CN114196028B - PAMAM-TPGS, and preparation method and application thereof - Google Patents

PAMAM-TPGS, and preparation method and application thereof Download PDF

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CN114196028B
CN114196028B CN202111586684.4A CN202111586684A CN114196028B CN 114196028 B CN114196028 B CN 114196028B CN 202111586684 A CN202111586684 A CN 202111586684A CN 114196028 B CN114196028 B CN 114196028B
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polyethylene glycol
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郭钫元
焦云龙
杨根生
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Zhejiang University of Technology ZJUT
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Abstract

The invention provides a dendritic polymer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS), a preparation method thereof and application thereof as a drug carrier in preparation of a pharmaceutical preparation. The dendritic polymer-vitamin E succinate polyethylene glycol 1000 nanoparticles prepared by a microchannel method, a film dispersion method and an emulsion solvent volatilization method respectively have the characteristics of easy industrialization, high drug loading capacity and stable system, can obviously improve the solubility of model drugs in water, realize controllable release, have obvious pharmacological activity, and are suitable for various drug administration modes such as intravenous injection.

Description

PAMAM-TPGS, and preparation method and application thereof
(I) the technical field
The invention relates to the technical field of preparation of a medicine high-molecular carrier and a medicine preparation, in particular to a dendritic polymer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) -based precursor material, a preparation method thereof and application thereof as a medicine carrier in preparation of a medicine preparation.
(II) background of the invention
Cancer is a problem to be solved urgently in the international medical field today. Chemotherapy is one of the three main methods for treating cancer, but most clinical anticancer drugs are lipophilic compounds, which have poor water solubility, easy metabolism in vivo and low specific selectivity for cancer cells, and need long-term, multiple or large-dose administration for treatment. Often presents serious toxic and side effects, anaphylactic reaction and drug resistance of tumor cells, and seriously influences the effectiveness and safety of clinical application of the drug. The nano drug-loading system is a new favorite in the field of drug delivery because the nano drug-loading system can effectively improve the solubility of drugs in water, realize the adjustability of drug release rate and blood concentration and reduce toxic and side effects, and meanwhile, the passive targeting characteristic of carrier drugs can be realized through controlling the size of a nanoparticle carrier (less than 200 nm) due to the existence of high permeability and retention effect (EPR effect) of solid tumors. However, clinical transformation of nano-formulations has progressed slowly, and tumor multidrug resistance (MDR) is one of the major causes of chemotherapy failure. Research finds that tumors have multiple signals, multiple links, multiple channels and a biological system compensation mechanism for comprehensive network management, which is one of the main reasons for generating drug resistance. The method of using multi-drug combination therapy is one of the most effective methods for inhibiting the multi-drug resistance of cancer cells at present. However, due to the limited internal space of the nano-carrier, the multi-drug combination therapy mostly causes the reduction of the loading amount of each drug. Meanwhile, the multi-drug co-loading often has the uncontrollable proportion of drug and drug release, and generates uncertainty on clinical treatment. If the drug is loaded in different nano carriers, the 'excess' carrier material can be caused to enter the organism, and a new challenge is brought to the biological safety. Therefore, there is an urgent need to develop a novel drug delivery platform to meet the clinical drug requirements.
Polyamidoamine dendrimers (PAMAMs) are one of the most functional nanopolymer structures at present, and have received scientific attention due to their unique properties such as monodispersity, solubility in water, high branching, and the presence of huge internal cavities. The PAMAM dendrimer is used as an excellent drug delivery platform and has the advantages of large drug loading capacity, strong water bonding capability, low toxicity and the like. The surface or the interior of the material contains a large number of primary amine and tertiary amine groups, which is beneficial to various targeted modifications and fluorescent grafting so as to improve the drug targeted delivery efficiency and perform fluorescent quantitative analysis. More interestingly, it was found that PAMAM dendrimers are not themselves inert, that cationic PAMAM dendrimers significantly reduce cell viability in a dose-dependent manner, induce apoptosis (upregulation of apoptosis markers (Bax, caspases-3,8 and 9), and Bcl-2 downregulation), and can replace nanomedicines under a variety of conditions. One of its most important biological effects is the ability to modulate gene expression patterns and interfere with the cell signaling pathways of the epidermal growth factor receptor family; in particular EGFR (epidermal growth factor receptor) and HER2. However, there are some difficulties in clinical application of dendrimer-based drug carriers, such as rapid clearance of dendrimers by the mononuclear cell phagocyte system (MPS) in vivo due to electropositivity of their surface and easy formation of protein corona on the surface.
Based on the continuing search for MDR, researchers have discovered that certain nonionic surfactants, such as Pluronic, tweens, span, and TPGS, are effective in reversing tumor MDR by (i) blocking substrate binding, (ii) altering cell membrane fluidity, (iii) reducing intracellular ATP levels, (iv) inhibiting efflux pump ATPase, (v) down-regulating P-gp levels, and the like, to inhibit P-gp activity. Among them, d- α -tocopheryl polyethylene glycol 1000 succinate (TPGS) has been approved by FDA as a widely developed adjuvant, and pharmacological studies have shown that TPGS has a broad spectrum of intrinsic toxicity to tumor cells, but no toxicity to normal cells or tissues. Meanwhile, TPGS can target mitochondria of tumor cells, causing mitochondria to malfunction by increasing the generation of Reactive Oxygen Species (ROS), thereby triggering apoptotic pathways. In addition, the existence of PEG fragment in TPGS structure can effectively prolong the circulation time in vivo.
