Tumor-targeted reduction response type carrier material and preparation method thereof
阅读说明：本技术 一种肿瘤靶向性的还原响应型载体材料及其制备方法 (Tumor-targeted reduction response type carrier material and preparation method thereof ) 是由 任丽莉 陈国广 赵淑庆 戴捷 唐雨辰 于 2021-08-26 设计创作，主要内容包括：一种肿瘤靶向性的还原响应型载体材料,由多嵌段聚合物在水中自组装形成的胶束组成,所述多嵌段聚合物为叶酸-聚乙二醇-ss-聚赖氨酸-二氢卟吩Ce6(FA-PEG-ss-PLL(-g-Ce6)),其亲水端为PEG,用以连接具有肿瘤靶向作用的叶酸,疏水端为PLL,两者通过二硫键进行连接,所形成的壳核结构,用以包裹疏水性的药物和Ce6。由于在肿瘤环境中,谷胱甘肽(GSH)的浓度高于正常组织,导致二硫键断裂,释放出抗癌药物和光敏剂,在激光照射下,二者发生化疗和光动力疗法双重响应的疗效。(A tumor-targeting reduction response type carrier material consists of micelles formed by self-assembly of multi-block polymers in water, wherein the multi-block polymers are folic acid-polyethylene glycol-ss-polylysine-chlorin Ce6(FA-PEG-ss-PLL (-g-Ce6)), the hydrophilic end of each multi-block polymer is PEG for connecting folic acid with a tumor targeting effect, the hydrophobic end of each multi-block polymer is PLL, the two multi-block polymers are connected through disulfide bonds, and the formed core-shell structure is used for wrapping hydrophobic drugs and Ce 6. Because the concentration of Glutathione (GSH) is higher than that of normal tissues in the tumor environment, disulfide bonds are broken, and anticancer drugs and photosensitizers are released, and under the irradiation of laser, the two have the curative effect of dual response of chemotherapy and photodynamic therapy.)
1. A tumor-targeting reduction-responsive carrier material is characterized by consisting of micelles formed by self-assembling multi-block polymers in water, wherein the multi-block polymers are folic acid-polyethylene glycol-ss-polylysine-chlorin Ce6, the hydrophilic end of each multi-block polymer is PEG, the hydrophobic end of each multi-block polymer is PLL, and the multi-block polymers are connected through disulfide bonds, so that folic acid targets folic acid receptors on tumor cells.
2. A tumor-targeting reduction-responsive carrier material according to claim 1, wherein: the micelle formed by self-assembly of the carrier material is a shell-core structure and is used for wrapping the hydrophobic drug and Ce 6.
3. A tumor-targeting reduction-responsive carrier material according to claim 1, wherein: the structural formula of the carrier material is as follows:
wherein n is more than or equal to 2, and x is more than or equal to 2.
4. The method for preparing a tumor-targeting reduction-responsive carrier material according to any one of claims 1 to 3, comprising the steps of:
(1) preparing anhydrous DMSO solution of folic acid, activating with DCC and NHS solution for 2-5 h, dropwise adding anhydrous DMSO solution of polyoxyethylene diamine while stirring, reacting for 24-48 h, dialyzing the reactant, and freeze-drying to obtain folic acid modified polyoxyethylene diamine solid product FA-PEG-NH2；
(2) Mixing FA-PEG-NH2Dissolving with succinic anhydride, dropwise adding a triethylamine solution of 1.5-10 times, reacting for 24-48 h, removing most of solvent by rotary evaporation, precipitating the concentrated solution with glacial ethyl ether, performing suction filtration, and drying at normal temperature in vacuum to obtain FA-PEG-COOH;
(3) dissolving FA-PEG-COOH in DMF, adding DCC and NHS solution to activate for 5-6 h, and mixing the materials in a molar ratio of 1: 5-20 of cystamine is dissolved, the cystamine solution is dropwise added into FA-PEG-COOH solution to react for 24-48 h at room temperature, the reactant is precipitated by ethyl glacial ether, and is dried after suction filtration to obtain FA-PEG-ss-NH2；
(4) Dissolving the solid obtained in the step (3) and zLL-NCA in anhydrous DMF according to a certain proportion, reacting for 48-72 h at 30-35 ℃, dialyzing the reactant, and freeze-drying to obtain FA-PEG-ss-PzlL;
(5) dissolving the product obtained in the step (4) with trifluoroacetic acid, adding a glacial bromic acid solution, carrying out ice-water bath, reacting for 1-4 h, precipitating the reactant with glacial ethyl ether, carrying out suction filtration, and freeze-drying to obtain FA-PEG-ss-PLL-NH2；
(6) Dissolving Ce6 in anhydrous DMF, adding EDC & NHS for activation for 2-5 h, slowly adding the solution into FA-PEG-ss-PLL solution for reaction for 24-48 h, dialyzing the reactant, and freeze-drying to obtain FA-PEG-ss-PLL (-g-Ce 6).
5. The method for preparing a tumor-targeting reduction-responsive carrier material according to claim 4, wherein the method comprises the following steps:folic acid and NH described in step (1)2-PEG-NH2The molar ratio of (A) to (B) is 2-6: 1; the reaction needs to be carried out at N2Under protection, aiming at isolating air; the dialysis is carried out by dialyzing the dialysis bag in pure water with the molecular weight cutoff of the dialysis bag being 500-1500.
