the application adopts an amphiphilic derivative OA-Met with activity similar to that of the metformin as a carrier material, self-assembles in water to construct a nano drug-loading platform, and in view of the poor single drug effect in the metformin, the combined drug use of the metformin and other drugs is realized through OA-Met micelles; researches show that the hydrophobic drug containing long unsaturated fatty carbon chains in the structure can form a stable mixed micelle with OA-Met through hydrophobic interaction and pi-pi conjugation, and meanwhile, other hydrophobic drugs can be encapsulated in the inner hydrophobic core, so that the combined treatment of the metformin and other drugs is realized, and the treatment effect is improved through different action mechanisms.
In view of the fact that the traditional cytotoxic drugs can promote tumor metastasis by inducing the tumor cell EMT process, triptolide (Trip) is selected to be encapsulated in mixed micelles to realize combined treatment; trip not only inhibits the proliferation of tumor cells, but also can reverse the abnormal expression of E-cadherin by inhibiting inflammatory signals (such as NF-kB), generate toxicity to CSCs and other ways to inhibit the EMT process of the tumor cells and prevent the occurrence of metastasis, and meanwhile, the Trip applied to micelles can improve the serious in vivo adverse reactions; in the invention, OA-Met and HDAC inhibitor pro-DHA are mixed, self-assembly is carried out in water, Trip is encapsulated to form a mixed micelle, HA is adsorbed on the surface of the micelle to realize targeted co-delivery of metformin, pro-DHA and Trip to in-situ tumors, and through different action mechanisms of the three drugs, the generation of EMT and metastasis is prevented while in-situ tumors are synergistically inhibited, and the comprehensive curative effect of metastatic tumors is improved.
The invention effectively inhibits the tumor metastasis by taking the EMT process generated in the in-situ tumor tissue as a target point for drug administration intervention.
More specifically, the present invention is to provide a novel,
in the invention, the first model drug is metformin, and the amphiphilic derivative OA-Met with similar pharmacodynamic activity can be used as a carrier material to form a micelle drug-carrying platform, so that the combined treatment of the metformin and other drugs is realized.
In the invention, the second model drug is a compound pro-DHA with HDAC inhibitory activity, which forms a mixed micelle through the hydrophobic effect and pi-pi conjugation with OA-Met unsaturated carbon chain to exert synergistic effect with metformin.
In the invention, the third model drug is Tripterygium wilfordii extract Trip, and the hydrophobic Trip can be effectively encapsulated in the hydrophobic core of the OA-Met and pro-DHA mixed micelle, so that the combined medication of the metformin, the pro-DHA and the Trip is realized.
In the invention, the adopted HA is adsorbed on the surface of the micelle through static electricity, and is targeted to tumor tissues through combining CD44, so that the effects of blocking EMT and preventing tumor metastasis are achieved, and the tumor growth is inhibited at the same time, thereby achieving the purpose of effectively treating metastatic tumors.
In the invention, the cells adopted are the three-negative breast cancer cell 4T1 of a mouse and the luciferase-labeled 4T1 cell (4T1-Luc) which are recognized in the field and are commercially available.
The mice used were female Balb/c mice, which are recognized in the art and commercially available.
The invention further provides a preparation method of the metformin-based multi-drug mixed micelle, and related preparation representation, in-vivo targeting evaluation, mechanism investigation and pharmacodynamic evaluation of EMT inhibition and tumor growth.
The invention proves that the multi-drug mixed micelle is successfully targeted to the in-situ tumor tissue through in-vivo distribution experiments.
MTT (methyl thiazolyl tetrazolium) experiments prove that the multi-drug mixed micelle has the capacity of inhibiting the proliferation of tumor cells; the in vitro CSCs microsphere balling proves that the multi-drug mixed micelle can inhibit the formation of CSCs; the effect of the multi-drug mixed micelle on up-regulating E-cadherin on inhibiting EMT is verified by immunohistochemical staining of tumor tissue sections.
The in-vivo pharmacodynamic experiment proves that the multi-drug mixed micelle can reduce the lung metastasis formation of in-situ breast cancer, inhibit the in-situ tumor growth and prolong the life cycle of tumor-bearing mice; in addition, the toxic and side effects of the Trip in vivo are improved, and the Trip has good in vivo medication safety.
Drawings
FIG. 1 is the structural formula of pro-DHA.