In conclusion, the method aims to effectively reverse the multidrug resistance of the tumor, overcome the defects of the multidrug combined anticancer therapy and improve the clinical curative effect. According to the advantages of PAMAM and TPGS materials, the invention provides a nano drug delivery system based on drug/material combination therapy, which comprises anticancer drugs, and then a nano drug delivery platform of drug/material combination therapy is respectively constructed by using a micro-channel continuous granulation technology, a film dispersion method and an emulsion solvent volatilization method. So as to improve the bioavailability of the medicament, reverse MDR and improve the clinical anticancer curative effect through the synergistic effect of the medicament and the carrier material.
Disclosure of the invention
The invention aims to provide a dendritic polymer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS), a preparation method thereof and application thereof as a drug carrier in preparation of a pharmaceutical preparation.
In order to achieve the purpose, the invention adopts the following technical scheme
In a first aspect, the present invention provides a dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS), said dendrimer-vitamin E succinate polyethylene glycol 1000 being prepared according to the following method:
(1) Dissolving vitamin E succinate polyethylene glycol 1000 (TPGS), succinic Anhydride (SA), N-diisopropylethylamine A (DIPEA) and 4-Dimethylaminopyridine (DMAP) in 1, 4-dioxane, reacting at room temperature for 8-48h under the protection of a protective atmosphere A (such as inert gas or nitrogen, preferably nitrogen protection), and performing aftertreatment on the obtained reaction liquid A to obtain TPGS-COOH;
the ratio of the vitamin E succinate polyethylene glycol 1000 to the succinic anhydride to the N, N-diisopropylethylamine A to the 4-dimethylaminopyridine is 1:2 to 20:0.1 to 1 (preferably 1;
(2) Dissolving the TPGS-COOH, N, N-diisopropylethylamine B (DIPEA), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) and PAMAM dendrimer polymer in the step (1) in dimethyl sulfoxide (DMSO), stirring until the TPGS-COOH, N, N ', N' -tetramethylurea hexafluorophosphate and PAMAM dendrimer polymer are completely dissolved, reacting at normal temperature for 8-48h (preferably 24 h) in a protective atmosphere B (such as inert gas or nitrogen, preferably nitrogen protection), and performing post-treatment on the obtained reaction liquid B to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000, namely PAMAM-TPGS;
the mass ratio of TPGS-COOH, N, N-diisopropylethylamine B, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) to PAMAM dendrimer is 1:1 to 10:1 to 10: 0.01-1 (preferably 1.
N, N-diisopropylethylamine A and N, N-diisopropylethylamine B are N, N-diisopropylethylamine, and A and B are only used for distinguishing N, N-diisopropylethylamine added at different stages, so that the description is convenient, and no other special meanings exist.
Further, the volume of the 1, 4-dioxane in the step (1) is 10-50 mL/g (preferably 32.7 mL/g) based on the mass of the vitamin E succinate polyethylene glycol 1000.
Further, the post-treatment A in the step (1) is as follows: and (3) removing the solvent from the obtained reaction solution A by rotary evaporation, re-dissolving dichloromethane to obtain a crude product, washing the crude product with a citric acid aqueous solution with the pH =5-6 (three times), washing the obtained organic phase with distilled water (three times), and carrying out rotary evaporation on the organic phase to obtain the TPGS-COOH.
Preferably, the volume of the dichloromethane is 5-30 mL/g (preferably 21.8 mL/g) based on the total mass of the vitamin E succinate polyethylene glycol 1000, succinic anhydride, N-diisopropylethylamine and 4-dimethylaminopyridine.
Further, the volume of the dimethyl sulfoxide (DMSO) in the step (2) is 5 to 50mL/g (preferably 24.6L/g) based on the mass of the TPGS-COOH.
Further, the post-treatment B in the step (2) is as follows: dialyzing the obtained reaction solution B in double distilled water by using a 1.4kDa dialysis bag for 1-10 days (preferably 4 days), and freeze-drying (preferably 12 hours) to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000 and PAMAM-TPGS.
In a second aspect, the present invention provides an application of the dendrimer-vitamin E succinate polyethylene glycol 1000 as a pharmaceutical carrier in the preparation of pharmaceutical preparations.
Particularly, the pharmaceutical preparation is recommended to be an anti-tumor pharmaceutical preparation, such as a pharmaceutical preparation for resisting A549 tumor cells.
Specifically, the invention provides three preparation methods of the pharmaceutical preparation.
1. A film dispersion method: the application is as follows: mixing the drug to be loaded with the dendritic polymer-vitamin E succinate polyethylene glycol 1000, dissolving in a volatile good solvent (such as methanol and ethanol, preferably methanol) to serve as a lipid phase, spin-drying, adding a TPGS aqueous solution to serve as a water phase, performing ultrasonic dissolution and centrifugation, and taking supernatant to serve as the drug preparation; namely aqueous solution containing dendritic polymer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) administration nanoparticles;
the mass ratio of the drug to be loaded to the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is 1; the volume of the volatile good solvent is 0.01-0.1mL/mg (preferably 0.05 mL/mg) based on the mass of the dendritic polymer-vitamin E succinate polyethylene glycol 1000; the concentration of TPGS in the TPGS aqueous solution is 0.01-5 mg/ml (preferably 0.2 mg/ml); the volume of the aqueous phase is 0.1-1mL/mg (preferably 0.4 mL/mg) based on the mass of dendrimer-vitamin E succinate polyethylene glycol 1000.
Further, the drug to be loaded such as the good volatile solvent which is insoluble in the solvent can be miscible with PAMAM-TPGS in the form of a solution, if the drug to be loaded adopted in the invention is curcumin, the curcumin is miscible in the form of an acetone solution of the curcumin, and the concentration of the acetone solution of the curcumin is 1-5 mg/mL (preferably 4 mg/mL).
Further, ultrasonic mixing is adopted for mixing and dissolving, and the ultrasonic mixing and dissolving time is 5-50 min, particularly preferably 20min.