6. The method for preparing a tumor-targeting reduction-responsive carrier material according to claim 4, wherein the method comprises the following steps: triethylamine solution is required to be added in the step (2) to be used as an acid-binding agent, and triethylamine and FA-PEG-NH2The molar ratio is 1.5-10: 1, the FA-PEG-NH2The molar ratio of the succinic anhydride to the succinic anhydride is 0.5-2: 1.
7. The method for preparing a tumor-targeting reduction-responsive carrier material according to claim 4, wherein the method comprises the following steps: to activate the carboxyl terminal of FA-PEG-COOH, NHS and DCC solution were added in step (3), and the FA-PEG-NH was2The molar ratio to cystamine was 1: 5 to 20.
8. The method for preparing a tumor-targeting reduction-responsive carrier material according to claim 4, wherein the method comprises the following steps: the FA-PEG-ss-NH in the step (4)2The molar ratio of the zLL-NCA to the NCA is 1: 15-20; the molecular weight cut-off of the dialysis bag is 3500-5000.
9. The synthetic route of tumor-targeting reduction-responsive carrier material according to claim 4, wherein: adding a glacial bromic acid solution to break amide bonds in the step (5); the volume of the glacial bromic acid is about 0.5-5 ml; adding NHS and DCC solution in the step (6) to activate the carboxyl terminal of the photosensitizer Ce 6; the molar ratio of the FA-PEG-ss-PLL to the Ce6 is 0.5-2: 1.
10. The use of the carrier material of claim 1 for the preparation of a tumor targeting medicament.
Technical Field
The invention relates to synthesis of a nano polymer carrier material, in particular to a tumor-targeted reduction response type carrier material, and belongs to the field of biomedical materials and nano pharmaceutical preparations.
Background
Cancer has become one of the major threats to human health. In recent years, the incidence and mortality of tumors have been on the rise. In the past decades, tremendous efforts have been made to combat cancer, making great progress in the field of cancer therapy. However, first-line treatment of cancer, such as surgery, chemotherapy, and radiation therapy, remains limited by significant side effects and systemic toxicity. Therefore, the development of emerging therapeutic modalities is essential for safer and effective treatment of cancer.
Photothermal, photodynamic and chemodynamic therapies are potential anticancer modalities due to their negligible invasiveness, low toxicity, high selectivity and minimal invasiveness. Among them, photodynamic therapy (PDT) is a new treatment modality for treating cancer by photodynamic action. PDT is achieved by the generation of Reactive Oxygen Species (ROS), such as singlet oxygen (I), using Photosensitizers (PI)1O2) Tumor cells were killed under light conditions. Chlorins are frequently used as photosensitizers due to their high extinction coefficient in the red region and high singlet oxygen quantum yield, of which chlorins e6(Chlorin e6, Ce6) is the most commonly used photosensitizer, which can generate ROS under 650 nm laser irradiation, thus being activated by near infrared light and rapidly eliminated from the body, and which can convert light energy into heat energy for tumor ablation. However, Studies by Zhang et al found that folic acid modified photosensitizer Ce6 can actively target tissues without damaging surrounding normal tissues, but it also causes photosensitizer aggregation, causing some toxic effects (Zhang, Li Yuan, He Ting. research progress of chemical modified photosensitizer [ J]Chemical reagents, 2014,36(11): 983-. Lidonghong et al use PEG as a bridge to link folate to Ce6, althoughThe problem of photosensitizer aggregation is solved, but the PEG modified nano-carrier has the defect of difficult release after reaching a target point (the photosensitizer folic acid-porphyrin targeting and photodynamic activity of the Lipocalin, Shanjunlin, Liujian storehouse and zang Tao. on HeLa cells of cervical cancer [ J)]Chinese journal of laser medicine 2010,19(05): 273-.
PEG is one of the most commonly used polymers, and is also one of the few polymers that has gained FDA approval for pharmaceutical and pharmaceutical applications. The molecules are hydrophilic and self-assemble into amphiphilic molecules when combined with hydrophobic polymers. Meanwhile, Polylysine (PLL) is a common polymer, which is a biomacromolecule having amino functional groups formed by connecting several tens of lysines, and is a biodegradable cationic polypeptide capable of interacting with cells and adhering to the cells.
Since PEG and PLL belong to biological macromolecules, in order to prevent the formation of large aggregates, the traditional approach is to make the molecular structure less rigid by inserting bridging groups (e.g., rotatable σ -bond-containing benzene rings) or flexible linkers (e.g., sulfide bonds), thereby preventing the stacking of long-range ordered molecules. Among them, one of the most commonly used flexible linkers is disulfide bond (s-s), which has almost perpendicular double bond angles and single dihedral angles, and plays an important role in improving structural flexibility and balancing intermolecular forces during molecular self-assembly.
