CN111956806B

CN111956806B - Drug carrier, micelle, medicament, and preparation method and application thereof

Info

Publication number: CN111956806B
Application number: CN202010916564.5A
Authority: CN
Inventors: 何勤; 卢正则; 龙洋; 李曼
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-09-03
Filing date: 2020-09-03
Publication date: 2023-04-07
Anticipated expiration: 2040-09-03
Also published as: CN111956806A

Abstract

The invention relates to the field of pharmaceutical preparations, and discloses a drug carrier, a micelle, a medicament, and a preparation method and application thereof; the drug carriers and micelles comprise: a) Hydrophilic moieties including hydrophilic heparinoids; b) A hydrophobic portion comprising one of a hydrophobic carotenoid and a vitamin a compound; wherein the hydrophilic and hydrophobic moieties are linked by a specific means; the drug carrier and the micelle not only have the functions of blocking the activation of an NF-kB signal path and an STAT3 signal path in an initial step in an inflammation feedforward loop, inhibiting the recruitment of bone marrow-derived system inhibitory cells in a tumor part and a remote organ, inhibiting the implantation of tumor cells in the remote organ and improving a local immunosuppressive microenvironment, thereby inhibiting tumor-related inflammation, the reconstruction of a niche before metastasis, the immune escape of tumor cells and strangling and killing circulating tumor cells; moreover, the best effect of inhibiting tumor and tumor metastasis can be realized by further encapsulating anti-tumor drugs respectively.

Description

Drug carrier, micelle, medicament, and preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical preparations, in particular to a drug carrier, a micelle, a medicament, and a preparation method and application thereof.

Background

Cancer seriously threatens the health of modern human beings, according to the statistical data of the national cancer center, about 392.9 ten thousands of people have cancer diseases in 2015 years in China, 7.5 people have confirmed diagnosis per minute on average, and the cancer diseases are in the trend of rising year by year. At present, the treatment means for cancer mainly include chemical drug therapy (chemotherapy), tumor radiotherapy (radiotherapy), surgical resection and the like. Among them, chemotherapy can directly kill tumor cells and has a certain degree of tumor inhibition, but has great toxic and side effects and a single action mechanism, and the treatment effect needs to be further improved. Therefore, if the tumor can be inhibited from different mechanisms in the chemotherapy process, the dosage required by chemical drugs can be reduced, the killing effect on the tumor is enhanced, and a better treatment effect is achieved.

Inflammation related to cancer plays a promoting role in the stages of generation, development, metastasis and the like of tumors, and the inflammation can be classified into exogenous inflammation and endogenous inflammation according to different causes. Exogenous inflammation refers to inflammation caused by external factors (such as bacterial endotoxin stimulation, mechanical injury, chemical induction, etc.), and colon cancer is a cancer typically promoted by exogenous inflammation. Endogenous inflammation refers to inflammation produced by nearby cells stimulated by proinflammatory factors secreted by cancerous cells during proliferation, and breast cancer is a cancer which is typically promoted by endogenous inflammation. In the process of inflammation development, inflammatory factors, chemokines, proteases and the like secreted by inflammatory cells and tumor cells can promote the growth, proliferation, invasion and metastasis of precancerous cells and cancer cells, thereby playing a role in promoting cancer. Therefore, the cancer-related inflammation is inhibited while chemotherapy is performed, and the further development of the tumor can be inhibited while directly killing the tumor cells. The combined treatment strategy of anti-inflammation and anti-tumor is helpful to improve the treatment effect on the tumor.

Multiple signaling pathways and numerous factors are involved in the development of inflammation. Among them, nuclear transcription factor-. Kappa.B (NF-. Kappa.B) signaling pathway is one of the most important signaling pathways. NF-. Kappa.B is generally present in inactive form in a wide variety of cells, and it can be activated by a variety of factors including proinflammatory tumor necrosis factor-alpha (TNF-. Alpha.); after entering into nucleus, the activated NF-kB can activate the expression of genes encoding inflammatory factors and promote the production of a plurality of downstream inflammatory factors, chemokines and enzymes, including TNF-alpha, interleukin-6 (IL-6), matrix metalloproteinase-9 (MMP-9), vascular Endothelial Growth Factor (VEGF) and the like. Wherein, TNF-alpha and IL-6 can be used as growth factors of precancerous cells and tumors and can further promote inflammatory reaction; MMP-9 plays an important role in the invasion process of tumor cells, while VEGF can promote the generation of new blood vessels in tumors, which both contribute to the growth and metastasis of tumors. Thus, it can be seen that the NF- κ B signaling pathway is associated with inflammation and cancer, and that its activation may contribute to the development and progression of tumors. Therefore, the treatment of cancer-related inflammation in the cancer treatment process is beneficial to improving the treatment effect, and the inhibition of the NF-kB signal channel can effectively inhibit the tumor-related inflammation, so as to inhibit the growth of the tumor.

Exogenous inflammation or infection and activation of endogenous oncogenes can cause inflammatory transcription factors such as NF-kappa B, STAT and the like in tumor cells to be activated, so that the tumor cells begin to generate substances such as chemokines, cytokines and prostaglandins and the like to promote the inflammatory cells such as: recruitment of macrophages, eosinophils, mast cells, neutrophils, and myeloid-derived suppressor cells. Then, the inflammatory cells and the inflammatory transcription factors in the stromal cells and tumor cells are activated to generate more chemokines, cytokines and prostaglandins, etc. to recruit more inflammatory cells. Thus, an Inflammatory closed Loop with continuous circulation amplification is formed, which is called an Inflammatory Feed-forward Loop (inflammation Feed-forward Loop), and mainly comprises the following links: the activation of inflammatory pivotal signal pathways such as NF-kappa B, STAT3 and the like; 2. multiple downstream inflammatory factor production; 3. inflammatory cells, including myeloid derived suppressor cells, were recruited. Among them, myeloid-derived suppressor cells (MDSCs) are a type of precursor cells of dendritic cells, neutrophils, and macrophages derived from bone marrow, which are prevented from differentiating and maturing in pathological conditions such as inflammatory environments, thus staying at an immature stage. It is an important population of recruited inflammatory cells with significant immunosuppressive effects. Since the use of antagonists is inefficient for a single inflammatory factor, multi-site blockade of the inflammatory feed-forward loop starting from both the initial signaling pathway and MDSC is the most efficient anti-inflammatory strategy.