Further, the temperature of the spinning is 25 to 60 ℃ (preferably 35 ℃), and the time of the spinning is 5 to 50min, particularly preferably 20min.
Preferably, the ultrasonic dissolution time is 5 to 150min, particularly preferably 90min.
The dendritic polymer PAMAM-TPGS administration nanoparticle prepared by the method has the average particle diameter of 10-150 nm, the polydispersity index (PDI) of less than 0.30 and the drug loading rate of 8-30%.
2. An emulsion solvent volatilization method: the application is as follows: mixing a drug to be loaded with the dendritic polymer-vitamin E succinate polyethylene glycol 1000, dissolving in a volatile good solvent (such as methanol and ethanol, preferably methanol), dropwise adding the obtained lipid phase into an aqueous solution (namely an aqueous phase) of TPGS, stirring for 1-3 h (magnetically) after dropwise adding, vacuum drying to remove an organic solvent, centrifuging, and taking a supernatant, namely the drug preparation; namely aqueous solution containing dendritic polymer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) administration nanoparticles;
the mass ratio of the drug to be loaded to the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is 1; the volume of the volatile good solvent is 0.01-0.1mL/mg (preferably 0.05 mL/mg) based on the mass of the dendritic polymer-vitamin E succinate polyethylene glycol 1000; the concentration of TPGS in the TPGS aqueous solution is 0.01-5 mg/ml (preferably 0.2 mg/ml); the volume of the aqueous phase is 0.1-1mL/mg (preferably 0.4 mL/mg) based on the mass of dendrimer-vitamin E succinate polyethylene glycol 1000.
Further, the drug to be loaded such as a good volatile solvent (such as methanol and ethanol) which is difficult to dissolve can be miscible with PAMAM-TPGS in the form of a solution, if the drug to be loaded adopted by the invention is curcumin, the curcumin is miscible in the form of an acetone solution of the curcumin, and the concentration of the acetone solution of the curcumin is 1-5 mg/mL (preferably 4 mg/mL).
Further, ultrasonic mixing is adopted for mixing and dissolving, and the ultrasonic mixing and dissolving time is 5-50 min, particularly preferably 20min.
The dendritic polymer PAMAM-TPGS administration nanoparticle prepared by the method has the average particle diameter of 10-50 nm, the polydispersity index (PDI) of less than 0.20 and the drug loading rate of 5-17%.
3. Microchannel continuous granulation technique: the application is as follows: the pharmaceutical formulation is prepared using a microchannel plate comprising a diameter of 300-420 μm (preferably 36)0 μm), a first sub-channel with a diameter of 530-650 μm (preferably 590 μm), a second sub-channel and a N with a diameter of 390-550 μm (preferably 470 μm) 2 A channel; the two ends of the main channel are respectively provided with a main channel inlet and a main channel outlet, and the main channel is also provided with a position N200-280 mm (preferably 240 mm) away from the main channel outlet 2 Entrance and two distances from said N 2 A first aqueous phase inlet and a second aqueous phase inlet having an inlet of 30 to 50mm (preferably 40 mm); said N is 2 Channel and said N 2 The first branch channel and the second branch channel are respectively connected with the first water phase inlet and the second water phase inlet;
mixing the drug to be loaded with the dendritic polymer-vitamin E succinate polyethylene glycol 1000, and dissolving in volatile good solvent (such as methanol, ethanol, preferably methanol) to obtain lipid phase, and using TPGS water solution as water phase; the mass ratio of the drug to be loaded to the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is 1; the volume of the volatile good solvent is 0.01-0.1mL/mg (preferably 0.05 mL/mg) based on the mass of the dendritic polymer-vitamin E succinate polyethylene glycol 1000; the concentration of TPGS in the TPGS aqueous solution is 0.01-5 mg/ml (preferably 0.2 mg/ml);
the lipid phase enters the microchannel plate from the inlet of the main channel at a flow rate of 1-10mL/h (preferably 2 mL/h), the aqueous phase enters the microchannel plate from the first branch channel and the second branch channel at a flow rate of 1-10mL/h (preferably 1 mL/h), respectively, nitrogen or inert gas (preferably nitrogen, the flow rate has little influence on the reaction, and the flow rate adopted by the invention is 0.25 mL/min) 2 The channel is communicated into the microchannel plate; collecting the mixed liquid flowing out of the outlet of the main channel, simultaneously stirring at constant speed for 10-60 min at room temperature, carrying out vacuum drying for 3-10 h, centrifuging at 3000-10000 r/min, and taking supernatant, namely the medicinal preparation; namely aqueous solution containing dendritic polymer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) administration nanoparticles.
Further, the drug to be loaded such as a good volatile solvent (such as methanol and ethanol) which is difficult to dissolve can be miscible with PAMAM-TPGS in the form of a solution, if the drug to be loaded adopted by the invention is curcumin, the curcumin is miscible in the form of an acetone solution of the curcumin, and the concentration of the acetone solution of the curcumin is 1-5 mg/mL (preferably 4 mg/mL).
Further, ultrasonic mixing is adopted for mixing and dissolving, and the ultrasonic mixing and dissolving time is 5-50 min, particularly preferably 20min.
The dendritic polymer PAMAM-TPGS administration nanoparticle prepared by the method has the average particle size of 10-50 nm and the polydispersity index (PDI) of 0. About 2 percent, and the drug loading is 5 to 17 percent.