Because the traditional drug therapy has the defects of systemic disorder and cell disorder in the transportation mode, high toxicity, low targeting property and the like, the preparation of a multifunctional carrier material is needed to be optimized. The functional block copolymer is often applied to the construction of a novel nano-drug carrier because of the advantages of higher stability, low critical micelle concentration, controllable size, functional modification and the like, the folic acid modified star-shaped end amino PEG-PLGA nano-micelle prepared by Zhaochun Xin et al has the advantages of active targeting of tumor tissues and better killing effect on tumor cells, but when the nano-micelle taking PEG-PLGA as the carrier is prepared, the used solvent is easily dissolved by PLGA, and has immune suppression or immune stimulation effect on organisms, so that the organisms are induced to generate immune reactions of different degrees (Magnetitum, Zhaoxin, Jinxu, Chen 26108, Zhangpu, Song dynasty, the preparation of the folic acid modified star-shaped end amino PEG-PLGA nano-micelle and the targeting effect of the tumor cells [ J ]. high school chemistry report, 2012,33(08):1854-1859.). The Weilu dew discloses the preparation of folic acid modified amphiphilic block copolymer with stimulation response, and the prepared nano carrier material is complex and has higher cost (Weilu. preparation of folic acid modified tumor cell responsive block copolymer and research on controlled release behavior of DOX [ D ] Shihezi university, 2018.).
Folic Acid (FA) is a small molecule vitamin. Many tumor cells have an overexpressed specific protein (folate receptor) on their cell membrane, which is underexpressed in normal tissues and overexpressed in tumor cells. Compared with other targeting receptors of tumor cells, the tumor cell targeting receptor has the advantages of low immunogenicity, easy modification, storage stability, compatibility with various organic and water-soluble reagents, low cost and the like. Therefore, the folic acid modified nano carrier material has the characteristic of targeted cancer cell delivery on the surface.
The preparation method disclosed by the invention is simple and controllable in preparation process, mild in condition and simple in raw material, and no report that the photosensitizer is used for marking the folic acid modified disulfide-linked diblock copolymer which is used as a carrier material to convert light energy into heat energy for tumor ablation and tumor cell targeting is found at present.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the prior technical condition, a tumor-targeted reduction-responsive carrier material is provided.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a tumor-targeting reduction-responsive carrier material is composed of micelles formed by self-assembling multi-block polymers in water, wherein the multi-block polymers are folic acid-polyethylene glycol-ss-polylysine-chlorin Ce6(FA-PEG-ss-PLL (-g-Ce6)), the hydrophilic end of each multi-block polymer is PEG, the hydrophobic ends of the multi-block polymers are PLL, the multi-block polymers are connected through disulfide bonds, and folic acid targets folic acid receptors on tumor cells.
The structure is shown as the following formula:
wherein n is more than or equal to 2, and x is more than or equal to 2;
the polymer folic acid-polyethylene glycol-ss-polylysine-chlorin Ce6(FA-PEG-ss-PLL (-g-Ce6)) is connected by a disulfide bond, wherein the hydrophilic end of the polymer folic acid-polyethylene glycol-ss-polylysine-chlorin Ce6 is PEG, and the hydrophobic end of the polymer folic acid-polyethylene glycol-ss-PLL (-g-Ce6) is PLL; the folate-targeted receptor is a single-chain glycoprotein anchored on glycerophosphatidylinositol in cell membranes, and is low in expression in normal tissues and high in expression on the surfaces of tumor cells.
The micelle formed by self-assembly of the carrier material is a shell-core structure, the amphiphilic block copolymer contains PEG (polyethylene glycol) at a hydrophilic end and PLL (phase locked loop) at a hydrophobic end, the core at the hydrophobic end is used for wrapping hydrophobic drugs and Ce6, and the shell at the hydrophilic end can enable the whole polymer micelle to be better dissolved in water due to the hydrophilicity of the shell at the hydrophilic end. In the tumor environment, the GSH reductase content is high, disulfide bonds in micelles are reduced to sulfydryl, cell membranes are penetrated, and the disulfide bonds are broken, so that the photosensitizer of the medicine is released.
A preparation method of a tumor targeting reduction response type carrier material comprises the following specific experimental steps:
(1) preparing anhydrous DMSO solution of folic acid, activating with DCC and NHS solution for 2-5 h, dropwise adding anhydrous DMSO solution of polyoxyethylene diamine while stirring, reacting for 24-48 h, dialyzing the reactant, and freeze-drying to obtain folic acid modified polyoxyethylene diamine solid product FA-PEG-NH2；
(2) Mixing FA-PEG-NH2Dissolving with succinic anhydride, dropwise adding a triethylamine solution of 1.5-10 times, reacting for 24-48 h, removing most of solvent by rotary evaporation, precipitating the concentrated solution with glacial ethyl ether, performing suction filtration, and drying at normal temperature in vacuum to obtain FA-PEG-COOH;
(3) dissolving FA-PEG-COOH in DMF, adding DCC and NHS solution to activate for 5-6 h, and mixing the materials in a molar ratio of 1: 5-20 of cystamine is dissolved, the cystamine solution is dropwise added into FA-PEG-COOH solution to react for 24-48 h at room temperature, the reactant is precipitated by ethyl glacial ether, and is dried after suction filtration to obtain FA-PEG-ss-NH2；
(4) Dissolving the solid obtained in the step (3) and zLL-NCA in anhydrous DMF according to a certain proportion, reacting for 48-72 h at 30-35 ℃, dialyzing the reactant, and freeze-drying to obtain FA-PEG-ss-PzlL;
(5) dissolving the product obtained in the step (4) with trifluoroacetic acid, adding a glacial bromic acid solution, carrying out ice-water bath, reacting for 1-4 h, precipitating the reactant with glacial ethyl ether, carrying out suction filtration, and freeze-drying to obtain FA-PEG-ss-PLL-NH2；
(6) Dissolving Ce6 in anhydrous DMF, adding EDC & NHS for activation for 2-5 h, slowly adding the solution into FA-PEG-ss-PLL solution for reaction for 24-48 h, dialyzing the reactant, and freeze-drying to obtain FA-PEG-ss-PLL (-g-Ce 6).