Fig. 2 is a result of characterization of metformin-based multi-drug mixed micelles, wherein,
panel A is a plot of particle size distribution and quantitation of OPTs, DOPTs and HOPTs,
panel B is a Zeta potential quantitation plot for OPTs, DOPTs and HOPTs,
panel C is a field emission electron micrograph of OPTs and HOPTs,
wherein: scalebar,100 nm.
Fig. 3 is the in vivo targeting results for metformin-based multi-drug mixed micelles, wherein,
panel A is a qualitative result of DiR fluorescence from major organs and tumors of mice in the OPTs, DOPTs and HOPTs groups,
panel B is a quantification of DiR fluorescence from major organs and tumors of mice in the OPTs, DOPTs and HOPTs groups, where P < 0.0001.
Fig. 4 is the in vitro toxicity results (Trip-related group tumor cell survival curves) of metformin-based multi-drug mixed micelles on breast cancer cells.
FIG. 5 is a graph of the effect of metformin-based multi-drug mixed micelles on inhibiting the in vitro beading of tumor stem cell microspheres, wherein,
FIG. A is a qualitative result of stem cell balling inhibition in the tumor stem cell induction medium for each administration group,
panel B is a calculation result of tumor sphere balling efficiency (MSFE) for inhibiting stem cell balling for each administration group in the tumor stem cell induction medium;
Scalebar,100μm.*P<0.05,**P<0.01,****P<0.0001.。
FIG. 6 is an evaluation of the effect of metformin-based multi-drug mixed micelles on the upregulation of E-cadherin expression in tumor tissues, wherein,
panel A shows the results of immunohistochemical staining of up-regulated E-cadherin expression in tumor tissues from each administration group,
panel B is the results of immunohistochemical quantitative analysis of the upregulated expression of E-cadherin in tumor tissues from each dosing group;
Scalebar,100μm.*P<0.05,**P<0.01,****P<0.0001。
fig. 7 is an evaluation of the efficacy of metformin-based multi-drug mixed micelles in an in situ breast tumor mouse model, wherein,
FIG. A is a volume growth curve of in situ breast tumor of mice of each administration group during the treatment process,
the graph B is the quantitative result of the in-situ breast tumor mass of each administration group of mice after the treatment is finished,
figure C is the result of biological self-luminescence of the lung metastasis in vitro of each administration group of mice after the treatment is finished,
figure D shows the results of the self-luminescence quantification of the ex vivo lung metastasis of mice in each administration group after the treatment,
FIG. E is a life cycle curve of tumor-bearing mice in each administration group;
*P<0.05,**P<0.01,***P<0.001,****P<0.0001。
fig. 8 is an in vivo safety evaluation of metformin-based multi-drug mixed micelles, wherein,
FIG. A is a graph showing the change in body weight of mice in each administration group during the course of treatment,
panel B is the H & E staining of major organ tissues of mice in the saline, Trip, HOTs, DOPTs and HOPTs groups after treatment;
note: scallebar, 200 μm.
The specific implementation mode is as follows:
example 1: preparation and characterization of mixed micelle based on metformin and multi-drug
Micelles (OPTs) which are mixed by OA-Met and pro-DHA and simultaneously entrap Trip are prepared by a nano-precipitation method. mu.L of Trip ethanol stock solution (1.5mg/mL) was weighed into an EP tube and the solvent was blown dry with a nitrogen blower. 2mg of pro-DHA was weighed into 500. mu.L of OA-Met ethanol stock (10mg/mL), added to an EP tube containing Trip, and the Trip was vortexed. The mixed solution was slowly dropped into 1mL of distilled water (500rpm) under stirring, and stirred at room temperature for 3 hours. And (3) evaporating ethanol under reduced pressure in a water bath at 25 ℃ to obtain OPTs micelle suspension, and storing at 4 ℃ for later use.
HA encapsulated OPTs (HOPTs) were prepared by electrostatic adsorption. Centrifuging the OPTs micelle suspension at 3000rpm for 5min, measuring 300 μ L of OPTs micelle suspension supernatant, slowly dripping into 2mL HA aqueous solution (0.8mg/mL) while stirring (500rpm), stirring at room temperature for 2 h to obtain HOPTs micelle suspension, and storing at 4 ℃ for later use. Dextran (Dextran) -encapsulated OPTs (DOPTs) were also prepared by electrostatic adsorption and used as a non-targeting polysaccharide micelle control for follow-up studies (including OPTs). The HA-coated OA-Met and pro-DHA mixed micelle HOPs (without Trip) and the HA-coated OA-Met-coated Trip micelle HOTs (without pro-DHA) can be used as control micelles lacking a certain component in HOPTs in subsequent researches, and the micelles are prepared by the nano-precipitation and electrostatic adsorption method, namely, the single Trip or the single pro-DHA is dissolved in an OA-Met ethanol stock solution for subsequent micelle preparation. DiR-labeled OPTs, DOPTs and HOPTs are prepared by dissolving DiR instead of Trip and pro-DHA together in OA-Met ethanol stock solution, and the rest preparation methods are the same as above.