Compared with the prior art, the invention has the following effective benefits: the average grain diameter of the dendritic polymer PAMAM-TPGS administration nanoparticle prepared by the invention is 10-120 nm, the polydispersity index (PDI) is less than 0.30, and the drug loading is 6-20%. When the PAMAM-TPGS nano-particles are prepared by a film dispersion method, the medicine-carrying nano-particles with different particle sizes are obtained by changing the preparation processes such as ultrasonic mixing time and the like, and the medicine-carrying amount is the highest; when PAMAM-TPGS nanoparticles are prepared by a microchannel continuous granulation technology, the obtained nano system has better stability and inferior drug loading, and is easy for industrialized production; when PAMAM-TPGS nanoparticles are prepared by an emulsion solvent volatilization method, the formed nano system has good stability, is not easy to precipitate and separate out, but has the lowest drug-loading rate. The dendritic polymer PAMAM-TPGS administration nanoparticles prepared by different methods obviously improve the solubility of model drugs in water, have slow release characteristics, have obvious anti-tumor effect, and are suitable for various administration modes such as intravenous injection and the like. The invention combines the two points, the grafting of TPGS not only reduces strong cell toxicity generated by positive charge of PAMAM, but also has the effect of inhibiting multi-drug resistance, and the curcumin (a natural medicine extract with good biocompatibility and certain effect in the aspects of tumor resistance, inflammation resistance, neocoronene resistance and the like) which also has the effect of inhibiting multi-drug resistance is coated by the hydrophobic bond, so that the effect of the synergistic enhancement system on the multi-drug resistance of tumors is realized.
(IV) description of the drawings
FIG. 1: a synthetic route of PAMAM-TPGS; wherein R is TPGS-CONH-.
FIG. 2 is a schematic diagram: method for producing PAMAM-TPGS 1 H-NMR。
FIG. 3: fourier transform infrared spectroscopy of PAMAM-TPGS
FIG. 4: a. microchannel plate installation schematic; b. microchannel plate structure schematic diagram
FIG. 5: TEM transmission electron micrograph
FIG. 6: malvern particle size distribution diagram
FIG. 7 is a schematic view of: nanoparticle solution release profile
FIG. 8: anti-proliferation effect chart of A549 cells
(V) detailed description of the preferred embodiments
The present invention is further illustrated by the following specific examples, but the scope of the invention is not limited thereto.
The model drugs in the examples below are exemplified by curcumin, which is purchased from alatin.
In the following examples, the finally obtained solution of dendrimer PAMAM-TPGS administration nanoparticles was measured for nanoparticle size and polydispersity index (PDI) by a particle sizer, the content of the model drug in the solution was measured by UV, and the Drug Loading (DL) was calculated.
Figure BDA0003428018990000051
W 1 : total model drug mass in nanoparticle solution
W 2 : quality of PAMAM-TPGS in nanoparticle solution.
In vitro release: in order to simulate the in vitro release of drug-loaded nanoparticles in a tumor microenvironment, a model drug DMSO solution (model drug/DMSO) and a model drug/PAMAM-TPGS nanoparticle are respectively diluted to 100 μ g/mL (drug concentration) with purified water, 2mL of suspension is taken to be put in a dialysis bag (MW =14000 Da), 200mL of phosphate buffer (pH 7.4, 0.01M) containing 0.5% of Tween 80 is taken as a release medium, sustained release is carried out in a constant-temperature shaking box (37 ℃,100 rpm), and 5mL of the release medium is taken out at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 100h, 124h and time points to determine the model drug concentration therein, and the same volume of fresh release medium is supplemented to ensure that the volume of the release medium is not changed. And (3) measuring the content Cn of the model drug in the release solution by using an ultraviolet spectrophotometer, and calculating the cumulative release rate of the drug according to a formula.
Figure BDA0003428018990000061
C n -the concentration of the model drug in the nth spot release medium, μ g/mL;
C i -the sum of the concentrations of the model drugs in the release medium after i spotting, μ g/mL;
v-total volume of release medium, mL;
vo-volume sampled each time, mL;
m Cur the mass of the model drug in the nanoparticle before release, mg.
Anti-proliferation assay for A549 cells
The anti-proliferation capacity of the drug-loaded nanoparticles on lung cancer cells A549 is measured and examined by an MTT method, the A549 cells in the logarithmic growth period are inoculated into a 96-well plate at 5000/well, and the culture is incubated for 24 hours in A5-CO2 incubator at 37 ℃. Model drug/DMSO and model drug/PAMAM-TPGS NPS were diluted to 0, 5, 10, 20, 40 and 60 μ g/mL with fresh medium and added to a 96-well plate containing a549 cells at 100 μ L per well in 5 replicates of each concentration. Fresh medium was set as a control group (cell survival rate 100%) and cell-free wells were blank. After 24 hours of incubation, 10. Mu.L of 5mg/ml MTT medium was added, incubation was continued for 4 hours, the old medium was discarded, 150. Mu.L of DMSO was added, shaking was carried out for 10 minutes, and the absorbance (OD value) was measured at a wavelength of 490nm on a microplate reader. The results are shown in FIG. 8
The cell survival rate calculation formula is as follows:
Figure BDA0003428018990000062
in the formula, OD Experimental group -OD values of experimental groups;
OD blank group -OD value of blank set;
OD control group -OD value of control group.
Reagents and abbreviations used in the examples
Vitamin E succinate polyethylene glycol 1000 TPGS
Succinic anhydride SA
N, N-diisopropylethylamine DIPEA
4-dimethylaminopyridine DMAP
1,4 dioxane
Methylene dichloride DCM
2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate HATU
N-hydroxy-5-norbornene-2, 3-dicarboximides HONB
N, N' -diisopropylcarbodiimide DIC
Dimethyl sulfoxide DMSO
Dendritic polymeric networks PAMAM
Example 1 Synthesis of TPGS-COOH
TPGS (0.3mmol, 459mg), SA (0.6mmol, 60mg), DIPEA (0.9mmol, 160. Mu.l) and DMAP (0.06mmol, 7.2mg) are dissolved in 15mL of 1, 4-dioxane, and the mixture is reacted for 24 hours at room temperature under the protection of nitrogen. Removing the solvent by rotary evaporation, dissolving with 10ml of cold dichloromethane (0 ℃) solvent, dripping into 250ml of diethyl ether (-20 ℃) to precipitate, centrifuging to obtain precipitate, placing in a vacuum drying oven to dry to constant weight to obtain TPGS-COOH, and obtaining 32mg of product with the final yield of 6.5%.