Folic acid and NH described in step (1)2-PEG-NH2In a molar ratio of 2 to up to6: 1; the reaction needs to be carried out at N2Under protection, aiming at isolating air; the dialysis is specifically that after dialysis is carried out in pure water for 24 hours by using a dialysis bag, the solution permeates a microporous filter membrane; the molecular weight cut-off of the dialysis bag is 500-1500.
Adding triethylamine solution, triethylamine and FA-PEG-NH into the mixture in the step (2)2The molar ratio is 1.5-10: 1, intended to act as an acid-binding agent; the FA-PEG-NH2The molar ratio of the succinic anhydride to the succinic anhydride is 0.5-2: 1.
Adding NHS and DCC solution in the step (3) in order to activate the carboxyl terminal of FA-PEG-COOH; the FA-PEG-NH2The molar ratio to cystamine was 1: 5 to 20.
The FA-PEG-ss-NH in the step (4)2The molar ratio of the zLL-NCA to the NCA is 1: 15-20; the molecular weight cut-off of the dialysis bag is 3500-5000.
Adding a glacial bromic acid solution in the step (5) for the purpose of breaking amide bonds; the volume of the glacial bromic acid is about 0.5-5 ml.
Adding NHS and DCC solution in the step (6) in order to activate the carboxyl terminal of the photosensitizer Ce 6; the molar ratio of the FA-PEG-ss-PLL to the Ce6 is 0.5-2: 1.
The carrier material is used for preparing tumor targeting drugs, and can be widely applied to various tumor parts in a targeted chemotherapy or targeted phototherapy body.
The tumor-targeted reduction responsive carrier prepared by the invention is used as an innovative carrier material and has the following advantages:
1. the tumor-targeting reduction-responsive carrier material prepared by the invention has the advantages of simple and easily-obtained raw materials of folic acid and polyoxyethylene diamine, mild preparation conditions and is an excellent anticancer drug-targeted carrier.
2. The two-block polymer connected by the folic acid modified disulfide bond of the carrier material prepared by the invention is beneficial to folic acid active targeting to a target tissue, and the reduction response of the reduction response type disulfide bond connected block copolymer is beneficial to the carrier material to break the disulfide bond according to the GSH concentration in a tumor environment, so that the drug is released without damaging surrounding tissues.
3. The tumor-targeting reduction-responsive carrier material prepared by the invention is beneficial to grafting a plurality of amino groups of polylysine on the carrier material with a plurality of photosensitizers Ce6, so that the drug-loading capacity and the encapsulation efficiency of the micelle are improved.
4. The tumor-targeting reduction-responsive carrier material prepared by the invention has good biocompatibility and does not cause cytotoxicity.
5. The drug-loaded micelle formed by self-assembly of the tumor-targeting reduction responsive carrier material has the effect of dual response of chemotherapy and photodynamic therapy.
Drawings
FIG. 1 shows the intermediate FA-PEG-NH prepared by the present invention2The nuclear magnetic resonance hydrogen spectrum of (a);
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of an intermediate FA-PEG-ss-PzlL prepared by the invention;
FIG. 3 is the NMR spectrum of FA-PEG-ss-PLL (-g-Ce6) prepared by the present invention;
FIG. 4 is a DOX standard graph;
FIG. 5 is a graph showing the release profiles of different micelles in pH7.4 PBS and pH7.4 PBS +10mM DTT buffer;
FIG. 6 is a cytotoxicity plot of two carrier materials;
FIG. 7 is a graph of cytotoxicity of different support materials in the presence or absence of laser irradiation;
FIG. 8 is a cytometric map of two different carrier materials.
Detailed Description
The present invention will be further illustrated with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and those equivalents may fall within the scope of the present invention defined by the appended claims.
