CN113679849B - High-targeting low-toxicity tumor microenvironment intelligent response type nano-carrier and preparation method thereof - Google Patents

High-targeting low-toxicity tumor microenvironment intelligent response type nano-carrier and preparation method thereof Download PDF

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CN113679849B
CN113679849B CN202110988890.1A CN202110988890A CN113679849B CN 113679849 B CN113679849 B CN 113679849B CN 202110988890 A CN202110988890 A CN 202110988890A CN 113679849 B CN113679849 B CN 113679849B
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任丽莉
陈国广
赵淑庆
魏杰
唐雨辰
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Abstract

The invention discloses a high-targeting low-toxicity tumor microenvironment intelligent response type nano-carrier and a preparation method thereof, wherein the nano-carrier consists of micelles with the particle size of 30-200nm, and the micelles are formed by self-assembling multi-block polymers in water. The multi-block polymer contains a hydrophobic end DSPE and a hydrophilic end PEG, and an acid response block PAE and a tumor vessel targeting peptide are coupled to the hydrophilic end PEG. PAE is hydrophobic in a neutral PH blood environment, and c (RGDyc) can be occluded in micelles, so that c (RGDyc) is prevented from acting with opsonin in blood, and clearance of reticuloendothelial system is reduced. Meanwhile, the hydrophilic end PEG and the hydrophobic end DSPE of the micelle are connected through disulfide bonds, the micelle is influenced by the reducibility of glutathione in tumor cells, and the disulfide bonds are broken to release the drug and kill the tumor cells. Enhance the endocytosis of tumor cells to the carrier and prevent the micelle from escaping from tumor tissues. The disulfide bonds reduce response in tumor tissues, so that the specific release of the chemotherapeutic drugs reduces the toxic and side effects of the chemotherapeutic drugs.

Description

High-targeting low-toxicity tumor microenvironment intelligent response type nano-carrier and preparation method thereof
Technical Field
The invention belongs to the technical fields of biological medicine and nano medicine, and particularly relates to an intelligent response type nano carrier with high targeting and low toxicity.
Background
Cancer is a malignant disease affecting human health, and the incidence of cancer in the world has increased at a rate of 3% -5% per year for nearly thirty years, severely jeopardizing the physical and mental health of people. Currently, surgery, radiation therapy and chemotherapy are the primary methods of clinical cancer treatment. The traditional antitumor drug has poor selectivity, kills normal cells as well as tumor cells, and generally has the problems of low solubility, short half-life and the like.
Nano-drug carriers present a number of obstacles in the delivery of drugs and undergo a complex process in vivo: 1) circulating in the blood and staying for a longer time 2) when the drug reaches the tumor tissue, it accumulates in the tumor tissue (EPR effect) 3) the nano-drug permeates in the tumor tissue to reach the tumor cells 4) rapidly enters into the tumor cells 5) rapidly releases the drug in the tumor cells. For example, some drug carriers have been surface modified with a more hydrophilic polymer to avoid rapid clearance of the reticuloendothelial system, to achieve better long circulation performance and to increase tumor site enrichment. However, research shows that the vectors are not easy to be endocytosed by tumor cells, and the tumor treatment effect is reduced. Meanwhile, some targeting drug carriers with good application prospects have good tumor cell endocytosis capability. Studies of PEG-DSPE micelles mediated by RGD peptide and tumor targeting thereof by Chen et al [ Chen Wangyan ], shanghai university of Chinese medicine, 2013 ], designed targeting peptides linked to DSPE-PEG blocks for targeted treatment of tumors. However, c (RGDyc) is directly exposed on the surface of the nano-particles, so that the nano-particles are easy to interact with opsonin in blood, and can be promoted to be rapidly cleared by reticuloendothelial systems under the action of opsonin, and immune response can be possibly caused, so that the anti-tumor curative effect of the nano-drug is greatly reduced.
Current research to address long circulation and endocytosis has focused mainly on hydrophilic-hydrophobic switching and hiding of targeting groups. After the nano-carrier enters blood circulation, the hydrophilic material on the surface of the carrier is used for increasing the circulation time of the carrier in blood circulation, reducing the clearance effect of a reticuloendothelial system on the carrier and improving the bioavailability. Meanwhile, the targeting group is exposed by utilizing the tumor microenvironment different from normal tissues, so that the endocytosis of the tumor cells to the carrier is enhanced. The research of the high-red army and function synergistic composite micelle as an anti-tumor nano drug carrier [ D ]. South-open university, 2014 ] utilizes two block polymers of PEG-PCL and RGD-PAE-PCL to form a mixed micelle. In the blood environment, hydrophilic PEG plays a long-circulating role, and PAE undergoes hydrophilic-hydrophobic conversion to expose a targeting group after reaching tumor tissues, so that the targeting effect of the block polymer is exerted. However, the preparation process of the mixed micelle is complex, and a large number of experiments are required to search the proportion of the block polymer.
