CN110664751A - PH responsive polymer nano micelle and preparation and application thereof - Google Patents
PH responsive polymer nano micelle and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a pH responsive polymer nano micelle, which is characterized in that C7A is used as a base material, a hydrophobic macromolecule PC7A polymer is prepared through RAFT polymerization, then PEG is used for chain extension of PC7A to obtain a PC7A-PEG polymer, VI and PBA monomers are used for chain extension of PC7A-PEG to obtain a block compound PC7A-PEG-VI-PBA, and finally, the block compound PC7A-PEG-VI-PBA is coordinated with quantum dots CdSeQDs to obtain a CdSeTe @ PC7A-PEG-VI-PBA polymer micelle. In vitro cytotoxicity research shows that the polymer nano-micelle has good pH responsiveness and biocompatibility, can be released in a tumor cell weak acid environment, and is expected to become a good drug delivery system.
Description
Technical Field
The invention relates to a pH responsive polymer nano micelle and a preparation method thereof; the invention also relates to application of the pH responsive polymer nano micelle as a drug delivery carrier, belonging to the field of nano drug-loaded carriers.
Background
In recent years, polymeric micelles have been considered as ideal nano drug delivery vehicles because they have Enhanced Permeability and Retention (EPR) effects, are capable of prolonging blood circulation and can be passively targeted to solid tumors. The design of polymer micelles is mainly based on stimuli-responsive nanomaterials. In the tumor microenvironment, the polymer micelle can undergo a series of changes (such as surface charge conversion, ligand activation and the like), so that efficient drug release is realized, and the tumor is ablated. A common method of constructing stimulus-responsive materials is to use responsive chemical bonds (e.g., disulfide, acetal, hydrazone, etc.). However, most chemical bonds have the disadvantages of low stability under physiological conditions and low sensitivity to the tumor microenvironment.
In order to make the nano-micelle have good stability under physiological conditions, the nano-micelle is generally prepared by adopting a special material of which non-responsive chemical bonds can carry out hydrophilic-hydrophobic conversion. To more accurately locate the distribution of the drug delivery system in the organism, fluorescent substances are often loaded into the system for imaging. Quantum dots have been widely used for bioluminescence imaging due to their particular advantages. The nano micelle has excellent performances of strong hydration performance, charge reversal, easy chemical modification and the like, and has longer circulation half-life and good biocompatibility. Therefore, the nano drug delivery system is expected to become an attractive drug delivery system.
Disclosure of Invention
The invention aims to provide a pH responsive polymer nano micelle and a preparation method thereof;
the invention also aims to research the drug loading capacity, the drug loading efficiency, the drug release performance and the in vitro cytotoxicity of the pH responsive polymer nano micelle, so that the pH responsive polymer nano micelle can be used as a drug delivery carrier for delivering tumor targeted drugs.
pH responsive polymer nano micelle and preparation thereof
The preparation method of the pH responsive polymer nano micelle comprises the following steps:
(1) the preparation of the C7A monomer compound comprises the steps of reacting 2- (hexamethylene imino) -ethyl methacrylate (C7A) with triethylamine and methacryloyl chloride in dichloromethane at room temperature for 20 ~ 24 hours, washing a reaction product with anhydrous sodium carbonate, and removing water with anhydrous magnesium sulfate to obtain the C7A monomer compound;
the molar ratio of the 2- (hexamethylene imino) -ethyl methacrylate to the triethylamine is 1: 1; the molar ratio of 2- (hexamethyleneimino) -ethyl methacrylate to methacryloyl chloride is 1: 1.
The structural formula of the obtained C7A monomer compound is as follows:
the NMR spectrum and NMR spectrum of the C7A monomer compound are shown in FIG. 2.
(2) Synthesizing a PC7A polymer, namely under the protection of nitrogen, in dimethyl sulfoxide, taking Azobisisobutyronitrile (AIBN) as an initiator, carrying out polymerization reaction on a C7A monomer compound and a RAFT reagent DMP for 20 ~ 24 hours at 50 ~ 60 ℃, dialyzing by using a dialysis bag with the molecular weight cutoff of 3000 after the polymerization is finished to remove impurities, and carrying out reduced pressure distillation to obtain the PC7A polymer;
the molar ratio of the C7A monomer compound to the RAFT reagent DMAP was 30: 1, and the amount of AIBN used was 0.3 ~ 0.4.4% of the molar amount of the C7A monomer compound.