Example 2 Synthesis of TPGS-COOH
TPGS (0.3mmol, 459mg), SA (0.6mmol, 60mg), DIPEA (0.9mmol, 160. Mu.l) and DMAP (0.06mmol, 7.2mg) are dissolved in 15mL of 1, 4-dioxane, and the mixture is reacted for 24 hours at room temperature under the protection of nitrogen. Removing the solvent by rotary evaporation, dissolving with 10ml of cold dichloromethane solvent, dripping into 250ml of glacial ethyl ether, precipitating, performing suction filtration under reduced pressure, placing the precipitate in a vacuum drying oven, drying to constant weight to obtain TPGS-COOH, and obtaining 25mg of product with the final yield of 5.1%. Example 3 Synthesis of TPGS-COOH
TPGS (0.3mmol, 459mg), SA (0.6mmol, 60mg), DIPEA (0.9mmol, 160. Mu.l) and DMAP (0.06mmol, 7.2mg) are dissolved in 15mL of 1, 4-dioxane, and the mixture is reacted for 24 hours at room temperature under the protection of nitrogen. Removing the solvent by rotary evaporation, dissolving the solvent by using 10ml of cold dichloromethane (0 ℃), adding citric acid aqueous solution (PH = 5-6), extracting for three times, washing for three times by using distilled water, collecting an organic phase, removing the organic solvent by rotary evaporation, drying the organic phase in a vacuum drying oven to constant weight to obtain TPGS-COOH, and obtaining 453mg of product with the final yield of 92.63%.
Example 4 Synthesis of PAMAM-TPGS
TPGS-COOH (0.15mmol, 244mg), HONB (0.18mmol, 33mg) and DIC (0.18mmol, 28. Mu.l) were dissolved in 6ml of methylene chloride, and reacted in an ice-water bath for 2 hours and at room temperature for 3 hours to obtain an activated ester of TPGS. The activated ester was spun dry, PAMAM (0.0075mmol, 106.5mg), DMAP (0.06mmol, 7.4 mg), DIPEA (0.6mmol, 105. Mu.l) were added and dissolved in 6ml of DMSO solution under nitrogen protection, and the reaction was carried out at room temperature for 72 hours. Dialyzing the reaction solution by using a 14kDa dialysis bag and double distilled water for 4d, and freeze-drying for 12h to obtain PAMAM-TPGS, wherein the obtained product is 87.3mg, and the final yield is as follows: 24.91 percent.
Example 5 Synthesis of PAMAM-TPGS
TPGS-COOH (0.15mmol, 244mg), DIPEA (0.45mmol, 78. Mu.l), HATU (0.18mmol, 68.5mg), and PAMAM (0.0075mmol, 106.5mg) were dissolved in 6ml of a DMSO solution and reacted at normal temperature for 24 hours under nitrogen protection. Dialyzing the reaction solution by using a 14kDa dialysis bag and double distilled water for 4d, and freeze-drying for 12h to obtain PAMAM-TPGS, and collecting a product 132.3mg, wherein the final yield is as follows: 37.75 percent.
Example 6 preparation of nanoparticles by thin film Dispersion
0.25ml of 4mg/ml curcumin acetone solution and 5mg of the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) prepared in example 7 are dissolved in 0.25ml methanol, mixed and dissolved in a round-bottomed flask by ultrasound for 20min to serve as a lipid phase, and then the lipid phase is spin-dried for 20min at 35 ℃; taking 0.2mg/ml TPGS aqueous solution as a water phase; adding 2ml of TPGS aqueous solution into a round-bottom flask, ultrasonically dissolving for 30min, then centrifuging at 6000r/min, and taking supernate to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) administration nanoparticle.
TABLE 1 preparation of nanoparticles by film Dispersion
Figure BDA0003428018990000071
Example 7 preparation of nanoparticles by thin film Dispersion
0.25ml of 4mg/ml curcumin acetone solution and 5mg of the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) prepared in example 7 are dissolved in 0.25ml methanol, mixed and dissolved in a round-bottomed flask for 20min by ultrasound as a lipid phase, and spin-dried at 35 ℃ for 20min; taking 0.2mg/ml TPGS aqueous solution as a water phase; adding 2ml of TPGS aqueous solution into a round-bottom flask, ultrasonically dissolving for 90min, then centrifuging at 6000r/min, and taking supernate to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) administration nanoparticles;
example 8 preparation of nanoparticles by emulsion solvent volatilization method
0.25ml of 4mg/ml curcumin acetone solution and 5mg of the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) prepared in example 7 are dissolved in 0.25ml of methanol and ultrasonically mixed for 20min to serve as a lipid phase; taking 0.2mg/ml TPGS water solution as a water phase; and (3) dripping 0.5ml of the obtained lipid phase into 2ml of the water phase, stirring for 3h after dripping, drying overnight in vacuum, centrifuging at 6000r/min, and taking supernatant to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) administration nanoparticle.