Disclosure of Invention
Based on the thought of high et al and on the prior background, in order to simplify the preparation process of the mixed micelle, the invention creatively utilizes the acryloyl chloride to modify PEG, and synthesizes an unreported polymer long chain, and connects an acid response block PAE with RGD with the PEG. Meanwhile, the phospholipid is used as the hydrophobic end of the block polymer, so that the block polymer has good biocompatibility. The long-chain polymer is connected and synthesized into a polymer through disulfide bonds by utilizing a hydrophilic-hydrophobic switching material PAE and a targeting group c (RGDyc) to form micelles. Can realize good biocompatibility and tumor targeting at the same time, and achieve the specific release of chemotherapeutic drugs in tumor cells, thus showing higher targeting and killing effects on the tumor cells.
In order to realize the scheme, the technical scheme of the invention is as follows: a reduction-responsive vector with occlusion targeting ligand, highly targeted to tumor tissue, having the structural formula:
wherein n is more than or equal to 2, and x is more than or equal to 2;
the compound shown in the formula I is cRGD-PAE-PEG-SS-DSPE, the Chinese name is cRGD-poly beta amino ester-polyethylene glycol-SS-distearoyl phosphatidylethanolamine, the block polymer is a whole long chain, wherein n is more than or equal to 2, x is more than or equal to 2, preferably n is in the range of 30-100, and x is in the range of 8-15, and the block polymer can respond to acid rapidly.
A high-targeting low-toxicity tumor microenvironment intelligent response type nano-carrier is composed of uniformly distributed micelles with the particle size of 30-200nm, wherein the micelles are formed by self-assembling multi-block polymers in water. The multiblock polymer is cRGD-poly beta amino ester-polyethylene glycol-SS-distearoyl phosphatidylethanolamine (cRGD-PAE-PEG-SS-DSPE), the hydrophilic end is PEG with a targeting group connected with a PH response block (PAE), wherein c (RGDyc) is tumor neovascular targeting peptide (the amino acid sequence of which is Ary-Gly-Asp-D-Tyr-Cys), and the hydrophobic end is DSPE. In a medium with pH of 7.4, the core is a core-shell structure, the core is a hydrophobic end DSPE, and the shell is a hydrophilic block with a targeting group cRGD.
The prepared block polymer DSPE-SS-PEG-PAE-cRGD and the drug are dissolved in methanol together, ultrasonic vortex is used for promoting dissolution, then the solution is distilled under the condition of room temperature, and the organic solvent is removed, so that a uniform film is formed. Dissolving, hydrating and ultrasonic treating with neutral water, and finally filtering with 0.45 μm organic filter membrane to obtain micelle.
The invention creatively utilizes PAE (poly beta amino ester) to occlude c (RGDyc) in blood circulation, reduces the clearance effect of blood circulation on carriers, and simultaneously in the blood environment with pH of 7.4, hydrophilic PAE can prolong the blood circulation time of nano medicines, when the PAE reaches tumor tissues, the PAE undergoes hydrophilic-hydrophobic conversion, and the exposure of c (RGDyc) can play a role of targeting tumor cells, which can be said to be two purposes. When the carrier enters into tumor cells, disulfide bonds can be broken by the carrier under the condition of high-concentration glutathione reduction, and the drug is specifically released to kill the tumor cells, so that the treatment effect of the chemotherapeutic drug is enhanced.
The particle size of the nano carrier DSPE-SS-PEG-PAE-cRGD in different pH environments is shown in figure 6, and when the pH is higher than 7.5, the particle size change is not obvious; between 7.5 and 6.5, the particle size is obviously increased, because tertiary amine groups in PAE are protonated, so that the hydrophilic and hydrophobic properties of the PAE are changed, the carrier structure is expanded, the particle size is increased, and the pH response performance of the material is shown.
PAE is hydrophobic in a neutral PH blood environment, and c (RGDyc) can be occluded in micelles, so that c (RGDyc) is prevented from acting with opsonin in blood, and clearance of reticuloendothelial system is reduced. Meanwhile, the hydrophilic end PEG and the hydrophobic end DSPE of the micelle are connected through disulfide bonds, the micelle is influenced by the reducibility of glutathione in tumor cells, and the disulfide bonds are broken to release the drug and kill the tumor cells. According to the invention, the c (RGDyc) is occluded in the hydrophobic core PAE of the micelle by the block long chain polymer, so that the c (RGDyc) is exposed in a tumor acid environment, the volume is increased, the endocytosis of tumor cells on the carrier is enhanced, and the micelle is prevented from escaping from tumor tissues. Meanwhile, the micelle is prepared by the long-chain polymer, and the process is simpler. The disulfide bonds reduce response in tumor tissues, so that the specific release of the chemotherapeutic drugs reduces the toxic and side effects of the chemotherapeutic drugs.