The results show that: the particle sizes of OPTs, DOPTs and HOPTs were 140.7 + -3.2 nm, 175.3 + -2.6 nm and 170.2 + -2.4 nm, respectively, and the corresponding PDIs were 0.210 + -0.014, 0.124 + -0.019 and 0.124 + -0.016, respectively. The Zeta potentials of the OPTs, DOPTs and HOPTs were 46.4 + -3.6 mV, -19.1 + -2.2 mV and-19.6 + -3.2 mV, respectively. The particle size increased and the potential changed from positive to negative indicating that the negatively charged polysaccharide was successfully adsorbed to the surface of the OPTs by electrostatic interaction. The field emission electron microscope analysis shows that the micelle HAs no obvious aggregation phenomenon, and compared with OPTs, the micelle HAs an obvious HA adsorption layer on the surfaces of HOPTs, thereby proving that HA is successfully adsorbed on the surfaces of the micelle OPTs.
Example 2: evaluation of in-situ breast tumor targeting in multi-drug mixed micelle based on metformin
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. And (3) carrying out intraperitoneal injection of 120 mu L of 5% chloral hydrate on female Balb/c mice for anesthesia, removing body hair of the third and fourth pairs of milk fat pads on the left side of the mice, inoculating cell suspension into the milk fat pads on the fourth pair of left sides of the mice, slowly moving out a needle head after the milk fat pads of the mice are obviously filled, smearing a proper amount of antibiotics on wounds, and continuously feeding the mice. When the tumor volume after inoculation is increased to 100-200mm3The mice were randomly divided into 3 groups,DiR-labeled OPTs, DOPTs and HOPTs (OPTs-DiR, DOPTS-DiR and HOPTs-DiR) were injected into 3 groups via tail vein, respectively, and the dose of DiR in vivo in each group was 1 mg/kg. After 24 hours, the mice are anesthetized, normal saline is subjected to heart perfusion, 4% Paraformaldehyde (PFA) perfusion is fixed, main organs (heart, liver, spleen, lung and kidney) and in-situ tumors are collected, in-vitro fluorescence images of each organ and tumor are collected by a small animal living body imaging system, and meanwhile, the DiR fluorescence intensity of the organs is semi-quantitatively analyzed.
The results show that: the antigen CD44 is highly expressed on the surface of tumor cells in tumor tissue, whereas HA HAs high affinity for CD 44. After the micelle is injected for 24 hours, HOPTs-DiR can actively target the tumor and highly accumulate in the tumor tissue, and the fluorescence intensity is strongest; because of the lack of HA, DOPTS-DiR and OPTs-DiR have significantly weaker fluorescence intensity in tumor tissues than HOPTs-DiR (P <0.0001), but because DOPTS-DiR HAs certain high permeability and retention Effect (EPR) and better in vivo pharmacokinetics property, the DOPTS-DiR HAs certain accumulation in tumors, and the surface of OPTs-DiR presents positive charge, the circulating time in vivo is short, the OPTs-DiR is easy to clear, and the accumulation in tumors is almost not generated. This experiment demonstrates that HOPTs can be significantly targeted to tumor tissues in order to be expected to exert their pharmacological effects.
Example 3: in-vitro toxicity investigation of metformin-based multi-drug mixed micelles on breast cancer cells
In vitro toxicity was investigated using MTT assay. 4T1 cells were plated at 1X 103Density per well was seeded in 96-well plates with 3 replicates per group. After 24 hours of culture, the supernatant was discarded, and OA-Met, pro-DHA, Trip, HOTs, DOPTs, HOPTs and serum-free medium were added to the culture medium at different concentration gradients, respectively, for co-incubation. After 24 hours 20uL of MTT solution was added to each well and incubation was continued for 4 hours. The supernatant was discarded and 150uL of DMSO was added, and formazan produced by surviving tumor cells was sufficiently dissolved by shaking for 10 minutes. Finally, detecting the absorbance of each hole under 570nm by using a multifunctional microplate reader, and calculating to obtain the IC of the administration group50Values and cell survival curves were plotted.