EXAMPLE 9 preparation of nanoparticles by Microchannel continuous granulation
Nanoparticles were prepared in the apparatus shown in fig. 4 a. The microchannel of the device is customized to Suzhou Central open Hengchang scientific instruments, inc., the width of the main channel of the microchannel is 360 mu m, and the length of the main channel is 340mm; the width of the branch channel is 590 mu m, and the distance from the inlet of the main channel is 60mm; n is a radical of 2 Width of the channel: 470 μm, and 40mm from the side channels. Microfluidic syringe pumps were from HA Harvard Apparatus.
0.25ml of 4mg/ml curcumin acetone solution and 5mg of the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) prepared in example 7 are dissolved in 0.25ml of methanol and ultrasonically mixed for 20min to serve as a lipid phase; taking 0.2mg/ml TPGS water solution as a water phase; loading a lipid phase into a 1mL needle tube and loading the needle tube on a flow rate pump, loading a TPGS aqueous phase into two 2mL needle tubes and loading the two solutions on the flow rate pump, enabling the two solutions to enter a microchannel plate by controlling the flow rate of the lipid phase of a main channel to be 1mL/min and the flow rates of the aqueous phases of two branch channels to be 1mL/min respectively, preparing CUR/PAMAM-TPGS nanoparticles under N2 protection (0.25 mL/min), simultaneously stirring the mixed solution flowing out of the microchannel at a constant speed for 30min at a constant temperature, carrying out vacuum drying for 3h, carrying out centrifugation at 600r/min, and taking supernate to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000 (PAMAM-TPGS) administration nanoparticles.
The results are shown in Table 2
Table 2 examples 7-9 Effect of different preparation methods on nanoparticle Properties
Figure BDA0003428018990000081
The above results show that the last ultrasonic mixing time of the film dispersion method has obvious influence on the state of the nanoparticles, and along with the increase of ultrasonic time, the nanoparticles can be gathered, the particle size is increased to 103nm by 37nm, and PDI is reduced, and the electric potential is increased, and supposedly under the ultrasonic action, the nanoparticles tend to the more uniform and stable state existence, but the corresponding can be because of the settlement of the part of the drug that the part loading state is not good, so that the drug loading capacity is reduced. Compared with an emulsion solvent volatilization method and a microchannel continuous granulation technology, the nano-particles prepared by a film dispersion method have higher drug loading, but the state is easy to change by ultrasonic time, and the nano-particles with proper particle size can be selected to prepare by controlling the factor. The three nanoparticle solutions were stored separately and observed for drug precipitation at intervals. As a result, it was found that: the nanoparticles prepared by the emulsifying solvent volatilization method and the microchannel continuous granulation technology are more stable and are not easy to separate out.
Fig. 5 and 6 are a transmission electron microscope image and a malvern particle size distribution image of the drug-loaded nanoparticles, respectively. It can be seen that the drug-loaded nanoparticles are uniform in size and spherical, the particle size is about 120nm, and the electron micrograph is consistent with the test result of a Malvern particle sizer.
Example 10 drug Release Profile of drug-loaded nanoparticles (nanoparticles prepared by film Dispersion method for example)
To simulate the in vitro release of drug-loaded nanoparticles in a tumor microenvironment, a DMSO solution of curcumin (Cur/DMSO, curcumin is insoluble in water, so DMSO is used as a solvent) and Cur/PAMAM-TPGS nanoparticles prepared in example 7 were diluted with purified water to a curcumin concentration of 100 μ g/mL, 2mL of suspensions were taken in dialysis bags (MW =14000 Da), 200mL of PBS buffer (pH 7.4, 0.01m) containing 0.5% tween 80 was used as a release medium, sustained release was performed in a constant temperature shaking chamber (37 ℃,100 rpm), 5mL of release medium was taken at time points of 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 100h, 124h, 148h, and 172h to determine the curcumin concentration therein, and the same volume of fresh release medium (PBS buffer) was supplemented to ensure that the volume of the release medium was constant. And (3) measuring the content Cn of the curcumin in the release solution by using an ultraviolet spectrophotometer, and calculating the cumulative release rate of the medicine according to a formula. The results are shown in FIG. 7.
Figure BDA0003428018990000091
/>
C n -the concentration of curcumin in the nth spot release medium, μ g/mL;
C i -the sum of the curcumin concentrations in the release medium after i spotting, μ g/mL;
v-total volume of release medium, mL;
vo-volume sampled each time, mL;
m Cur -curcumin mass in nanoparticles before release, mg.
As shown in figure 7, compared with a DMSO solution of curcumin, the PAMAM-TPGS nanoparticle loaded with curcumin has an obvious sustained-release effect in an in-vitro simulation environment and has a higher cumulative release rate.
In vitro anti-proliferation study of A549 cells (nanoparticles prepared by thin film dispersion method for example)
The anti-proliferation capacity of the drug-loaded nanoparticles on lung cancer cells A549 is measured and examined by an MTT method, the A549 cells in the logarithmic growth period are inoculated into a 96-well plate at 5000/well, and the culture is incubated for 24 hours in A5-CO2 incubator at 37 ℃. Cur/DMSO, PAMAM-TPGS NPS and CUR/PAMAM-TPGS NPS were diluted to 0, 5, 10, 20, 40 and 60. Mu.g/mL with fresh medium and added to a 96-well plate containing A549 cells, 100. Mu.L per well, in 5 replicates at each concentration. Fresh medium was set as a control group (cell viability: 100%) and cell-free wells were blank. After 24 hours of incubation, 10. Mu.L of 5mg/ml MTT medium was added, incubation was continued for 4 hours, the old medium was discarded, 150. Mu.L of DMSO was added, shaking was carried out for 10 minutes, and the absorbance (OD value) was measured at a wavelength of 490nm on a microplate reader. The results are shown in FIG. 8
The cell survival rate calculation formula is as follows:
Figure BDA0003428018990000092
in the formula, OD Experimental group -OD value of experimental group;
OD blank group -OD value of blank set;
OD control group -OD value of control group.