The carrier can wrap the chemotherapeutic drugs such as DOX and the like in the nano-carrier, is particularly suitable for drugs with poor water solubility and large toxic and side effects, and can wrap the chemotherapeutic drugs in the hydrophobic inner core of the polymer, so that the water solubility and bioavailability of the drugs are improved. When the medicine is in blood circulation, the medicine is not easy to leak, and the toxic and side effects of the chemotherapeutic medicine on normal tissues are reduced.
The preparation method of the intelligent response type nano-carrier for the microenvironment of the tumor with high targeting and low toxicity comprises the following steps:
1)HO-PEG-SS-NH 2 is synthesized by (a)
1 to 50 parts of HO-PEG-COOH, 1 to 80 parts of NHS (N-hydroxysuccinimide), 1 to 60 parts of DCC (dicyclohexylcarbodiimide), and 1 to 30 parts of DMAP (4-dimethylaminopyridine) are dissolved in a three-necked flask containing DMF (N, N-dimethylformamide), and the mixture is dissolved in a solvent containing DMF 2 And (3) carrying out ice water bath reaction for 3-8h under the protection condition to activate carboxyl. 1-800 parts of cystamine hydrochloride and 1-1500 parts of triethylamine are weighed and dissolved in DMF for desalting. Slowly dripping DMF dissolved with cystamine into a three-mouth bottle, and adding N 2 And reacting for 18-36h at room temperature under the protection condition. Precipitating with glacial ethyl ether after the reaction is finished, and vacuum drying to obtain the structural polymer shown in the formula II, wherein the structural polymer is abbreviated as HO-PEG-SS-NH 2 。
Wherein n is more than or equal to 2;
2) Synthesis of DSPE-SS-PEG-OH
1-40 parts of OH-PEG-SS synthesized by the method, 1-45 parts of DCC (N, N-dicyclohexylcarbodiimide) and 1-35 parts of DSPE-COOH (distearoyl phosphatidyl ethanolamine modified carboxyl) are taken and dissolved in chloroform, the reaction is carried out for 2-6 hours at room temperature, the reaction solution is washed by water after the reaction is finished, the reaction solution is dried by anhydrous sodium sulfate and removed by rotary evaporation, the crude product is eluted by an anion exchange column through ammonia water to obtain the structural polymer shown in the formula III, and the structural polymer shown in the formula III is abbreviated as DSPE-SS-PEG-OH.
Wherein n is more than or equal to 2;
3) Synthesis of DSPE-SS-PEG-AC
Dissolving 1-30 parts of DSPE-SS-PEG-OH, 1-60 parts of triethylamine and 1-50 parts of acryloyl chloride in methylene dichloride under the ice bath condition, reacting for 2-6 hours at room temperature, filtering to remove white precipitate after the reaction is finished, extracting filtrate with 1mol/L dilute hydrochloric acid for three times, adding anhydrous sodium sulfate and sodium carbonate into an organic phase, and filtering to obtain the structural polymer shown in the formula IV, wherein the structural polymer is abbreviated as DSPE-SS-PEG-AC.
Wherein n is more than or equal to 2;
4) Synthesis of DSPE-SS-PEG-PAE-NHS
1-20 parts of DSPE-SS-PEG-AC, 1-600 parts of 1, 6-hexanediol diacrylate (HDD) and 1-480 parts of 1, 3-bis (4-piperidinyl) propane (TDP) are dissolved in dichloromethane, and the mixture is dissolved in N 2 Reacting at 30-60deg.C for 2-4 days under protection, adding 1-45 parts of N-acryloyloxy succinimide, reacting for 18-36 hr, and precipitating with diethyl ether to obtain structural polymer of formula VThe compound is abbreviated as DSPE-SS-PEG-PAE-NHS.
Wherein n is more than or equal to 2, and x is more than or equal to 2;
5) Synthesis of DSPE-SS-PEG-PAE-cRGD
Dissolving 1-15 parts of DSPE-SS-PEG-PAE-NHS, 1-40 parts of cRGD and 1-50 parts of triethylamine obtained by the reaction in a mixed solution of chloroform and DMSO, reacting for 8-10 hours at 20-50 ℃, removing the chloroform by rotary evaporation after the reaction is finished, dialyzing in ultrapure water, changing water for 3-5 times a day, dialyzing for three days, and freeze-drying to obtain the structural polymer shown in the formula I, wherein the structural polymer is abbreviated as DSPE-SS-PEG-PAE-cRGD.