Example 1
(1)FA-PEG-NH2Synthesizing: 451.99mg Folic Acid (FA), 1690.26mg Dicyclohexylcarbodiimide (DCC) and 647.99mg N-hydroxy-carbodiimide (DCC) were weighed outSuccinimide (NHS) was dissolved in 20mL anhydrous dimethyl sulfoxide (DMSO) in N2Reacting for 4 hours at room temperature in a dark place under protection to obtain an activated FA solution; 5.12g of NH are weighed2-PEG-NH2Dissolved in 10mL DMSO, 3mL anhydrous triethylamine was added and the activated FA solution was slowly added dropwise to the NH2-PEG-NH2In solution, N2Reacting at room temperature in dark place for 48h under protection, dialyzing with 1000D dialysis bag for 48h, and freeze-drying to obtain 2.14g FA-PEG-NH2。
(2) FA-PEG-COOH Synthesis: 1.58g of FA-PEG-NH were weighed2Adding 0.42g succinic anhydride and 0.51g 4-Dimethylaminopyridine (DMAP) into a 100mL round-bottom flask, adding 30.00mL dioxane solution, dropwise adding 0.58mL triethylamine solution, magnetically stirring for 24h, and then rotationally evaporating dioxane; extracting the residue with 100mL of 0.1M hydrochloric acid and ethyl acetate respectively, discarding the organic phase, repeatedly extracting for 3 times, extracting the aqueous phase with 100mL of Dichloromethane (DCM) for 3 times, combining the organic phases, drying with anhydrous sodium sulfate, performing suction filtration, performing rotary evaporation on the filtrate with a rotary evaporator to remove most of DCM, retaining about 5-10 mL of DCM, adding 150-200 mL of glacial ethyl ether for precipitation for 12h, performing suction filtration, and performing vacuum drying at normal temperature to obtain 1.20g of FA-PEG-COOH.
(3)FA-PEG-ss-NH2Synthesizing: 1.00g of FA-PEG-COOH, 123.00mg of DCC, 70.00mg of NHS and 18.30mg of DMAP were weighed out and dissolved in 30mLN, N-Dimethylformamide (DMF), N2Under protection, performing ice-water bath reaction for 5 hours to obtain an activated FA-PEG-COOH solution; 867.26mg of cystamine is weighed and dissolved in 10mL of DMF, and the activated FA-PEG-COOH solution is slowly added into the cystamine solution dropwise, and N2Reacting at room temperature for 24h under protection, performing suction filtration, reserving about 5-10 mL of filtrate by using a rotary evaporator, adding 150-200 mL of glacial ethyl ether for precipitation for 12h, and drying after suction filtration to obtain 1.01g of FA-PEG-ss-NH2。
(4) FA-PEG-ss-PzlL synthesis: weighing 1.01g of FA-PEG-ss-NH2And 2.91g N (. epsilon.) -benzyloxycarbonyl-L-lysine-N-carboxylic anhydride (zLL-NCA) were dissolved in 30mL of DMF, reacted at 35 ℃ for 72 hours, and then the reaction product was dialyzed with 3500D dialysis bag for 24 hours, and the solution was passed through a 0.45 μm filter and lyophilized to obtain 810.14mg of FA-PEG-ss-PzlL.
(5)FA-PEG-ss-PLL-NH2Synthesizing: weighing 160.00mg of FA-PEG-ss-Pzll, dissolving in 10mL of trifluoroacetic acid, adding 0.5mL of hydrogen bromide 33 wt% glacial acetic acid solution, reacting at 0 ℃ in an ice water bath for 1h, adding 150-200 mL of glacial ethyl ether for precipitation for 12h, carrying out suction filtration on the ethyl ether, dissolving the filter residue in 10mL of DMF, putting the dissolved filter residue into alkaline solution with pH of 9.0, dialyzing in 3500D dialysis bag for 4-5 h, dialyzing in 3500D dialysis bag with pure water for 24h, and freeze-drying to obtain 131.68mg of FA-PEG-ss-PLL-NH2。
(6) FA-PEG-ss-PLL (-g-Ce6) Synthesis: 28.83mg of chlorin e6(Ce6), 62.10mg of EDC and 46.04mg of NHS were weighed out and dissolved in 10mL of anhydrous DMF, N2Reacting for 4 hours under protection to obtain an activated Ce6 solution; 100.00mg of FA-PEG-ss-PLL was dissolved in 20mL of anhydrous DMF, and the activated Ce6 solution was slowly added dropwise to the FA-PEG-ss-PLL solution, N2Reacting at room temperature for 48h under protection, dialyzing with 3500D dialysis bag for 48h, and freeze-drying to obtain 65.83mg FA-PEG-ss-PLL (-g-Ce 6).
Table 1 synthetic yield of example 1
Step (ii) of
(1)
(2)
(3)
(4)
(5)
(6)
Theoretical yield
2.50g
1.58g
1.22g
1.01g
160mg
79.88mg
Actual yield
2.14g
1.20g
1.01g
810.14mg
131.68mg
65.83mg
Yield of
85.60％
75.95％
82.78％
79.97％
82.30％
82.41％
The total yield of the final product was 29.19%.
Example 2
(1)FA-PEG-NH2Synthesizing: weighing 662.10mg FA, 2475.96mg DCC and 949.2mg NHS dissolved in 20mL anhydrous DMSO in N2Reacting for 4 hours at room temperature in a dark place under protection to obtain an activated FA solution; weighing 7.50g NH2-PEG-NH2Dissolved in 10mL DMSO, 3mL anhydrous triethylamine was added and the activated FA solution was slowly added dropwise to the NH2-PEG-NH2In solution, N2Reacting at room temperature in dark place for 48h under protection, dialyzing with 1000D dialysis bag for 48h, and freeze-drying to obtain 3.02g FA-PEG-NH2. (2) FA-PEG-COOH Synthesis: 2.37g of FA-PEG-NH were weighed2Adding 0.50g succinic anhydride and 0.51g DMAP into a 100mL round-bottom flask, adding 30.00mL dioxane solution, dropwise adding 0.58mL triethylamine solution, magnetically stirring for 24h, and then rotationally steaming out dioxane; extracting the residue with 100mL of 0.1M hydrochloric acid and ethyl acetate, discarding the organic phase, repeating the extraction 3 times, extracting the aqueous phase with 100mL of DCM 3 times, combining the organic phases, and extracting with anhydrous Na2SO4Drying, performing suction filtration, performing rotary evaporation on the filtrate by using a rotary evaporator to remove most DCM, retaining about 5-10 mL of DCM, adding 150-200 mL of glacial ethyl ether for precipitation for 12h, and performing vacuum drying at normal temperature after suction filtration to obtain 2.12g of FA-PEG-COOH.