The result of fig. 8 shows that the blank PAMAM-TPGS nanoparticle without drug loading has a certain inhibition effect on tumor cells, has obvious cytotoxicity on a549 cells when reaching about 200 μ g/mL, and compared with the free curcumin group, the CUR/PAMAM-TPGS nanoparticle has obvious cytotoxicity, which indicates that the drug-loaded nanoparticle can better kill tumors.

Claims (18)

1. The application of dendritic polymer-vitamin E succinate polyethylene glycol 1000 as a drug carrier in preparing a pharmaceutical preparation is characterized in that the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is prepared by the following method:
(1) Dissolving vitamin E succinate polyethylene glycol 1000, succinic anhydride, N-diisopropylethylamine A and 4-dimethylaminopyridine in 1, 4-dioxane, reacting at room temperature for 8-48h under a protective atmosphere A, and performing aftertreatment on the obtained reaction liquid A to obtain TPGS-COOH;
the ratio of the vitamin E succinate polyethylene glycol 1000 to the succinic anhydride to the N, N-diisopropylethylamine A to the 4-dimethylaminopyridine is 1:2 to 20:0.1 to 1;
(2) Dissolving the TPGS-COOH, the N, N-diisopropylethylamine B, the 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate and the PAMAM dendrimer in the step (1) in dimethyl sulfoxide, stirring until the TPGS-COOH, the N, N-diisopropylethylamine B and the 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate are completely dissolved, reacting for 8-48h at normal temperature in a protective atmosphere B, and performing post-treatment on the obtained reaction liquid B to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000;
the mass ratio of TPGS-COOH, N, N-diisopropylethylamine B, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate to PAMAM dendrimer is 1:1 to 10:1 to 10:0.01 to 1;
the application is as follows: mixing a drug to be loaded with the dendritic polymer-vitamin E succinate polyethylene glycol 1000, dissolving in a volatile good solvent, spin-drying, adding TPGS aqueous solution, ultrasonically dissolving, centrifuging, and taking supernatant, namely the drug preparation;
the mass ratio of the drug to be loaded to the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is 1 to 20; the volume of the volatile good solvent is 0.01-0.1mL/mg based on the mass of the dendritic polymer-vitamin E succinate polyethylene glycol 1000; the concentration of TPGS in the TPGS aqueous solution is 0.01 to 5mg/ml; the volume of the TPGS aqueous solution is 0.1-1mL/mg based on the mass of the dendrimer-vitamin E succinate polyethylene glycol 1000.
2. The use of claim 1, wherein: the protective atmosphere A and the protective atmosphere B are respectively and independently inert gas or nitrogen.
3. The use of claim 1, wherein: the volume of the 1,4 dioxane in the step (1) is 10 to 50mL/g based on the mass of the vitamin E succinate polyethylene glycol 1000.
4. The use according to claim 1, characterized in that in step (1) the post-treatment a is: and (3) removing the solvent from the obtained reaction solution A by rotary evaporation, redissolving dichloromethane to obtain a crude product, washing the crude product with a citric acid aqueous solution with the pH =5-6, washing an organic phase with distilled water, and carrying out rotary evaporation on the obtained organic phase to obtain the TPGS-COOH.
5. The use of claim 1, wherein: and (3) in the step (2), the volume of the dimethyl sulfoxide is 5 to 50mL/g based on the mass of the TPGS-COOH.
6. The use according to claim 1, characterized in that the post-treatment B in step (2) is: and dialyzing the obtained reaction solution B in double distilled water by using a 1.4kDa dialysis bag for 1 to 10 days, and freeze-drying to obtain the dendritic polymer-vitamin E succinate polyethylene glycol 1000.
7. The application of dendritic polymer-vitamin E succinate polyethylene glycol 1000 as a drug carrier in preparing a pharmaceutical preparation is characterized in that the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is prepared by the following method:
(1) Dissolving vitamin E succinate polyethylene glycol 1000, succinic anhydride, N-diisopropylethylamine A and 4-dimethylaminopyridine in 1, 4-dioxane, reacting at room temperature for 8-48h under a protective atmosphere A, and performing aftertreatment on the obtained reaction liquid A to obtain TPGS-COOH;
the ratio of the vitamin E succinate polyethylene glycol 1000 to the succinic anhydride to the N, N-diisopropylethylamine A to the 4-dimethylaminopyridine is 1:2 to 20:0.1 to 1;
(2) Dissolving the TPGS-COOH, the N, N-diisopropylethylamine B, the 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate and the PAMAM dendrimer in the step (1) in dimethyl sulfoxide, stirring until the TPGS-COOH, the N, N-diisopropylethylamine B and the 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate are completely dissolved, reacting for 8-48h at normal temperature in a protective atmosphere B, and performing post-treatment on the obtained reaction liquid B to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000;
the mass ratio of TPGS-COOH, N, N-diisopropylethylamine B, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate to PAMAM dendrimer is 1:1 to 10:1 to 10:0.01 to 1;
the application is as follows: mixing a drug to be loaded with the dendritic polymer-vitamin E succinate polyethylene glycol 1000, dissolving in a good volatile solvent, dropwise adding the obtained lipid phase into a TPGS aqueous solution, stirring for 1-3 h after dropwise adding, vacuum drying to remove an organic solvent, centrifuging, and taking a supernatant, namely the drug preparation;
the mass ratio of the drug to be loaded to the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is 1 to 20; the volume of the volatile good solvent is 0.01-0.1mL/mg based on the mass of the dendritic polymer-vitamin E succinate polyethylene glycol 1000; the concentration of TPGS in the TPGS aqueous solution is 0.01 to 5mg/ml; the volume of the TPGS aqueous solution is 0.1-1mL/mg based on the mass of the dendrimer-vitamin E succinate polyethylene glycol 1000.