As a further improvement of the technical scheme, the preferable conditions of the synthesis step are as follows:
said HO-PEG-COOH as described in step 1): NHS: DCC: DMAP: cystine: the feed ratio of triethylamine was 10:12:12:3:120:240. the time for activating carboxyl is 5 hours, the activation temperature is 0 ℃, and the reaction time is 24 hours. The feed liquid ratio of the HO-PEG-COOH to the DMF is 0.025mmol/ml.
Said step 2) HO-PEG-SS-NH 2 : DSPE-COOH: DCC: the feed ratio of NHS is 1:1:1:1, reacting for 3 hours at room temperature, wherein the HO-PEG-SS-NH is 2 The feed liquid ratio of the aqueous ammonia to the chloroform is 0.024mmol/ml, and the volume fraction of the aqueous ammonia is 0.1%.
The DSPE-SS-PEG-OH in the step 3): triethylamine: the charging ratio of the acrylic chloride is 10:15:15, reacting for 3 hours at room temperature, wherein the feed liquid ratio of DSPE-SS-PEG-OH to dichloromethane is 0.179mmol/ml.
The DSPE-SS-PEG-AC in step 4): 1, 6-hexanediol diacrylate (HDD): 1, 3-bis (4-piperidinyl) propane (TDP): the feed ratio of N-acryloyloxy succinimide is 1:11:11:1.5, the reaction time is 72h, the reaction temperature is 40 ℃, the reaction is continued for 24h after adding N-acryloyloxy succinimide, and the feed liquid ratio of DSPE-SS-PEG-AC to dichloromethane is 0.0125mmol/ml.
The DSPE-SS-PEG-PAE-NHS in the step 5): triethylamine: c (RGDyc) with a feed ratio of 10:25:15, the reaction time is 9h, the reaction temperature is 45 ℃, water is changed for 4 times a day, and the feed liquid ratio of the mixed solution of DSPE-SS-PEG-PAE-NHS, chloroform and DMSO is 1.25 mu mol/ml.
The equation for DSPE-SS-PEG-PAE-RGD synthesis is:
the working mechanism of the invention is as follows:
the block polymer carrier DSPE-SS-PEG-PAE-cRGD is used for entrapping the medicine, enters the blood circulation, and PAE shows hydrophobicity under the condition of pH7.4, and cRGD is occluded in the hydrophobic block of the polymer and in the hydrophobic core of the micelle. When the drug-loaded nano-carrier enters tumor tissues, tertiary ammonia in PAE is protonated under the acidic condition to carry out hydrophilic-hydrophobic conversion, cRGD is exposed, and the endocytosis of tumor cells on the nano-carrier is enhanced. After the drug-loaded nano-carrier enters tumor cells, disulfide bond in the block polymer is promoted to be broken due to higher glutathione level in the tumor cells, and the drug is released. Thus greatly reducing the toxicity of the chemotherapeutic drugs to normal tissues and enhancing the therapeutic effect of the chemotherapeutic drugs.
The beneficial effects are that:
the anti-tumor targeting nano-drug carrier has specific targeting effect on tumor cells by targeting groups; drug delivery systems have responsive release capabilities, release relatively rapidly under reducing conditions, and little release in normal tissues. Has better targeting effect and killing effect on tumor cells.
The preparation method adopts fewer synthesis steps and milder reaction conditions to prepare the whole polymer long chain, and the synthesized polymer long chain can be used for preparing micelles very easily, and the preparation process is simple. Meanwhile, the prepared polymer micelle has higher acid response performance, and the particle size change can be generated when the pH is less than 7.5 and the polymer micelle is incubated for 30min to release the drug, so that the polymer micelle can quickly respond compared with other occlusion targeting peptide technical routes.
Drawings
FIG. one shows an infrared spectrum of the nano-carrier DSPE-SS-PEG-PAE-cRGD of the present invention.
FIG. 2 shows 1HNMR of the nano-carrier DSPE-SS-PEG-PAE-cRGD.
FIG. three shows titration curves of the nanocarrier DSPE-SS-PEG-PAE-cRGD of the present invention.
And fourth, the particle size diagram of the micelle formed by the nano carrier DSPE-SS-PEG-PAE-cRGD.
FIG. five is a graph showing particle size of micelle formed by nano-carrier DSPE-SS-PEG-PAE-cRGD under the condition of DTT reduction.
FIG. six is a graph showing particle size of the nano-carrier DSPE-SS-PEG-PAE-cRGD micelle under different pH environments.
FIG. seven shows cytotoxicity test of the nano-carrier DSPE-SS-PEG-PAE-cRGD micelle of the invention on 4T1 cells.
FIG. eight shows the experiment of the toxicity of the nano-carrier DSPE-SS-PEG-PAE-cRGD micelle to normal cells L929.
FIG. nine is an electron microscope image of 4T1 cells uptake of the nanocarrier DSPE-SS-PEG-PAE-cRGD micelles of the invention.