Meanwhile, the inflammation also has great promotion effect on the metastasis and recurrence of the tumor, which are two main causes of death of cancer patients. The data indicate that 40% of cancer patients relapse within 5 years after surgery, and more than 90% of cancer patients eventually die from tumor metastases. In the medical context of rapid development of targeted and immunotherapy, surgery remains an essential means of treating solid tumors. However, metastasis and recurrence after tumor resection still result in very low patient survival. Specifically, postoperative metastasis recurrence is a complex multifactorial regulatory process, such as: 1. solid tumors before resection induce the reconstitution of far-end organ inflammatory microenvironment (PMN), which is beneficial to the implantation and colonization of Circulating Tumor Cells (CTCs); 2. the residual tumor tissue after resection has an immune tolerance mechanism; 3. surgical resection may cause a rapid increase in the number of CTCs. However, the strategy of single-site regulation of the above factors is difficult to effectively control the postoperative metastatic recurrence process. Therefore, a means of simultaneously inhibiting PMN reconstitution, controlling tumor cell immune escape and strangling CTCs is a promising and challenging postoperative anti-tumor metastasis and recurrence strategy.

In view of the above, there is a need for a method that can not only perform multi-site blocking on the inflammation feedforward loop, but also simultaneously inhibit PMN reconstitution, control tumor cell immune escape and strangle CTCs.

Disclosure of Invention

One of the objectives of the present invention is to overcome the deficiencies of the prior art and provide a drug carrier to at least achieve the effects of multi-site blocking of the inflammation feedforward loop, inhibition of PMN reconstitution, control of tumor cell immune escape, and strangulation of CTCs.

The above purpose is realized by the following technical scheme: a drug carrier comprising:

a) Hydrophilic moieties including hydrophilic heparinoids;

b) A hydrophobic portion comprising one of a hydrophobic carotenoid and a vitamin a compound;

preferably, the hydrophilic heparin compounds comprise one or more of low molecular heparin and derivatives thereof, common heparin, undaria pinnatifida polysaccharide and derivatives thereof, basil polysaccharide and derivatives thereof, fucoidan polysaccharide and derivatives thereof and fondaparinux sodium;

more preferably, the hydrophilic heparin compound is low molecular heparin or a derivative thereof;

more preferably, the low molecular heparin comprises one of heparitin sodium, enoxaparin sodium, nadroparin calcium and dalteparin sodium;

more preferably, the low molecular weight heparin is enoxaparin sodium;

preferably, the hydrophobic carotenoid comprises one of carotene and derivatives thereof, lycopene and derivatives thereof, lutein and derivatives thereof, and astaxanthin and derivatives thereof;

more preferably, the hydrophobic carotenoid is astaxanthin;

preferably, the connection mode of the hydrophilic part and the hydrophobic part comprises one or more of an amido bond, an ester bond, a pH sensitive hydrazone bond, an imine bond, an alcohol ether bond, a disulfide bond and a monothioether bond;

more preferably, the linking means is an ester bond.

Preferably, the drug carrier is micron-sized or nano-sized;

more preferably, the particle size of the drug carrier is 30 to 300nm;

more preferably, the particle size of the drug carrier is 90 to 150nm.

The second purpose of the present invention is to provide a method for preparing the above drug carrier, comprising the following steps:

the method comprises the following steps:

s1, mixing an activating agent, a reaction solvent I and the hydrophilic heparin compound, and performing an activation reaction to obtain an activation solution;

preferably, the activating agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and 4-dimethylaminopyridine;

preferably, the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide to the 4-dimethylaminopyridine is 16-8;

preferably, the mass ratio of the hydrophilic heparin compound to the activating agent is 3-6:8-15;

preferably, the reaction solvent I is an aprotic polar solvent;

more preferably, the reaction solvent I is formamide;

preferably, the activation reaction is carried out under the condition of keeping out light;

more preferably, the time of the activation reaction is 0.5 to 12 hours, and most preferably 3 hours;

more preferably, the temperature of the activation reaction is 15 to 35 ℃, preferably 25 ℃;

s2, dissolving the hydrophobic carotenoid or vitamin A compound in a reaction solvent II to obtain a mixed solution;

preferably, the reaction solvent II is an aprotic polar solvent;

more preferably, the reaction solvent II is N, N-dimethylformamide;

s3, dropwise adding the activating solution into the mixed solution, and performing substitution reaction to obtain a reaction solution;

preferably, the substitution reaction is carried out under the conditions of gas protection and light protection;

more preferably, the gas is argon;

more preferably, the temperature of the substitution reaction is 15 to 35 ℃, preferably 30 ℃;

more preferably, the time of the substitution reaction is 12 to 96 hours, most preferably 48 hours;

preferably, the ratio of the structural unit of the hydrophilic heparin compound to the molar weight of the hydrophobic carotenoid or vitamin A compound is 1-10;

s4, purifying the reaction solution through organic solvent precipitation to prepare gel-like precipitate;

preferably, the organic solvent comprises one or more of methanol, ethanol, propanol, acetone, methyl ethyl ketone, diethyl ketone, dichloromethane and chloroform;

more preferably, the organic solvent is acetone;

more preferably, the volume ratio of the reaction solution to acetone is 1:1-10;

more preferably, the volume ratio of the reaction solution to acetone is 1:3;

and S5, dialyzing and drying the gel sample precipitate by using pure water to prepare the drug carrier.