(3)FA-PEG-ss-NH2Synthesizing: 1.50g of FA-PEG-COOH, 185.70mg of DCC, 103.60mg of NHS and 19.12mg of DMAP were weighed out and dissolved in 30mL of DMF, N2Under protection, performing ice-water bath reaction for 5 hours to obtain an activated FA-PEG-COOH solution; 1324.84mg of cystamine is weighed and dissolved in 10mL of DMF, and the activated FA-PEG-COOH solution is slowly added into the cystamine solution dropwise, and N2Reacting at room temperature for 24h under protection, performing suction filtration, reserving the filtrate by using a rotary evaporator for about 5-10 mL, adding 150-200 mL of glacial ethyl ether for precipitation for 12h, performing suction filtration, and performing freeze drying to obtain 1.29g of FA-PEG-ss-NH2。
(4) FA-PEG-ss-PzlL synthesis: weighing 1.04g of FA-PEG-ss-NH2And 3.00g zLL-NCA in 30mL DMF, reaction at 35 ℃ for 72h, dialysis of the reaction product in 3500D dialysis bag for 24h, filtration of the solution through 0.45 μm filter, and freeze-drying to obtain 875.28mg of FA-PEG-ss-PzLL.
(5)FA-PEG-ss-PLL-NH2Synthesizing: weighing 244.00mg of FA-PEG-ss-PzlL, dissolving in 10mL of trifluoroacetic acid, adding 0.5mL of hydrogen bromide 33 wt% glacial acetic acid solution, reacting at 0 ℃ in an ice water bath for 1h, adding 150-200 mL of glacial ethyl ether for precipitation for 12h, carrying out suction filtration on the ethyl ether, dissolving the filter residue in 10mL of DMF, dialyzing in alkaline solution with the pH of 9.0 for 4-5 h after dissolving, dialyzing with 3500D pure water for 24h, and freeze-drying to obtain 198.80mg of FA-PEG-ss-PLL-NH2。
(6) FA-PEG-ss-PLL (-g-Ce6) Synthesis: 44.75mg of Ce6, 92.02mg of EDC and 69.05g of NHS were weighed out and dissolved in 10mL of anhydrous DMF, N2Reacting for 4 hours under protection to obtain activated Ce6 solutionLiquid; 150.00mg of FA-PEG-ss-PLL was dissolved in 20mL of anhydrous DMF, and the activated Ce6 solution was slowly added dropwise to the FA-PEG-ss-PLL solution, N2Reacting at room temperature for 48h under protection, dialyzing with 3500D dialysis bag for 48h, and freeze-drying to obtain 112.53mg of FA-PEG-ss-PLL (-g-Ce 6).
Table 2 synthetic yield of example 2
Step (ii) of
(1)
(2)
(3)
(4)
(5)
(6)
Theoretical yield
3.66g
2.37g
1.50g
1.04g
244.00mg
150.00mg
Actual yield
3.02g
2.12g
1.29g
875.28g
198.80mg
112.53mg
Yield of
82.43％
89.45％
86.00％
84.16％
81.47％
75.02％
The total yield of the final product of the invention is 32.62%.
The nuclear magnetic resonance hydrogen spectra of the synthesized intermediate and the carrier material are shown in figures 1-3.
FIG. 1 shows FA-PEG-NH2Is/are as follows1HNMR analysis
The strong signal peak at a chemical shift of 3.5ppm is the repeat unit (-CH) of PEG2-a proton characteristic peak of O-; the signal peaks at chemical shifts 2.4-2.7ppm are-CH at different positions on FA2Characteristic peak of protons; the signal peak at chemical shift 7.7ppm and 6.7ppm is the proton characteristic peak of the benzene ring on FA; the signal peak at chemical shift 8.7ppm is the proton characteristic peak on FA.
FIG. 2 shows the method of preparing FA-PEG-ss-PzlL1HNMR analysis
The strong signal peak with the chemical shift of 3.5ppm is the proton characteristic peak of the repeating unit (-CH2-O-) of PEG; the signal peak at the chemical shift of 2.4-2.7ppm is the proton characteristic peak of-CH 2 at different positions on FA; chemical shifts 1.3-1.7ppm, 3.0ppm, 5.0ppm, 7.3ppm are characteristic peaks of protons of H in the PzlL repeat unit.