Detailed Description
The following description of the present invention is provided with reference to the accompanying drawings, but is not limited to the following description, and any modifications or equivalent substitutions of the present invention without departing from the spirit and scope of the present invention shall be covered in the protection scope of the present invention.
Example 1
The embodiment provides a method for synthesizing a nano carrier, which sequentially comprises the following steps:
1) 1 part of HO-PEG-COOH, 1.2 parts of NHS (N-hydroxysuccinimide), 1.2 parts of DCC (dicyclohexylcarbodiimide) and 0.3 part of DMAP (4-dimethylaminopyridine) are dissolved in a three-necked flask containing DMF, and the mixture is subjected to ice-water bath under N 2 The reaction is carried out for 5 hours under the protection condition to activate carboxyl. 6 parts of cystamine hydrochloride and 12 parts of triethylamine are weighed out and dissolved in DMF for desalting. Slowly dripping DMF dissolved with cystamine into a three-mouth bottle, and adding N 2 The reaction is carried out for 24 hours at room temperature under the protection condition. Precipitating with glacial ethyl ether after the reaction is finished, and vacuum drying to obtain polymer HO-PEG-SS-NH 2 . The feed liquid ratio of the HO-PEG-COOH to the DMF is 0.025mmol/ml.
2) Taking 1 part of the synthesized OH-PEG-SS-NH 2 1 part of DCC (N, N-dicyclohexylcarbodiimide), 1 part of DSPE-COOOH (distearoyl phosphatidylethanolamine modified carboxyl) and 1 part of NHS are dissolved in chloroform to react for 3 hours at room temperature, the reaction solution is washed by water after the reaction is finished, the reaction solution is dried by anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and the crude product is eluted by an anion exchange column through ammonia water to obtain the polymer DSPE-SS-PEG-OH. The OH-PEG-SS-NH 2 The feed liquid ratio of the aqueous ammonia to the chloroform is 0.024mmol/ml, and the volume fraction of the aqueous ammonia is 0.1%.
3) Dissolving 1 part of DSPE-SS-PEG-OH synthesized by the method, 1.5 parts of triethylamine and 1.5 parts of acryloyl chloride in methylene dichloride under ice bath condition, reacting for 3 hours at room temperature, filtering to remove white precipitate after the reaction is finished, extracting filtrate with 1mol/L dilute hydrochloric acid for three times, adding anhydrous sodium sulfate and sodium carbonate into an organic phase, and filtering to obtain a polymer DSPE-SS-PEG-AC. The feed liquid ratio of DSPE-SS-PEG-OH to dichloromethane is 0.179mmol/ml.
4) 1 part of DSPE-SS-PEG-AC synthesized in the above, 11 parts of 1, 6-hexanediol diacrylate (HDD), 11 parts of 1, 3-bis (4-piperidinyl) propane (TDP) were taken and dissolved in methylene chloride under N 2 Reacting for 3 days at 40 ℃ under the protection condition, adding 1.5 parts of N-acryloyloxy succinimide (NHS) for further reacting for 1 day, and precipitating by diethyl ether to obtain the polymer D SPE-SS-PEG-PAE-NHS, wherein the feed liquid ratio of DSPE-SS-PEG-AC to dichloromethane is 0.0125mmol/m l.
5) 1 part of DSPE-SS-PEG-PAE-NHS, 1.5 parts of cRGD and 2.5 parts of Triethylamine (TEA) obtained by the reaction are dissolved in a mixed solution of chloroform and DMSO, the mixture is reacted for 9 hours at 45 ℃, chloroform is removed by rotary evaporation after the reaction, dialysis is carried out in ultrapure water by using a dialysis bag with the molecular weight of 8000, water is changed for 4 times per day, after three days of dialysis, the polymer DSPE-SS-PEG-PAE-cRGD is obtained by freeze drying, and the feed liquid ratio of the mixed solution of DSPE-SS-PEG-PAE-NHS, chloroform and DMSO is 1.25 mu mol/ml. The structural formula of the prepared intelligent response type nano-carrier for the microenvironment of the tumor with high targeting and low toxicity is shown as follows:
in this embodiment, n=50 and x=10. The size of n can seriously affect the solubility of the polymer, the ratio of n to x, and the drug carrying capacity of the polymer micelle, and the ratio of hydrophilic end to hydrophobic end in the example of the invention is 1:2.
The IR spectrum of vector DSPE-SS-PEG-PAE-cGRD is shown in FIG. 1.
FIG. 1 is an infrared spectrum of the nano-carrier DSPE-SS-PEG-PAE-cRGD of the invention, and the infrared peak is marked. Wherein the C-H stretching vibration of PEG is 2919 and 2851cm -1 C-O stretching vibration is 1112cm -1 The C-N telescopic vibration of PBAE is 1184cm -1 Carbonyl and C-N vibration of amide are 1727 cm and 1560cm respectively -1 。
Vector DSPE-SS-PEG-PAE-cRGD 1 The HNMR analysis chart is shown in fig. 2.