The invention also aims to provide the application of the drug carrier in the preparation of antitumor drugs.

The fourth purpose of the present invention is to provide a micelle, which comprises the above drug carrier, and water or a pharmaceutically acceptable aqueous medium.

The fifth purpose of the invention is to provide the application of the micelle in the preparation of antitumor drugs.

The sixth purpose of the invention is to provide a medicament containing the medicament carrier, which also comprises an anti-tumor medicament;

preferably, the anti-tumor drug comprises one or more of actinomycin D, daunorubicin, adriamycin and derivatives thereof, paclitaxel and derivatives thereof, camptothecin and derivatives thereof, 5-fluorouracil, vincristine, curcumin, quercetin and emodin;

more preferably, the antitumor drug is adriamycin and/or paclitaxel;

more preferably, the anti-tumor drug is adriamycin.

Preferably, the particle size of the drug is 100 to 160nm.

The seventh purpose of the invention is to provide a method for preparing the medicament, which is to load the anti-tumor medicament on the hydrophobic part of the medicament carrier through physical action;

preferably, the loading method comprises one or more of an emulsification ultrasonic rotary evaporation method, a dialysis method, a solvent injection method, a direct dissolution method and a solvent volatilization method;

more preferably, the loading method is an emulsification ultrasonic rotary evaporation method;

preferably, the solvent used in the method comprises one or more of dichloromethane, water, methanol, ethanol, chloroform and tetrahydrofuran;

more preferably, the solvent comprises one or more of methanol, chloroform, dichloromethane and tetrahydrofuran;

more preferably, the solvent is methanol and dichloromethane;

more preferably, the volume ratio of the methanol to the dichloromethane is 1;

more preferably, the volume ratio of methanol to methylene chloride is 1:2.

The eighth purpose of the invention is to provide the application of the medicament in preparing anti-tumor medicaments.

It is worth noting that the inventors have successfully constructed a drug carrier and micelle which are stable, efficient, suitable for drug loading and simple in preparation method through extensive and intensive research for many years. More preferably, the constructed drug carrier has the blank carrier function of carrying other anti-tumor drugs, and the carrier also has the functions of anti-inflammation and anti-cancer cell transfer.

There is provided a pharmaceutical carrier having anti-inflammatory and anti-cancer cell metastasis effects, a micelle having anti-inflammatory and anti-cancer cell metastasis effects, an anti-inflammatory, anti-tumor and anti-tumor cell metastasis agent, and methods of their preparation and use, said pharmaceutical carrier, micelle comprising a hydrophilic portion and a hydrophobic portion, and optionally a tumor targeting component. After entering blood circulation, the drug carrier and the medicament can be firstly combined with P-selectin on the blood platelet, thereby inhibiting circulating tumor cells in the blood from obtaining blood platelet coats and inhibiting the transfer cascade process of the blood platelet coats. Can also be combined with high-expression P-selectin on the surfaces of vascular endothelial cells which are activated by inflammation at a tumor part and a microenvironment part before metastasis to realize inflammatory targeting and simultaneously antagonize an adhesion cascade process in the recruitment process of MDSCs. The nano micelle releases drugs through a lysosome way, ester bonds connected with hydrophilic and hydrophobic segments are broken under the acid environment in the lysosome, the micelle is disintegrated, the entrapped drugs are released, meanwhile, the hydrophobic segment astaxanthin can also play a relevant action mechanism of anti-inflammation, an inflammation feedforward loop is cut off by blocking NF-kB and STAT3 signal paths, MDSCs are prevented from being further recruited, the action mechanism of anti-ROS can antagonize the immunosuppressive action of the recruited MDSCs, and the effects of regulating the microenvironment of tumors and metastasis parts and inhibiting the metastasis generation are jointly played from a plurality of targets.

In addition, the inventors found in the research that the mechanism of heparin anti-metastasis is based on P-selectin/PSGL-1 (P-selectin ligand), and based on this mechanism, the metastasis cascade of tumor cells can be inhibited, and the recruitment of myeloid-derived suppressor cells (MDSCs) in the pre-metastatic microenvironment, i.e., the adhesion cascade existing during the recruitment of MDSCs, can be inhibited.

The astaxanthin at the hydrophobic segment can effectively block an inflammation Feed-Forward Loop (inflammation Feed-Forward Loop) by acting on two Inflammatory pivot signal channels of NF-kB and STAT3, and inhibit the recruitment of MDSCs. The anti-ROS mechanism can effectively antagonize the immunosuppressive action of MDSCs in the microenvironment and the tumor part before metastasis, so the action mechanism of the hydrophilic and hydrophobic segments has a synergistic effect. Specifically, the antioxidant action mechanism of astaxanthin mainly comes from a large number of conjugated alkene structures in the structure of astaxanthin, and oxygen free radicals, namely ROS (reactive oxygen species) can be effectively eliminated. The abundant production of ROS is the important reason for the immunosuppressive effects of MDSCs. Therefore, astaxanthin can effectively antagonize immunosuppression caused by MDSCs at the focus based on the ROS resistant effect of the astaxanthin. Meanwhile, ROS can stimulate inflammatory reaction of local tissues, promote generation of a series of inflammatory factors, and can lead to recruitment of more MDSCs through an inflammation feed-forward loop, so that astaxanthin can effectively antagonize recruitment of MDSCs and immunosuppressive functions of MDSCs through an antioxidant action mechanism of astaxanthin. On one hand, astaxanthin can cut off an inflammation feedforward loop by blocking NF-kB and STAT3 inflammatory signal pathways; on the other hand, the tissue inflammatory response can be reduced by reducing the ROS level, the generation of inflammatory factors can be reduced, and the recruitment of MDSCs can be antagonized by blocking an inflammation feedforward loop.