FIG. 3 shows the preparation of FA-PEG-ss-PLL- (-g-Ce6)1HNMR analysis
The signal peak at the chemical shift of 11.5ppm is the proton characteristic peak of-OH on Ce 6; the signal peak with chemical shift of 8.0ppm is the proton characteristic peak of-NH-; a proton characteristic peak of a repeating unit (-CH2-O-) of the strong signal peak PEG at a chemical shift of 3.5 ppm; the strong signal peak at the chemical shift of 2.4-2.7ppm is the proton characteristic peak of-CH 2 at different positions on FA; the signal peaks at chemical shifts 1.3-1.7ppm, 2.7-3.1ppm are characteristic peaks of protons of H in the PzlL repeat unit.
Example 3
Drug loading and encapsulation efficiency experiments
Weighing 5.00mg of mPEG-b-PzlL and 1.00mg of Doxorubicin (DOX), dissolving in 1ml of methanol solution, performing ultrasonic treatment for 10-15 min, stirring for 4h, and performing rotary evaporation. 10ml of purified water was added and stirred overnight to form mPEG-b-PzlL drug loaded micelle solution.
Weighing 5.00mg of FA-PEG-ss-PLL- (-g-Ce6) and 1.00mg of adriamycin (DOX) and dissolving in 1ml of methanol solution, carrying out ultrasonic treatment for 10-15 min, stirring for 4h in the dark and then carrying out rotary evaporation. Adding 10ml of purified water, and stirring overnight in the dark to form FA-PEG-ss-PLL- (-g-Ce6) drug-loaded micelle solution.
(1) Chromatographic conditions
A chromatographic column: TIANHETM Kromasil C18 column (4.6 mm. times.250 mm, 5 μm)
Detection wavelength: 252nm
Mobile phase: acetonitrile: pH7.4 phosphate buffer (25:75)
Column temperature: 35 deg.C
Flow rate: 1.0mL/min
Sample introduction amount: 10 μ L
(2) Determination of the Standard Curve
Accurately weighing 5mg of adriamycin standard, adding a small amount of methanol solution, placing the mixture into a 25mL volumetric flask, dissolving the mixture by using ultrasound, and continuously dropwise adding methanol until the volume reaches the scale, thus obtaining the adriamycin stock solution with the concentration of 200 mug/mL. The doxorubicin stock solution was diluted with methanol at different concentration gradients: 200, 150, 100, 50, 20, 5, 1 mug/mL of adriamycin stock solution with different concentrations. After passing through a 0.45 mu m microporous filter membrane, injecting the sample into a sample injection vial, and waiting for the determination by a high performance liquid chromatograph. After the measurement, the standard curve of doxorubicin in methanol was plotted with the horizontal axis representing the concentration of doxorubicin and the vertical axis representing the peak area by integration using chromatography software, as shown in fig. 4.
(3) Drug loading and encapsulation efficiency determination
The prepared mPEG-b-PzlL drug-loaded micelle solution and FA-PEG-ss-PLL- (-g-Ce6) drug-loaded micelle solution are subjected to ultrasonic operation, so that the core-shell structure of the micelle is destroyed, and the entrapped adriamycin is released from the micelle. The micelle solution is filtered through a 0.45um microporous membrane, and the Drug Loading Capacity (DLC) and the Encapsulation Efficiency (EE) are measured by High Performance Liquid Chromatography (HPLC), and the formula is as follows:
the result of the high performance liquid chromatography determination is as follows: the drug loading rate of the mPEG-b-PzlL micelle solution is 5.0 percent, and the encapsulation rate is 73.4 percent; the drug loading rate of the FA-PEG-ss-PLL- (-g-Ce6) micelle solution is 15.3%, and the encapsulation rate is 95.8%.
Example 4
Ce6 graft content determination experiment
2ml of the prepared FA-PEG-ss-PLL- (-g-Ce6) micelle solution is taken and put into a cuvette, 2ml of PBS solution is taken as a reference and put into another cuvette, the absorbance of Ce6 at the full wavelength is measured by an ultraviolet-visible spectrophotometer, and the grafting rate of Ce6 is measured according to the absorbance (660 nm). The Ce6 grafting ratio was calculated to be 4.76%.
Example 5
In vitro drug release assay using reverse dynamic dialysis
The prepared mPEG-b-PzlL drug-loaded micelle solution and FA-PEG-ss-PLL- (-g-Ce6) drug-loaded micelle solution (prepared in example 3) are divided into two parts, the two parts are transferred into a dialysis bag with a certain size, the two ends of the dialysis bag are fastened by cotton ropes and placed in centrifuge tubes respectively filled with 30ml of release medium (PBS or PBS containing 10 mMDTT). The entire release system was placed in a constant temperature shaker (shaker parameters 37 ℃, 100rpm) and sampled at predetermined time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48, 60, 72h) taking 1ml of release medium each time and replenishing with 1ml of fresh medium in the centrifuge tube. After a sample passes through a 0.45um microporous filter membrane, the sample is injected into a sample injection vial, HPLC is adopted for determination, and the cumulative release rate is calculated according to the following formula:
as shown in fig. 5, the other four solutions exhibited significant controlled release characteristics compared to the free doxorubicin solution. However, the FA-PEG-ss-PLL- (-g-Ce6) micellar solution loaded with DOX has obvious long-acting sustained and controlled release characteristics in tumor environment.