FIG. 2 shows the nano-carrier DSPE-SS-PEG-PAE-cRGD of the present invention 1 HNMR, wherein DSPE methyl, methylene chemical shifts are shown at δ=0.85, 1.23ppm, disulfide chemical shifts are shown at δ=2.88 ppm, peg methyl methylene chemical shifts are shown at δ=3.51, 3.36ppm, pae and two methylene chemical shifts adjacent to the characterizing carbonyl group are shown at δ=4.02, 1.61ppm, and two methylene chemical shifts adjacent to tertiary ammonia are shown at δ=2.88, 1.90ppm, respectively.
Example 2 acid-base titration experiments of vector DSPE-SS-PEG-PAE-cRGD
5mg of copolymer was dissolved in 10ml of deionized water, and 0.1 mol.L was used -1 HCL was adjusted to pH3.0. By 0.1 mol.L -1 Titration was performed with 5. Mu.L of NaOH solution each time, the pH change of the solution was measured with a pH meter, the pH change each time was recorded, and a titration curve was drawn. The control group, naCL, was operated as described above. FIG. 3 is a titration curve of inventive nanocarrier DSPE-SS-PEG-PAE-cRGD, showing a significant decrease in slope of the curve over a pH range of 6.8-9.7 compared to NaCl nanocarrier, in which the carrier has a significant effectIs used for buffering.
EXAMPLE 3 preparation of micelles by the vector DSPE-SS-PEG-PAE-cRGD
Precisely weighing 5mg of carrier, adding 1mg of DOX into 20ml eggplant-shaped bottle, adding 5ml of methanol, dissolving for 5min, removing organic solvent by rotary evaporation at normal temperature, and placing into a vacuum drying oven. Then 10ml of purified water is added, hydration is carried out for 30min under the condition of 100rpm, and the medicine carrying micelle is obtained through an organic filter membrane with the thickness of 0.45 mu m. FIG. 4 shows that the block polymer forms micelle with average particle diameter of 113.2nm, PDI of 0.228, and particle diameter of reasonable distribution in water as measured by Markov particle size analyzer.
Example 4 reduction response of vector DSPE-SS-PEG-PAE-cRGD micelle
A micelle solution of 0.5mg/ml was prepared with a pH6.8 acetate buffer containing 10mM DTT, and the particle size distribution was measured by DLS after 24 hours.
FIG. 5 is a graph showing particle size of micelle formed by the nano-carrier DSPE-SS-PEG-PAE-cRGD under the DTT reduction condition. It can be found that new small-sized micelles are formed under reducing conditions, compared to fig. 4, demonstrating the presence of disulfide bonds.
Example 5 particle size Change of Carrier DSPE-SS-PEG-PAE-cRGD at different pH
The change in particle size was measured after incubation of freshly prepared blank micelles in PBS buffer at different pH values for 30 min. FIG. 6 shows the change in particle size of the nanocarrier DSPE-SS-PEG-PAE-cRGD of the present invention under different pH environments. The results show that when the pH is higher than 7.5, the particle size change is not obvious; between 7.5 and 6.5, a significant increase in particle size occurs due to protonation of the tertiary amine groups in the PAE, which changes the hydrophilicity and hydrophobicity, thus expanding the support structure, resulting in an increase in particle size. Indicating that the material has pH response performance
EXAMPLE 6 cytotoxicity assay of vector DSPE-SS-PEG-PAE-cRGD
The experimental groups were: a: doxorubicin solution group (DOX); b: doxorubicin-entrapped polymeric micelle group (PEG-SS-DSPE/DOX); c: polymeric micelle (RGD-PAE-PEG-SS-DSPE/DOX) mediated by RGD peptide and entrapped with doxorubicin.
After 4T1 cells and L929 cells in the logarithmic growth phase were digested with 0.25% trypsin, single cell suspensions were prepared in RPMI1640 medium, and then inoculated into 96-well plates, the number of cells per well being about 1X 10 4 Three complex holes are provided. After 24 hours incubation, the culture solution is changed into the above physical mixed solution or micelle solution with different concentrations and incubation is continued for 4 hours. After 4h incubation the solution was changed to MTT solution (0.5 mg/ml,200 ul/well) and incubation was continued for 4h; after 4h incubation of the cells with MTT solution, the solution in the wells was removed, then 150 μl dmso was added to each well and the formazan crystals were dissolved by shaking 5 mi. The absorbance at 630nm was measured for each well with a microplate reader and cell viability was calculated according to the following formula:
wherein A is sample For each experimental group, absorbance value, A control Absorbance values for PBS blank.