In conclusion, the scheme adopts low molecular weight heparin and astaxanthin to construct a micelle type drug carrier capable of aiming at a tumor related inflammation feedforward loop. In particular, astaxanthin as a hydrophobic fragment thereof can block the activation of the NF-kB signal pathway and the STAT3 signal pathway in the initial step of a feedforward loop, namely 'pinching head'; low molecular weight heparin as a hydrophilic fragment antagonizes the recruitment of a major population of neutrophil and myeloid-derived suppressor cells in inflammatory cells at the tumor site and in distant organs, i.e., "tail-out", through P-selectin/PSGL-1 interactions; thus, the micelle carrier can block the tumor-related inflammation feed-forward loop in a head-to-tail manner. Meanwhile, the astaxanthin serving as the hydrophobic fragment has extremely excellent antioxidant activity, so that the content of Reactive Oxygen Species (ROS) in a tumor part and a remote organ can be obviously reduced, and the effects of further inhibiting MDSC recruitment and improving a local immunosuppressive microenvironment are achieved.

The particle size of the product micelle can be adjusted by adjusting the feeding ratio of the hydrophilic and hydrophobic segments in the synthesis method, the reaction time, the reaction temperature and other conditions. The reaction is carried out according to the synthesis conditions strictly described in the patent, and the obtained product is in a reasonable range. Similarly, the method and conditions for drug loading have also been screened. During reaction, the proportion of the astaxanthin of the hydrophobic fragment is properly reduced or the reaction time is shortened, so that the particle size of a product micelle is increased by 10-200 nm, conversely, the proportion of the astaxanthin of the hydrophobic fragment is increased, so that the particle size is reduced by 10-50 nm, and when the increased or reduced proportion exceeds a certain range, the obtained copolymer cannot self-assemble in an aqueous solution to form the micelle. When the drug carrier is used as a blank carrier, the particle size can reach 120nm, the hydrophobic core of the drug carrier can be used for loading various hydrophobic chemotherapeutic drugs (such as DOX), the particle size can reach 130nm after drug loading, and the drug can be passively accumulated through the EPR effect of solid tumors and delivered to tumor parts.

It is worth mentioning that in addition to passive targeting, the heparin shell is based on a P-selectin mechanism and has an active targeting effect on vascular endothelial cells activated at the focal site.

The inventor finally determines the two substances from a plurality of alternative components through a large number of experiments, and directly and effectively connects the two substances to construct a novel efficient and stable drug carrier which can be self-assembled into micelles in aqueous solution.

The invention has the beneficial effects that:

1. the drug carrier and the micelle have the functions of blocking the activation of an NF-kB signal path and an STAT3 signal path in an initial step in a feedforward loop, inhibiting the recruitment of bone marrow-derived system inhibiting cells in a tumor part and a remote organ, inhibiting the implantation of tumor cells in the remote organ and improving a local immunosuppressive microenvironment, so that tumor-related inflammation, PMN reconstruction, tumor cell immune escape and strangulation of CTCs are inhibited; and the drug carrier is directly connected with the hydrophilic part and the hydrophobic part of the micelle, so that the construction steps are simple and convenient, the structure is simple, the particles are uniform, and the stability is excellent.

2. The hydrophobic parts of the drug carrier and the micelle can package the anti-tumor drug through physical action, and compared with the drug loading through chemical coupling, the method has the advantages of simpler operation, stronger controllability in industrial production and wider variety of applicable drug loading; after the anti-tumor drug is encapsulated, the optimal effects of inhibiting tumors and tumor metastasis can be realized.

Drawings

FIG. 1 is a schematic diagram of the synthesis of the LMWH-AST copolymer of example 1;

FIG. 2 is a NMR hydrogen spectrum of the LMWH-AST copolymer of example 2 part 1;

FIG. 3 is an infrared spectrum of the LMWH-AST copolymer of example 2 part 2;

FIG. 4 is a transmission electron microscope image of each micelle and a particle size distribution map of LANPs in example 2, section 3;

FIG. 5 is a graph of the stability of LANPs in example 2, part 4 and LA/DOX NPs in example 5, part 2 in 50% fetal calf serum;

FIG. 6 is a graph of the release profile of each group of example 6, part 1, at different pH conditions;

FIG. 7 is a graph showing the effect of lung implantation of groups of anti-4T 1 tumor cells of example 6, part 2;

FIG. 8 is a graph of the effect of the groups of example 6, part 3, in reducing MDSCs recruitment to the lungs and tumors of mice;

FIG. 9 is a graph of the effect of the groups of example 6 section 4 on blocking the NF-. Kappa.B and STAT3 inflammatory signaling pathways;

FIG. 10 is a graph of the effect of the groups in section 5 of example 6 in inhibiting ROS levels in 4T1 breast cancer cells;

FIG. 11 is a graph of the effect of each group in section 6 of example 6 on the inhibition of ROS levels in 4T1 breast cancer cells in the lungs and tumors of mice;

FIG. 12 is a graph showing the effect of anti-tumor cell metastasis in each organ in each group in section 7 of example 6;

FIG. 13 is a graph of evaluation indices for each of the groups of anti-metastatic treatments in section 7 of example 6.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following.

Example 1 Synthesis of Low molecular heparin and hydrophobic Carotenoid copolymer

The purpose of this example is to illustrate the synthesis of low molecular weight heparin and hydrophobic carotenoid copolymers, and characterization of low molecular weight heparin and hydrophobic carotenoid copolymers.