The results show that: IC of OA-Met by itself on 4T1 cells50IC of free pro-DHA with a value of 7.88. mu.g/mL50Values of 5.01. mu.g/mL, theyAll have tumor cell toxicity of microgram grade. IC of free Trip50IC of Trip in Trip-Encapsulated micelles (HOTs, DOPTs, HOPTs) with a value of 47.96ng/mL50The values are respectively changed into 37.05ng/mL, 27.62ng/mL and 18.8ng/mL, and the cytotoxicity is respectively improved by 1.29, 1.74 and 2.55 times compared with that of the free Trip. The results show that OA-Met, pro-DHA and Trip have certain toxic effects on tumor cells, wherein the Trip has the strongest effect and mainly has toxic effect on the tumor cells in the micelle. The combined application micelle (HOTs) of the Trip and the OA-Met and the combined application micelle (DOPTs and HOPTs) of the three drugs of the Trip, the OA-Met and the pro-DHA can obviously improve the cytotoxicity of the Trip to tumor cells, and show the synergistic effect of drug combination. In addition, due to the targeting effect of HOPTs on tumor cells and the combined effect of the three medicines, the toxic effect of HOPTs is stronger than that of non-targeting micelle DOPTs and two-medicine combined micelle HOTs, and is respectively improved by 1.47 times and 1.97 times. The strong toxic effect of HOPTs on tumor cells can significantly inhibit the growth of in situ tumor tissues.
Example 4: evaluation of effect of multi-drug mixed micelle for inhibiting in-vitro balling of tumor stem cell microspheres based on metformin and calculation of MSFE
The in vitro balling experiment of the tumor stem cell microsphere is a mature and widely used method for evaluating the CSCs inhibition effect, the balling of the tumor cells is stimulated in a special serum-free stem cell induction culture medium, and the effect of inhibiting the dryness of the tumor cells is represented by examining the size and the shape of the balling and MSFE. In a clean bench, 10mL of B-27Supplement (50X) is added into 490mL of DMEM/F12 culture medium, and after uniform mixing, a proper amount of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) are added, so that the final concentration of the two cytokines is 20 ng/mL. Finally, adding a proper amount of penicillin-streptomycin double-antibody solution to ensure that the antibiotic concentration is 100U/mL and 100ug/mL respectively, and storing the obtained tumor stem cell induction culture medium at 4 ℃. 4T1 cells were cultured in tumor stem cell medium at 2X 104/cm2The density of (D) is inoculated in a 24-well low-adsorption (Ultra-low Attachment) plate, and culture mediums of OA-Met, pro-DHA, Trip, HOTs, HOPs, DOPTs and HOPTs (the Trip administration concentration is 10ng/mL) are respectively added at the same timeThe solution was co-incubated with non-dosed stem cell culture medium, 3 replicates per group. After 5 days of incubation, the well plates were placed under a microscope to take a photograph and count stem cell tumor spheres with a diameter greater than 50 μm and calculate the MSFE of each group;
the results show that: the non-dosed PBS group formed distinct tumor spheres of larger diameter with the largest MSFE of each group. Free OA-Met, pro-DHA and Trip group cells can also form more obvious tumor spheres, and compared with PBS, the diameter is smaller, the tumor spheres are more dispersed, and MSFE is also obviously reduced (P < 0.01). Compared with a single administration group, the diameter and MSFE of the combined medicinal micelle group tumor spheres of HOPs, HOTs, DOPTs and HOPTs are further reduced (P <0.01), the tumor spheres are more dispersed, and dispersed single cells without spheres are also clear and visible, wherein the combined micelle HOPTs of the three compounds have better effect (P <0.0001) than the combined micelle HOPs and HOTs of the two compounds. In addition, because of the tumor targeting property of HOPTs, the inhibition effect of HOPTs is obviously better than that of DOPTs (P < 0.05). The EMT process of tumor cells can promote the generation of CSCs, and the CSCs have strong survival differentiation and self-renewal capacity and are important reasons for tumorigenesis, metastasis and relapse. In this example HOPTs are effective in inhibiting CSCs, and are an effective strategy for inhibiting tumor growth and preventing metastasis.