The results in FIG. 7 show that the nano-carrier DSPE-SS-PEG-PAE-cRGD entrapped DOX has stronger killing effect on 4T-1 cells compared with free DOX and non-targeting carrier groups, the cell survival rate of the targeting carrier group of the 5 mu g dose group is only 30%, compared with the non-targeting carrier group, the cell survival rate of the targeting carrier group is greatly reduced, and the carrier enhances the chemotherapy effect of the drug.
The results in fig. 8 show that the carrier DSPE-SS-PEG-PAE-cRGD entrapped DOX has less cytotoxicity and no killing effect compared with the free doxorubicin group and the non-targeting carrier group, and the carrier is not easy to leak, safe and nontoxic, and has good biocompatibility.
EXAMPLE 7 vector DSPE-SS-PEG-PAE-cRGD cell uptake assay
Breast cancer 4T1 cells were selected as model cells for cell uptake experiments. Taking 4T1 cells in logarithmic growth phase, digesting with 0.25% pancreatin, centrifuging to remove supernatant, adding RPMI1640 culture solution to prepare cell suspension, and adding 200 μl (about 1×10 per well 4 ) The cell suspension is added into a 96-well plate, incubated for 24 hours in a 37 ℃ incubator, and then discardedRemoving the original culture medium, adding free doxorubicin and drug-loaded micelle (the equivalent concentration of doxorubicin is 5 mug/mL L) respectively, and continuously incubating for 24 hours. The supernatant was discarded, the cells were washed three times with PBS at 4℃to remove residual drug, the cells were fixed with 4% paraformaldehyde, and stained with hoechst dye for 15min. Cells were observed for uptake using CLSM and photographed.
FIG. 9 is an electron microscope image of the targeting effect of the nanocarrier DSPE-SS-PEG-PAE-cRGD on cells, wherein the targeting vector group has better targeting effect on tumor cells compared with the free doxorubicin and red fluorescence of a plurality of cells of the non-targeting vector group.

Claims (10)

1. The intelligent response type nano carrier for the tumor microenvironment with high targeting and low toxicity is characterized by comprising micelles formed by self-assembly of multi-block polymers in water, wherein the multi-block polymers are distearoyl phosphatidylethanolamine (DSPE) -SS-polyethylene glycol (PEG) -poly beta amino ester (PAE) -targeting groups, the hydrophilic end of the multi-block polymers is PEG, and the hydrophobic end of the multi-block polymers is DSPE connected through disulfide bonds; the hydrophilic end is attached to a pH responsive block PAE bearing a targeting group.
2. The intelligent response type nano-carrier for the tumor microenvironment with high targeting and low toxicity according to claim 1, wherein the particle size of the micelle is 30-200nm in a neutral environment and becomes larger in an acidic environment.
3. The high-targeting low-toxicity tumor microenvironment intelligent response type nano-carrier according to claim 1, wherein the targeting group is tumor neovascularization targeting peptide c (RGDyc) and the amino acid sequence is Ary-Gly-Asp-D-Tyr-Cys.
4. The intelligent response type nano-carrier for the microenvironment of the tumor with high targeting and low toxicity according to claim 1, wherein the nano-carrier is a structural polymer shown in a formula I,
wherein n is greater than or equal to 2 and x is greater than or equal to 2.
5. The intelligent response type nano-carrier for the microenvironment of the tumor with high targeting and low toxicity according to claim 4, wherein the block polymer is a whole long chain, n is between 30 and 100, and x is between 8 and 15.