1. Preparation of a copolymer of enoxaparin sodium and astaxanthin:

astaxanthin (25.0 mg) was precisely weighed and dissolved in 24.0mLN of N-Dimethylformamide (DMF) at room temperature. In addition, 50.0mg of low molecular weight heparin (enoxaparin sodium) was precisely weighed, dissolved in 3.0mL of formamide under heating in a water bath at 60 ℃, and then 60.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 36.0mg of N-hydroxysuccinimide (NHS) and 10.0mg of 4-Dimethylaminopyridine (DMAP) were added, and stirred and activated for 3 hours at 25 ℃ in the dark. After the activation, the astaxanthin solution was added dropwise at a rate of 2 drops/s (about 40. Mu.L/s) at a stirring speed of 600rpm, and the mixture was reacted under protection of argon (Ar) at 30 ℃ for 48 hours in the dark. After the reaction was completed, the reaction solution was added to 0 ℃ acetone in a volume 3 times that of the reaction solution, sufficiently shaken, and centrifuged at 4000rpm at normal temperature. Discarding supernatant after centrifugation, draining the obtained precipitate, adding 25mL of boiled and cooled deionized water, stirring for dissolving, centrifuging at 9500rpm at normal temperature, transferring supernatant into a dialysis bag with cut-off molecular weight of 1000Da, and dialyzing in boiled and cooled deionized water for 48h. The obtained solution was lyophilized to obtain brick-red LMWH-AST copolymer powder, which was dissolved in water to form micelle LANPs (FIG. 4A).

2. Preparation of a copolymer of enoxaparin sodium and dihydroxylycopene:

dihydroxy lycopene 25.0mg was weighed out precisely and dissolved in 24.0ml of N-Dimethylformamide (DMF) at room temperature. In addition, precisely weighing 50.0mg of low molecular weight heparin (enoxaparin sodium), dissolving in 3.0mL of formamide under the heating condition of 60 ℃ in water bath, adding 60.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 36.0mg of N-hydroxysuccinimide (NHS) and 10.0mg of 4-Dimethylaminopyridine (DMAP), and stirring and activating for 3h at 25 ℃ in the dark. After activation, the mixture is added into the dihydroxylycopene solution dropwise at a speed of 2 drops/s (about 40 mu L/s) and a stirring speed of 600rpm, and the mixture is reacted for 48 hours at 30 ℃ in a dark place under the protection of argon (Ar). After the reaction was completed, the reaction solution was added to 0 ℃ acetone in a volume 3 times that of the reaction solution, sufficiently shaken, and centrifuged at 4000rpm at normal temperature. Discarding supernatant after centrifugation, draining the obtained precipitate, adding 25mL of boiled and cooled deionized water, stirring for dissolving, centrifuging at 9500rpm at normal temperature, transferring supernatant into a dialysis bag with cut-off molecular weight of 1000Da, and dialyzing in boiled and cooled deionized water for 48h. The obtained solution was lyophilized to obtain LMWH-DIH (DIH = dihydroxylopene, the hydrophilic segment being kept as enoxaparin sodium, the hydrophobic segment being more dihydroxy lycopene, carotenoid derivative) copolymer powder, which was dissolved in water to form micelles LD NPs (fig. 4C).

3. Preparation of enoxaparin sodium and all-trans retinoic acid copolymer:

25.0mg of all-trans retinoic acid was precisely weighed and dissolved in 24.0mL of N-Dimethylformamide (DMF) at room temperature. In addition, 50.0mg of low molecular weight heparin (enoxaparin sodium) was precisely weighed, dissolved in 3.0mL of formamide under heating in a water bath at 60 ℃, and then 60.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 36.0mg of N-hydroxysuccinimide (NHS) and 10.0mg of 4-Dimethylaminopyridine (DMAP) were added, and stirred and activated for 3 hours at 25 ℃ in the dark. After the activation, the solution was added dropwise to the all-trans retinoic acid solution at a rate of 2 drops/s (about 40. Mu.L/s) at a stirring speed of 600rpm, and the mixture was reacted under protection of argon (Ar) at 30 ℃ in the dark for 48 hours. After the reaction was completed, the reaction solution was added to 0 ℃ acetone in a volume 3 times that of the reaction solution, sufficiently shaken, and centrifuged at 4000rpm at normal temperature. Discarding supernatant after centrifugation, draining the obtained precipitate, adding 25mL of boiled and cooled deionized water, stirring for dissolving, centrifuging at 9500rpm at normal temperature, transferring supernatant into a dialysis bag with cut-off molecular weight of 1000Da, and dialyzing in boiled and cooled deionized water for 48h. The obtained solution was lyophilized to obtain LMWH-ATRA (ATRA = All-trans-retinic-acid, hydrophilic fragment is kept as enoxaparin sodium, hydrophobic fragment is more All-trans retinoic acid, vitamin a derivative) copolymer powder, which was dissolved in water to form micelle LAt NPs (fig. 4D).

Example 2 structural characterization of Low molecular heparin and hydrophobic Carotenoid copolymers (example 1 copolymer)

The hydrogen spectrum of the LMWH-AST copolymer confirms that:

6.0mg of the LMWH-AST sample powder after freeze-drying is taken, precisely weighed and respectively dissolved in 0.5mL of D2O and DMSO-D6. 3.0mg of LMWH and 3.0mg of AST were weighed out and dissolved in 0.5mL of D2O and 0.5mL of DMSO-D6, respectively, as reference samples. Hydrogen nuclear magnetic resonance (1H-NMR) analysis was performed at 400 MHz.

The results are shown in fig. 2, and the LMWH-AST sample forms forward micelles in D2O by comparing with the LMWH and AST maps, wherein a series of peaks between δ =3.5 and 5.5ppm are hydrogen peaks on the medium carbon chain skeleton of LMWH, and δ =6.5ppm is a hydrogen peak on the medium carbon chain skeleton of AST. As described above, the 1H-NMR results confirmed the successful synthesis of LANPs.

The infrared spectrum of the LMWH-AST copolymer confirms that:

the structure of the LMWH-AST copolymer obtained after purification was identified by infrared spectroscopy (IR).