8. The use of claim 7, wherein: the protective atmosphere A and the protective atmosphere B are respectively and independently inert gas or nitrogen.
9. The use of claim 7, wherein: the volume of the 1, 4-dioxane in the step (1) is 10 to 50mL/g based on the mass of the vitamin E succinate polyethylene glycol 1000.
10. The use according to claim 7, characterized in that the post-treatment A in step (1) is: and (3) removing the solvent from the obtained reaction solution A by rotary evaporation, redissolving dichloromethane to obtain a crude product, washing the crude product with a citric acid aqueous solution with the pH =5-6, washing an organic phase with distilled water, and carrying out rotary evaporation on the obtained organic phase to obtain the TPGS-COOH.
11. The use of claim 7, wherein: and (3) in the step (2), the volume of the dimethyl sulfoxide is 5 to 50mL/g based on the mass of the TPGS-COOH.
12. The use according to claim 7, characterized in that the post-treatment B in step (2) is: and dialyzing the obtained reaction solution B in double distilled water by using a 1.4kDa dialysis bag for 1 to 10 days, and freeze-drying to obtain the dendritic polymer-vitamin E succinate polyethylene glycol 1000.
13. The application of dendritic polymer-vitamin E succinate polyethylene glycol 1000 as a drug carrier in preparing a pharmaceutical preparation is characterized in that the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is prepared by the following method:
(1) Dissolving vitamin E succinate polyethylene glycol 1000, succinic anhydride, N-diisopropylethylamine A and 4-dimethylaminopyridine in 1, 4-dioxane, reacting at room temperature for 8-48h in a protective atmosphere A, and performing aftertreatment A on the obtained reaction liquid A to obtain TPGS-COOH;
the weight ratio of the vitamin E succinate polyethylene glycol 1000, succinic anhydride, N-diisopropylethylamine A to 4-dimethylaminopyridine is (1): 2 to 20:0.1 to 1;
(2) Dissolving the TPGS-COOH, the N, N-diisopropylethylamine B, the 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate and the PAMAM dendrimer in the step (1) in dimethyl sulfoxide, stirring until the TPGS-COOH, the N, N-diisopropylethylamine B and the 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate are completely dissolved, reacting for 8-48h at normal temperature in a protective atmosphere B, and performing post-treatment on the obtained reaction liquid B to obtain the dendrimer-vitamin E succinate polyethylene glycol 1000;
the mass ratio of TPGS-COOH, N, N-diisopropylethylamine B, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate to PAMAM dendrimer is 1:1 to 10:1 to 10:0.01 to 1;
the application is as follows: the pharmaceutical preparation is prepared using a microchannel plate comprising a main channel having a diameter of 300-420 μm, a first side channel having a diameter of 530-650 μm, a second side channel, and an N having a diameter of 390-550 μm 2 A channel; the two ends of the main channel are respectively provided with a main channel inlet and a main channel outlet, and the main channel is also provided with N at a position 200-280mm away from the main channel outlet 2 Entrance and two distances from said N 2 A first aqueous phase inlet and a second aqueous phase inlet having inlets of 30-50 mm; said N is 2 Channel and said N 2 The first branch channel and the second branch channel are respectively connected with the first water phase inlet and the second water phase inlet;
mixing a drug to be loaded with the dendritic polymer-vitamin E succinate polyethylene glycol 1000 in a volatile good solvent to obtain a lipid phase, and taking a TPGS aqueous solution as a water phase; the mass ratio of the drug to be loaded to the dendritic polymer-vitamin E succinate polyethylene glycol 1000 is 1 to 20; the volume of the volatile good solvent is 0.01-0.1mL/mg based on the mass of the dendritic polymer-vitamin E succinate polyethylene glycol 1000; the concentration of TPGS in the TPGS aqueous solution is 0.01 to 5mg/ml;
the lipid phase enters the microchannel plate from the inlet of the main channel at a flow rate of 1-10mL/h, the aqueous phase enters the microchannel plate from the first branch channel and the second branch channel at a flow rate of 1-10mL/h, respectively, and nitrogen or inert gas enters the microchannel plate from the N 2 The channel is communicated into the microchannel plate; collecting the mixed liquid flowing out of the main channel outlet, simultaneously stirring at a constant speed for 10 to 60min at room temperature, vacuum drying for 3 to 10h, centrifuging at 3000 to 10000r/min, and taking the supernatant, namely the medicinal preparation.
14. The use of claim 13, wherein: the protective atmosphere A and the protective atmosphere B are respectively and independently inert gas or nitrogen.
15. The use of claim 13, wherein: the volume of the 1, 4-dioxane in the step (1) is 10 to 50mL/g based on the mass of the vitamin E succinate polyethylene glycol 1000.
16. The use according to claim 13, characterized in that in step (1) the post-treatment a is: and (3) removing the solvent from the obtained reaction solution A by rotary evaporation, redissolving dichloromethane to obtain a crude product, washing the crude product with a citric acid aqueous solution with the pH =5-6, washing an organic phase with distilled water, and carrying out rotary evaporation on the obtained organic phase to obtain the TPGS-COOH.
17. The use of claim 13, wherein: and (3) in the step (2), the volume of the dimethyl sulfoxide is 5 to 50mL/g based on the mass of the TPGS-COOH.
18. The use according to claim 13, characterized in that the post-treatment B in step (2) is: and dialyzing the obtained reaction liquid B in double-distilled water by using a 1.4kDa dialysis bag for 1 to 10 days, and freeze-drying to obtain the dendritic polymer-vitamin E succinate polyethylene glycol 1000.
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