6. The method for preparing the intelligent response type nano-carrier for the microenvironment of the tumor with high targeting and low toxicity according to any one of claims 1 to 5, which is characterized by comprising the following steps:
1)HO-PEG-SS-NH 2 is synthesized by (a)
1 to 50 parts of HO-PEG-COOH, 1 to 80 parts of NHS (N-hydroxysuccinimide), 1 to 60 parts of DCC (dicyclohexylcarbodiimide), and 0.1 to 30 parts of DMAP (4-dimethylaminopyridine) are dissolved in a three-necked flask containing DMF (N, N-dimethylformamide), and the mixture is dissolved in N 2 Carrying out ice water bath reaction for 3-8h under the protection condition to activate carboxyl;
1-800 parts of cystamine hydrochloride and 1-1500 parts of triethylamine are weighed and dissolved in DMF for desalting; slowly dripping DMF dissolved with cystamine into a three-mouth bottle, and adding N 2 Reacting for 18-36h at room temperature under the protection condition; precipitating with glacial ethyl ether after the reaction is finished, and vacuum drying to obtain the structural polymer shown in the formula II, wherein the structural polymer is abbreviated as HO-PEG-SS-NH 2 ；
Wherein n is more than or equal to 2;
2) Synthesis of DSPE-SS-PEG-OH
Taking 1-40 parts of the synthesized OH-PEG-SS-NH 2 1-45 parts of DCC, 1-40 parts of DSPE-COOH (distearoyl phosphatidyl ethanolamine modified carboxyl) and 1-35 parts of NHS are dissolved in chloroform to react for 2-6 hours at room temperature, the reaction liquid is washed by water after the reaction is finished,drying with anhydrous sodium sulfate, removing solvent by rotary evaporation, and eluting the crude product with ammonia water by an anion exchange column to obtain a structural polymer shown in formula III, wherein the structural polymer is abbreviated as DSPE-SS-PEG-OH;
wherein n is more than or equal to 2;
3) Synthesis of DSPE-SS-PEG-AC
Dissolving 1-30 parts of the synthesized DSPE-SS-PEG-OH, 1-60 parts of triethylamine and 1-50 parts of acryloyl chloride in methylene dichloride under the ice bath condition, reacting for 2-6 hours at room temperature, filtering to remove white precipitate after the reaction is finished, extracting filtrate with 1mol/L dilute hydrochloric acid for three times, adding anhydrous sodium sulfate and sodium carbonate into an organic phase, and filtering to obtain a structural polymer shown in a formula IV, wherein the structural polymer is abbreviated as DSPE-SS-PEG-AC;
wherein n is more than or equal to 2;
4) Synthesis of DSPE-SS-PEG-PAE-NHS
1-20 parts of DSPE-SS-PEG-AC, 1-600 parts of 1, 6-hexanediol diacrylate (HDD) and 1-480 parts of 1, 3-bis (4-piperidinyl) propane (TDP) are dissolved in dichloromethane, and the mixture is dissolved in N 2 Reacting for 2-4 days at 30-60 ℃ under the protection condition, adding 1-45 parts of N-acryloyloxy succinimide to react for 18-36 hours, and precipitating by diethyl ether to obtain the structural polymer shown in the formula V, wherein the structural polymer shown in the formula V is abbreviated as DSPE-SS-PEG-PAE-NHS;
wherein n is more than or equal to 2, and x is more than or equal to 2;
5) Synthesis of DSPE-SS-PEG-PAE-cRGD
1-15 parts of DSPE-SS-PEG-PAE-NHS, 1-40 parts of c (RGDyc) and 1-50 parts of triethylamine are taken and dissolved in a mixed solution of chloroform and DMSO, the mixture is reacted for 8-10 hours at 20-50 ℃, chloroform is removed by rotary evaporation after the reaction, dialysis is carried out in ultrapure water, water is changed for 3-5 times a day, after three days of dialysis, the structural polymer shown in the formula I is obtained by freeze drying, and the structural polymer shown in the formula I is abbreviated as DSPE-SS-PEG-PAE-cRGD.
7. The method of preparing nanocarriers of claim 6, wherein the HO-PEG-COOH of step 1): NHS: DCC: DMAP: cystine: the feed ratio of triethylamine was 10:12:12:3:120:240, a step of; the time for activating carboxyl is 5 hours, the activation temperature is 0 ℃, and the reaction time is 24 hours; the feed liquid ratio of the HO-PEG-COOH to the DMF is 0.025mmol/ml.
8. The method of claim 6, wherein the step 2) comprises HO-PEG-SS-NH 2 : DSPE-COOH: DCC: the feed ratio of NHS is 1:1:1:1, reacting for 3 hours at room temperature, wherein the HO-PEG-SS-NH is 2 The feed liquid ratio of the ammonia water to the chloroform is 0.024mmol/ml, and the volume fraction of the ammonia water is 0.1%;
the DSPE-SS-PEG-OH in the step 3): triethylamine: the charging ratio of the acrylic chloride is 10:15:15, reacting for 3 hours at room temperature, wherein the feed liquid ratio of DSPE-SS-PEG-OH to dichloromethane is 0.179mmol/ml.
9. The method of claim 6, wherein the DSPE-SS-PEG-AC in step 4) is: 1, 6-hexanediol diacrylate (HDD): 1, 3-bis (4-piperidinyl) propane (TDP): the feed ratio of N-acryloyloxy succinimide is 1:11:11:1.5, the reaction time is 72h, the reaction temperature is 40 ℃, the reaction is continued for 24h after adding N-acryloyloxy succinimide, and the feed liquid ratio of DSPE-SS-PEG-AC to dichloromethane is 0.0125mmol/ml.
10. The method of claim 6, wherein the DSPE-SS-PEG-PAE-NHS in step 5): triethylamine: c (RGDyc) with a feed ratio of 10:25:15, the reaction time is 9h, the reaction temperature is 45 ℃, water is changed for 4 times a day, and the feed liquid ratio of the mixed solution of DSPE-SS-PEG-PAE-NHS, chloroform and DMSO is 1.25 mu mol/ml.
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