The result is shown in figure 3, and an infrared spectrum C picture of the LMWH-AST copolymer of the product not only has all main characteristic peaks of the LMWH in the picture A, but also can see an AST characteristic peak of 1650cm < -1 > in the picture B, and an ester bond characteristic peak of 1729cm < -1 > is newly added, which indicates the successful connection of the AST and the LMWH.

Particle size determination of LMWH-AST copolymer:

and taking a proper amount of the obtained LMWH-AST copolymer, and dissolving the LMWH-AST copolymer into a LANPs sample of 2mg/mL by using deionized water. The sample is prepared by adopting a phosphotungstic acid negative dyeing method, and the morphological characteristics of the nanoparticles are observed under a Transmission Electron Microscope (TEM).

The results are shown in fig. 4A, where the LANPs obtained have a distinct spherical morphology. Meanwhile, the particle size of LANPs was measured by DLS method using a laser particle sizer, and as shown in fig. 4B, LANPs had a narrow particle size distribution range, uniform particle size, and an average particle size of about 120nm.

Stability testing of LMWH-AST copolymers in aqueous solution to form micelles (LANPs):

2mg of LMWH-AST copolymer is dissolved in 1ml of PBS to prepare a LANPs solution, the LANPs solution is uniformly mixed with Fetal Bovine Serum (FBS) which is filtered by a microfiltration membrane with the aperture of 0.22 mu m in advance according to the volume ratio of 1:1, the final volume ratio of serum is 50%, 3 mixture samples are prepared in parallel for each group, and the mixture samples are incubated for 48 hours in a shaker at 37 ℃ and the rotating speed of 75 rpm. The particle size of the nanoparticles in each set of samples was determined by DLS method at various time points (0 h, 1h, 2h, 4h, 8h, 12h, 24h and 48 h) using a laser particle sizer, as shown in fig. 5.

The results show that LANPs incubated in 50% FBS for 48h showed no significant change in particle size, indicating good serum stability.

Example 3 Synthesis of a copolymer of fondaparinux sodium and astaxanthin

Astaxanthin (25.0 mg) was precisely weighed and dissolved in 24.0mLN of N-Dimethylformamide (DMF) at room temperature. In addition, 50.0mg of fondaparinux sodium was precisely weighed, dissolved in 3.0mL of formamide under heating in a water bath at 60 ℃, and then 60.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 36.0mg of N-hydroxysuccinimide (NHS) and 10.0mg of 4-Dimethylaminopyridine (DMAP) were added, stirred and activated for 3 hours at 25 ℃ in the dark. After the activation, the astaxanthin solution was added dropwise at a rate of 2 drops/s (about 40. Mu.L/s) at a stirring speed of 600rpm, and the mixture was reacted under protection of argon (Ar) at 30 ℃ for 48 hours in the dark. After the reaction was completed, the reaction solution was added to 0 ℃ acetone in a volume 3 times that of the reaction solution, sufficiently shaken, and centrifuged at 4000rpm at normal temperature. Discarding supernatant after centrifugation, draining the obtained precipitate, adding 25mL of boiled and cooled deionized water, stirring for dissolving, centrifuging at 9500rpm at normal temperature, transferring supernatant into a dialysis bag with cut-off molecular weight of 1000Da, and dialyzing in boiled and cooled deionized water for 48h. Freeze-drying the obtained solution to obtain FON-AST (FON = Fondaparinux, the hydrophobic fragment is astaxanthin, the hydrophilic fragment is Fondaparinux sodium, and artificially synthesized heparin analogues) copolymer powder, and dissolving the copolymer powder in water to form micelle FoANPs (figure 4E).

Example 4 Synthesis of fucoidan and astaxanthin copolymers

Astaxanthin (25.0 mg) was precisely weighed and dissolved in 24.0mLN of N-Dimethylformamide (DMF) at room temperature. 50.0mg of fucoidan was precisely weighed, dissolved in 3.0mL of formamide under heating in a water bath at 60 ℃, and then 60.0mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 36.0mg of N-hydroxysuccinimide (NHS) and 10.0mg of 4-Dimethylaminopyridine (DMAP) were added, and stirred and activated at 25 ℃ for 3 hours in the dark. After the activation, the astaxanthin solution was added dropwise at a rate of 2 drops/s (about 40. Mu.L/s) at a stirring speed of 600rpm, and the mixture was reacted under protection of argon (Ar) at 30 ℃ for 48 hours in the dark. After the reaction was completed, the reaction solution was added to 0 ℃ acetone in a volume 3 times that of the reaction solution, sufficiently shaken, and centrifuged at 4000rpm at normal temperature. Discarding supernatant after centrifugation, draining the obtained precipitate, adding 25mL of boiled and cooled deionized water, stirring for dissolving, centrifuging at 9500rpm at normal temperature, transferring supernatant into a dialysis bag with cut-off molecular weight of 1000Da, and dialyzing in boiled and cooled deionized water for 48h. Freeze-drying the obtained solution to obtain FUC-AST (FUC = Fucoidan, the hydrophobic fragment is astaxanthin, the hydrophilic fragment is Fucoidan, and natural heparin analogue) copolymer powder, and dissolving in water to form micelles FuANPs (FIG. 4F).

Example 5 Synthesis of DOX-carrying Agents (LA/DOX NPs)

1. The drug is loaded (taking the loaded adriamycin as an example), the hydrophobic drug is uniformly loaded by adopting an ultrasonic emulsification-rotary evaporation method:

1.0mg of doxorubicin hydrochloride is taken, precisely weighed, dissolved in 0.8mL of anhydrous methanol, then 0.867 μ L of triethylamine is added, the mixture is stirred at the rotating speed of 300rpm for 6 hours under the condition of keeping out of the sun to remove the hydrochloric acid, and then 1.6mL of dichloromethane is added to be uniformly mixed. Adding the mixture into 20.0mg of LMWH-AST copolymer powder, fully mixing, incubating at room temperature for 10min, adding 3mL of 0.9% physiological saline, ultrasonically emulsifying (100W, 5s/5s, 10min) by a probe, rotationally evaporating on a rotary evaporator at 45 ℃ water bath condition to remove all organic phases, and performing probe ultrasonic (100W, 5s/5s, 10min) to obtain a medicament (LA/DOX NPs) solution loaded with adriamycin.