Example 6
Cytotoxicity test
(1) Cytotoxicity evaluation of blank micelles
Weighing 5.00mg of mPEG-b-PzlL, dissolving in 1ml of methanol solution, performing ultrasonic treatment for 10-15 min, stirring for 4h, and performing rotary evaporation. 10ml of purified water was added and stirred overnight to form a blank micellar solution of mPEG-b-PzlL.
Weighing 5.00mg of FA-PEG-ss-PLL- (-g-Ce6) and dissolving in 1ml of methanol solution, carrying out ultrasonic treatment for 10-15 min, stirring for 4h in dark place and then carrying out rotary evaporation. 10ml of purified water was added and stirred overnight in the dark to form a FA-PEG-ss-PLL- (-g-Ce6) blank micellar solution.
Cytotoxicity evaluation was carried out on two blank micelles, mPEG-b-PzlL and FA-PEG-ss-PLL- (-g-Ce6) by MTT method. And 4T1 cells are revived, passaged and frozen. Cell suspensions were seeded in 96-well plates. The prepared blank micelles (concentration: 500. mu.g/mL) were diluted at different concentrations: 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL. 100 μ L of blank micellar solutions with different concentrations were added to a 96-well plate mixed with cell suspension, each set having 3 duplicate wells. After 20. mu.L of each 5mg/ml of a prepared MTT solution was added to the cells, the cells were cultured until blue-violet formazan appeared at the bottom, and after the supernatant liquid in each well was discarded, a proper amount of DMSO was added to each well, and the absorbance of each group was measured at 540nm using an ultraviolet-visible spectrophotometer, and the Survival Rate of each group of cells was measured based on the absorbance (Cell Survival Rate, CSR).
As shown in fig. 6: the survival rate of both micelles was not less than 90%, indicating that they were not cytotoxic.
(2) Cytotoxicity evaluation of drug-loaded micelles
The cytotoxicity evaluation of the drug-loaded micelles was carried out by the MTT method. After the diluted and counted cell suspension is inoculated to a 96-well plate, the cell suspension is placed in an incubator for continuous culture. After the wall is attached, the supernatant in each well is discarded, and 100 mu L of DOX & Ce6, DOX & Ce6+ laser irradiation group, DOX @ FA-PEG-ss-PLL- (-g-Ce6) and DOX @ FA-PEG-ss-PLL- (-g-Ce6) + laser irradiation group with the drug concentration of 0.1 mu g/mL, 1 mu g/mL, 5 mu g/mL, 10 mu g/mL and 50 mu g/mL are respectively added. Blank, negative and positive control groups were set simultaneously, each group having 3 replicate wells. After incubating the cells for 24h, adding 20 mu L of a pre-prepared MTT solution into each hole, continuously incubating in an incubator, removing the supernatant, adding a proper amount of DMSO, oscillating, detecting the absorbance of each group by using a microplate reader, and determining the cytotoxicity of the 4T1 cells in each group according to the absorbance.
As shown in fig. 7: when the laser irradiation group and the laser irradiation-free group are compared, the survival rate of the 4T1 cells of the corresponding laser irradiation group is lower than that of the laser irradiation-free group, which shows that under the laser irradiation, the killing effect of the 4T1 cells caused by the simultaneous action of the chemotherapeutic drug and the photosensitizer is higher than that of the single chemotherapeutic drug.
Example 7
Cell uptake assay
After counting the cells, the cells were inoculated into a confocal cell culture dish, and 2mL of cell suspension was added to each well, followed by continuous culture. After discarding supernatant in each well, cells were washed with sterile PBS solution, and free drug at 2. mu.g/mL and two different drug-loaded micelles, namely DOX @ mPEG-b-PzlL and DOX @ FA-PEG-ss-PLL- (-g-Ce6, were added, respectively. After incubating the cells in the incubator for 4h, the supernatant in each well was discarded and the cells were washed with sterile PBS solution. An appropriate amount of paraformaldehyde (4%) was added to each well to fix the cells, the fixative was discarded after 15min, and the cells were washed again with sterile PBS solution. In dark place, appropriate amount of DAPI was added to each culture dish for staining, the staining agent was discarded, the cells were washed with sterile PBS solution, and the uptake of each group of cells was observed under laser confocal microscope oil lens as shown in fig. 8:
(a) DOX @ mPEG-b-PzlL; (b) DOX @ FA-PEG-ss-PLL- (-g-Ce 6); (c) free DOX. After incubating free DOX with 4T1 cells for 4h, the free DOX is transported to nucleus and combined with DNA in the cell sap quickly; the fluorescence intensity of cytoplasm and nucleus of the cells treated by DOX @ mPEG-b-PzlL is relatively weak, so that the cells enter the cells due to passive diffusion, and the DOX is released slowly; the fluorescence intensity of the cells treated by DOX @ FA-PEG-ss-PLL- (-g-Ce6) is obviously stronger than that of DOX @ mPEG-b-PzlL, and the fluorescence intensity of cell nuclei is higher than that of cytoplasm, so that compared with the DOX @ mPEG-b-PzlL, the DOX @ FA-PEG-ss-PLL- (-g-Ce6) has the advantages that more DOX can be transported to the cell nuclei by FA with an active targeting effect, and the effect of actively targeting tumor cells is achieved.