Example 5: effect investigation of multi-drug mixed micelle up-regulating tumor tissue E-cadherin expression based on metformin
The expression condition of E-cadherin in-situ tumor tissue is examined through immunohistochemical staining of the tumor tissue of the mice. Firstly, an in-situ breast tumor mouse model is established by adopting the method of example 2, on the 6 th day after in-situ tumor inoculation, the mice are randomly divided into 8 groups, 4 mice in each group are respectively injected with physiological saline, OA-Met, pro-DHA, Trip, HOTs, HOPs, DOPTs and HOPTs (the dosage of the Trip is 0.7mg/kg) through tail veins, and the drugs are given for 5 times every 2 days. Anesthesia 2 days after administration, cardiac perfusion with physiological saline, perfusion with 4% PFA to collect tumor tissue, fixation with 4% PFA for 1-2 days, and gradient dehydration in 15% and 30% sucrose solutions. The tumor tissue was paraffin-embedded and frozen at-80 ℃. Paraffin-embedded tissue was cut into sections of 10 μm thickness and subjected to immunohistochemical analysis of E-cadherin. The results of the protein immunohistochemical staining were qualitatively analyzed using an inverted fluorescence microscope and the immunohistochemical sections were semi-quantitatively analyzed using imagej1.46.
The results show that: the expression level of E-cadherin in the tumor tissue of the normal saline control group is very low, and the free OA-Met, pro-DHA and Trip groups slightly increase the expression level of E-cadherin in the tumor tissue due to lack of targeting and the like, but the promotion effect is not obvious compared with the normal saline group. Compared with the free drug group, the HOPs, HOTs, DOPTs and HOPTs drug combination micelle group remarkably increases the expression level of E-cadherin (P <0.05), wherein the HOPTs of the three-drug combination micelle have more obvious promotion effect (P <0.0001) compared with HOPs and HOTs. In addition, the expression promoting effect of tumor-targeting micelle HOPTs is more significant (P <0.01) compared with DOPTs. The EMT process in tumor tissues is a key step of tumor cell invasion and metastasis, wherein the reduction of the expression of intercellular adhesion molecule E-cadherin is a main marker of EMT, which reduces the adhesion among tumor cells and promotes the invasiveness of the cells. HOPTs in this example effectively inhibit the EMT process by promoting the expression of E-cadherin.
Example 6: evaluation of Effect of metformin-based Multi-drug Mixed micelles on inhibiting in situ tumor growth while preventing metastasis
An in-situ breast cancer mouse model is established by adopting a 4T1-Luc cell line, and the establishment method is the same as that of example 2. When the tumor volume in situ reaches 100mm3On the left and right, the mice were randomly divided into 8 groups of 10 mice each, and the mice were injected with physiological saline, OA-Met, pro-DHA, Trip, HOTs, HOPs, DOPTS, and HOPTs, respectively, through the tail vein (the amount of Trip administered was 0.7mg/kg), from day 0, 1 dose was administered every 3 days, and the tumor volume in situ was measured using a vernier caliper. After 27 days, 5 mice per group were randomly anesthetized and perfused with normal saline, major organs (heart, liver, spleen, lung, kidney) and tumor tissues were removed and weighed. In addition, the taken lung tissue was immediately immersed in a D-fluorescein solution (0.5mg/mL) for 5 minutes, a biological self-luminescence qualitative image of the lung tissue was collected by a biopsy imager, and the self-luminescence was semi-quantitatively analyzed to compare the effect of inhibiting the generation of metastasis by each administration group. 5 mice remained in each groupContinuously feeding with GraphPad
Figure BDA0002556351860000101
6.02 software to perform life analysis and to plot life curves.