Stability testing of LA/DOX NPs solutions:

the LA/DOX NPs solution was mixed uniformly at a volume ratio of 1:1 with Fetal Bovine Serum (FBS) previously filtered through a 0.22 μm pore size microporous membrane to give a final serum volume of 50%, 3 samples of the mixture were prepared in parallel for each group and incubated at 75rpm for 48h in a 37 ℃ shaker. The particle size of the nanoparticles in each set of samples was determined by DLS method at various time points (0 h, 1h, 2h, 4h, 8h, 12h, 24h and 48 h) using a laser particle sizer, as shown in fig. 5.

The results show that LA/DOX NPs incubated in 50% FBS for 48h showed no significant change in particle size, indicating good serum stability.

Example 6 Effect verification

1. Drug-loaded agents release under different pH conditions:

preparing LA/DOX NPs solution with DOX content of 100 mu g/mL according to the method; while setting free DOX as the reference group. Putting 1mL of sample into a dialysis bag (MWCO 1000 Da) each time, fastening the opening of the bag, and respectively soaking the bag into PBS (release medium) with 50mL of pH value of 5.0, 6.8 or 7.4, wherein 3 groups of parallel samples are arranged below each pH value; incubating in a shaker at 37 ℃ for 48h at 75 rpm; each set was sampled at predetermined time points (0.5 h, 1h, 2h, 4h, 8h, 12h, 24h and 48 h) with 200. Mu.L of release medium while continuing incubation with an equal volume of isothermal medium. The obtained DOX release sample was measured for fluorescence value using a fluorescence spectrophotometer under the conditions of Ex =488nm, em =555nm, and the drug content and release rate in the release medium were calculated, and a release curve was plotted as shown in fig. 6.

2. In vivo anti-implantation experiment:

mouse breast cancer cells (4T 1 cells) were cultured in vitro, harvested, suspended in PBS, and a solution of 5 (6) -carboxydiacetoxyfluorescein succinimidyl ester (CFSE) was added to a final concentration of 20. Mu.M at 37 deg.CIncubate in cell incubator for 15min to stain. After staining was completed, the cells were washed 2 times with PBS and made to have a cell density of 2X 10 using PBS ⁶ Individual cells/mL of cell suspension. 12 female Balb/c mice (body weight: about 20g,5 Zhou Zhouling) were randomly divided into 5 groups of 3 mice each, and PBS, LMWH solution, LANPs solution, foANPs and FuANPs (60 mg/kg) were injected into the tail vein, respectively, and after 0.5h, 100. Mu.L of CFSE-stained cell suspension was injected into the tail vein. The mice were sacrificed 0.5h after injection of the cell suspension, frozen sections were prepared by collecting lung tissues after heart perfusion, and implantation of CFSE-stained tumor cells in the lung was observed with a laser confocal microscope (excitation wavelength Ex =496nm and emission wavelength Em =516nm for CFSE).

As shown in FIG. 7, it can be seen that the tumor cells were heavily implanted in the lung of the mice in PBS group and formed into clumps; the LMWH group and each micelle group mice have obviously reduced lung implantation tumor cell number and are distributed in a single cell, which shows that the micelle taking the hydrophilic heparin compound and the derivative thereof as the hydrophilic segment has the function of obviously inhibiting the implantation of the circulating tumor cells in the distal organs (lungs) in vivo.

3. In vivo anti-recruitment experiment:

culturing mouse breast cancer cell (4T 1 cell) in vitro, and making into cell density of 2 × 10 with PBS ⁶ Individual cells/mL of cell suspension, 12 SPF-grade female Balb/c mice (5 weeks old, body weight about 20 g) were anesthetized, the right side hair on the back of the body was shaved, and 2X 10 cells were injected subcutaneously into each mouse ⁵ The cells were randomly and evenly divided into 4 groups of 3 cells, and 3 mice of the same kind were used as controls. On 8 days after cell inoculation, each group of mice was administered with 0.2mL (75 mg/kg) of PBS, LMWH, AST or LANPs solution via tail vein, then administered once every 3 days for 4 times, sacrificed on the day following the last administration, lung and tumor samples were taken, homogenized to prepare single cell suspension, stained with MDSC feature marker CD11b antibody and Gr-1 antibody, and detected by flow cytometry.

The result is shown in fig. 8, the micelle can obviously reduce the ratio of MDSC in the lung and the tumor part of the tumor-bearing mouse, and the micelle can effectively inhibit the recruitment of MDSC in the lung and the tumor part.

4. In vitro detection of signal pathway blockade experiments:

mouse breast cancer cells (4T 1 cells) are cultured in vitro and evenly inoculated in 6-well plates, 3 multiple wells are arranged below each group, serum-free culture medium containing PBS, LMWH, AST or LANPs (100 mu g/mL) is added into each well, and after incubation for 24 hours in a cell incubator at 37 ℃, cell total protein of each well is extracted and analyzed by a Western-blot method.

The results are shown in fig. 9, after incubation with LANPs, two inflammatory signaling pathways, NF- κ B and STAT3, in mouse breast cancer cells and MDSCs were significantly inhibited.