The results show that: compared with the normal saline group, the free OA-Met, pro-DHA and Trip group has slightly reduced tumor volume and quality and no obvious curative effect due to the reasons of no targeting, quick clearance in the free drug body and the like. HOPs, HOTs, DOPTs and HOPTs drug combination micelle group can obviously inhibit the tumor volume (P <0.01) and the tumor mass (P <0.01), wherein the tumor volume (P <0.05) and the tumor mass (P <0.05) of the HOTs are less than that of the HOPs, which indicates that the cytotoxicity Trip has important function of inhibiting the tumor growth. Compared with HOTs, the micelle HOPTs combined with the three compounds has better effect (P <0.0001) in inhibiting in-situ tumor growth, and the tumor volume (P <0.05) and the tumor mass (P <0.05) of the targeting HOPTs group are smaller than those of the DOPTs group. For the inhibition effect of lung metastasis, only 3 mice in the HOPTs group have weak lung metastasis and the inhibition effect is strongest, and all mice in other groups have more serious lung metastasis. The physiological saline group has the most serious lung metastasis, the free drug group slightly inhibits the lung metastasis, the inhibition effect of HOPs and HOTs is stronger than that of the free drug group (P <0.05) but weaker than that of HOPTs (P <0.001), and the effect of DOPTs is also weaker than that of HOPTs (P < 0.05). Furthermore, survival studies showed that mice in the HOPTs group had the longest mid-term survival (60 days), while those in the saline, OA-Met, pro-DHA, Trip, HOPs, HOTs, DOPTs groups had mid-term survival of 33, 35, 34, 46, 48 and 52 days, respectively. This example demonstrates that HOPTs effectively inhibit tumor growth while preventing tumor metastasis, thereby significantly extending the survival of mice.
Example 7: metformin-based in vivo safety evaluation of multi-drug mixed micelles
In example 6 treatment of mouse in situ breast cancer with HOPTs, body weight changes were recorded for each group of mice every 3 days from day 0. After day 27, the removed major organs were paraffin-embedded and H & E stained, and the damage of micelles to major organ tissues was analyzed with an inverted microscope.
The results show that: the free Trip group showed a more significant weight loss later in the treatment period, while the other groups had different weight gains with the weight remaining steady. In addition, H & E staining analysis of the sections of the major organs (heart, liver, spleen, lung and kidney) of the physiological saline, free Trip, HOTs, DOPTs and HOPTs groups shows that compared with the physiological saline group, the liver sections of the Trip group have obvious liver injury and inflammatory cell infiltration, the liver injury and inflammatory cell infiltration of the DOPTs group are obviously reduced compared with the Trip, and the HOPTs and HOTs groups have no obvious organ injury. The results show that the Trip has strong toxic and side effects (hepatotoxicity) in vivo when being singly used, and OA-Met and pro-DHA have no obvious toxicity. The encapsulating of the Trip in the targeting micelle (such as HOPTs) can reduce the toxic and side effect of the Trip when being singly used, and proves the safety of the HOPTs when being used in vivo.

Claims (6)

1. A multi-drug mixed micelle based on metformin is characterized in that an amphiphilic derivative OA-Met is used as a micelle material, the micelle material is mixed with a histone deacetylase inhibitor pro-DHA, self-assembly is carried out in water, triptolide is entrapped to form cationic micelle OPTs, and anionic Hyaluronic Acid (HA) is adsorbed on the surface of the cationic micelle OPTs through electrostatic adsorption, so that the multi-drug mixed micelle is constructed.
2. The metformin-based multi-drug mixed micelle according to claim 1, wherein said histone deacetylase inhibitor pro-DHA, propofol (pro) and docosahexaenoic acid (DHA) are covalently linked via an ester bond to obtain a compound pro-DHA having HDAC inhibitory activity. The metformin-based multi-drug mixed micelle according to claim 1, wherein said multi-drug mixed micelle delivers three drugs, metformin, pro-DHA and Trip, simultaneously.
3. The metformin-based multi-drug mixed micelles of claim 1, wherein said OA-Met self-assembles in water and simultaneously mixes with pro-DHA to form mixed micelles by the hydrophobic interaction and pi-pi conjugation between the OA-Met and pro-DHA unsaturated carbon chains, and at the same time, the hydrophobic drug Trip is entrapped in the hydrophobic core to obtain cationic micelle OPTs.
4. The metformin-based multi-drug mixed micelle according to claim 1, wherein the in situ tumor targeting ingredient used in said mixed micelle is Hyaluronic Acid (HA), which specifically binds to CD44 highly expressed on the surface of breast cancer cells.
5. The metformin-based multi-drug mixed micelle according to claim 1, wherein said mixed micelle is prepared into targeted micelle HOPTs by adsorbing HA to the surface of OPTs through electrostatic adsorption of the positive charge of OA-Met and the negative charge of HA.
6. Use of metformin-based multi-drug mixed micelles of claim 1, in which HA-encapsulated HOPTs are targeted to tumor tissues highly expressing CD44, inhibiting the effect of EMT in tumor tissues, reducing the occurrence of tumor metastasis, while HOPTs inhibit the growth of tumors, treating metastatic tumors.
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