5. In vitro active oxygen detection experiment:

culturing mouse breast cancer cells (4T 1 cells) in vitro, uniformly inoculating the cells in a 6-well plate, arranging 3 multiple wells below each group, adding a serum-free culture medium containing PBS, LMWH, AST, DIH, ATRA, LANPs, LD NPs and LAt NPs (100 mu g/mL) into each well, incubating the cells in a 37 ℃ cell incubator for 24h, detecting the active oxygen level (showing fluorescence intensity) in each group of cells by using an active oxygen detection kit,

the results are shown in FIG. 10. The results show that the hydrophobic segments (namely carotenoid, vitamin A substances and derivatives thereof) of the micelles and the micelles constructed based on the hydrophobic segments can obviously inhibit the reactive oxygen level in breast cancer cells.

6. And (3) detecting ROS content experiment:

culturing mouse breast cancer cell (4T 1 cell) in vitro, and making into cell density of 2 × 10 with PBS ⁶ cells/mL cell suspension 15 SPF female Balb/c mice (5 weeks old, body weight about 20 g) were anesthetized, the right hair on the back of the body was shaved, and 2X 10 cells were injected subcutaneously into each mouse ⁵ The cells were randomly and evenly divided into 5 groups of 3 cells each, and 3 mice of the same kind were used as controls. On 8 days after cell inoculation, each group of mice was administered with 0.2mL of PBS, LMWH, AST, LANPs (75 mg/kg) or LA/DOX NPs (DOX content 3 mg/kg) solution via tail vein, and then administered once every 3 days for 4 times, mice were sacrificed the next day after the last administration, lung and tumor samples were taken, and ROS in tissues were quantified by ethidium dioxide staining.

The result is shown in fig. 11, the LANPs micelle can obviously reduce the content of ROS in the lung and the tumor part of the tumor-bearing mouse.

7. Carrying out a tumor-bearing mouse lung metastasis experiment;

culturing mouse breast cancer cell (4T 1 cell) in vitro, and making into cell density of 2 × 10 with PBS ⁶ cells/mL cell suspension 18 SPF female Balb/c mice (5 weeks old, approximately 20g body weight) were anesthetized, the right side of the body was shaved, and 2X 10 cells were injected subcutaneously into each mouse ⁵ Each cell was randomly divided into 6 groups of 3 mice each, and 3 mice of the same kind were used as controls. On 8 days after cell inoculation, each group of mice is respectively administered with 0.2mL of PBS, LMWH, AST, DOX, LANPs (75 mg/kg) or LA/DOX NPs (DOX content 3 mg/kg) solution through tail vein, then is administered once every 3 days for 4 times, mice are sacrificed on 45 days after tumor establishment, lung samples are taken out, and histochemical analysis is carried out by adopting hematoxylin-eosin staining method.

The results are shown in fig. 12 and 13, the micelle and the drug-loaded micelle can obviously reduce the pulmonary metastasis of tumor-bearing mice, and the drug-loaded micelle has the best treatment effect on tumors.

The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A micelle, comprising: a pharmaceutical carrier, and water or a pharmaceutically acceptable aqueous medium;

the drug carrier includes:

a) The hydrophilic part is low molecular heparin;

b) The hydrophobic part is one of hydrophobic carotenoid and all-trans retinoic acid;

the low molecular heparin comprises one of heparitin sodium, enoxaparin sodium, nadroparin calcium and dalteparin sodium;

the hydrophobic carotenoid is astaxanthin;

the hydrophilic part and the hydrophobic part are connected in an ester bond mode.

2. Micelle according to claim 1, wherein the particle size of the drug carrier is 30-300nm.

3. A medicament comprising the pharmaceutical carrier of any one of claims 1~2 further comprising an antineoplastic drug;

the antitumor drug comprises one or more of actinomycin D, daunorubicin, adriamycin, paclitaxel, camptothecin, 5-fluorouracil, vincristine, curcumin, quercetin and emodin.

4. The agent according to claim 3, wherein the particle size of the agent is 100 to 160nm.

5. A method of making the drug carrier of any one of claims 1~2 comprising the steps of:

the activating agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 4-dimethylaminopyridine;

the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide to the 4-dimethylaminopyridine is 16 to 8, and the mass ratio is (4 to 1);

the mass ratio of the hydrophilic heparin compound to the activating agent is 3 to 6;

the reaction solvent I is formamide;

the activation reaction is carried out under the condition of keeping out of the light;

the activation reaction time is 0.5 to 12h;

the temperature of the activation reaction is 15 to 35 ℃;

the reaction solvent II is N, N-dimethylformamide;

the substitution reaction is carried out under the conditions of gas protection and light protection;

the gas is argon;

the temperature of the substitution reaction is 15 to 35 ℃;

the time of the substitution reaction is 12 to 96h;

the ratio of the structural unit of the hydrophilic heparin compound to the molar weight of the hydrophobic carotenoid or vitamin A compound is 1-10;

the organic solvent comprises one or more of methanol, ethanol, propanol, acetone, methyl ethyl ketone, diethyl ketone, dichloromethane and chloroform;

when the organic solvent is acetone, the volume ratio of the reaction liquid to the acetone is 1 to 10; the volume ratio of the reaction liquid to the acetone is 1:3;

6. A method of preparing a medicament as claimed in claim 3, wherein the antineoplastic drug is loaded on the hydrophobic portion of the drug carrier by physical action;

the loading method comprises one or more of an emulsification ultrasonic rotary evaporation method, a dialysis method, a solvent injection method, a direct dissolution method and a solvent volatilization method;

the solvent used in the method comprises one or more of dichloromethane, water, methanol, ethanol, chloroform and tetrahydrofuran;

when the solvent is methanol and dichloromethane, the volume ratio of the methanol to the dichloromethane is 1.1 to 10, and the volume ratio of the methanol to the dichloromethane is 1:2.

7. Use of a pharmaceutical carrier according to claim 1 or 2 for the preparation of an anti-tumor medicament.

8. Use of the micelle of claim 1 or 2 for the preparation of an anti-tumor drug.

9. Use of the agent of claim 3 for the preparation of an anti-tumor medicament.