The structural formula of RAFT reagent 2- (dodecylthio thiocarbonylthio) -2-methylpropanoic acid (DMP) is as follows:
the NMR spectrum of the PC7A polymer is shown in FIG. 3, and the structural formula is:
(3) synthesizing a PC7A-PEG polymer, namely under the protection of nitrogen, in dimethyl sulfoxide solution, taking Azobisisobutyronitrile (AIBN) as an initiator, carrying out polymerization reaction on PC7A and polyethylene glycol methyl methacrylate (PEG) for 20 ~ 24 hours at 50 ~ 60 ℃, after the polymerization is finished, dialyzing by using a dialysis bag with the molecular weight cutoff of 3000 to remove impurities, and carrying out reduced pressure distillation to obtain the PC7A-PEG polymer;
the molar ratio of the PC7A polymer to polyethylene glycol methyl methacrylate (PEG) was 1:15, and the amount of AIBN initiator used was 20 ~ 25% of the molar amount of the PC7A polymer.
The NMR spectrum of the PC7A-PEG polymer is shown in FIG. 4, and the structural formula is:
(4) the compound PC7A-PEG-VI-PBA is synthesized by reacting PC7A-PEG with 1-Vinylimidazole (VI) and 4-vinylphenylboronic acid (PBA) in dimethyl sulfoxide solution at 50 ~ 60 ℃ for 20 ~ 24 hours under the protection of nitrogen by using Azobisisobutyronitrile (AIBN) as an initiator, dialyzing by using a dialysis bag with the molecular weight cutoff of 3000 after polymerization is completed to remove impurities, and distilling under reduced pressure to obtain the PC7A-PEG-VI-PBA polymer.
The molar ratio of 1-Vinylimidazole (VI) to PC7A-PEG was 10: 1. The structural formula of the 1-Vinyl Imidazole (VI) is as follows:
the molar ratio of 4-vinylphenylboronic acid (PBA) to PC7A-PEG was 5: 1. The structural formula of 4-vinylphenylboronic acid (PBA) is:
FIG. 5 is a NMR hydrogen spectrum of a prepared PC7A-PEG-VI-PBA polymer. From FIG. 51It can be seen from the H-NMR spectrum that the parent isThe ratio of hydrophobicity (molecular weight) is 1: 1; in PC7A-PEG-VI-PBA1The proton signals of 1-Vinylimidazole (VI) and 4-vinylphenylboronic acid (PBA) are visible in the H NMR spectrum. The material content and the molecular weight are calculated by integration at δ 4.0. Mn =9664.06g/mol (PC 7A)22-PEG10-VI5-PBA2). The structural formula is as follows:
(5) the preparation of the CdSeTe @ PC7A-PEG-VI-PBA polymer comprises the steps of dissolving PC7A-PEG-VI-PBA in DMSO, adding an aqueous solution of CdSeTe QDs, stirring for 20 ~ 24 hours, dialyzing in distilled water for 20 ~ 24 hours by a dialysis bag with the molecular weight cutoff of 3000 to remove the uncoordinated CdSeQDs, and carrying out vacuum distillation to obtain the CdSeTe @ PC 7A-PEG-VI-PBA.
The mass ratio of PC7A-PEG-VI-PBA to CdSeTe QDs is 4: 1.
The structural formula of the CdSeTe @ PC7A-PEG-VI-PBA is as follows:
wherein a =22, b =10, c =5, d = 2; number average molecular weight Mn =9664.06 g/mol;representing CdSeTeQDs.
FIG. 6 is a graph showing the UV absorption spectrum of CdSeTe @ PC 7A-PEG-VI-PBA. As can be seen from FIG. 6, the characteristic peak around 580nm indicates the presence of free CdSeTe QDs, and 535nm indicates the presence of CdSeTe @ PC 7A-PEG-VI-PBA. The absorption peak after coordination of the CdSeTe QDs is blue shifted by 45 nm compared with free CdSeTe QDs, because the coordination of the CdSeTe QDs with imidazole leads to n → pi transition.
Second, PC7A-PEG-VI-PBA performance test
1. pH responsiveness of PC7A-PEG-VI-PBA
Equal concentrations of PC7A-PEG-VI-PB solution were removed and placed in a series of tubes. And respectively adding phosphate buffer solutions with different pH values into each test tube, and respectively testing and recording the Ze-Ta potential value of the mixed liquid in each test tube after uniformly stirring by ultrasonic.
FIG. 7 is a graph of the pH responsiveness of PC7A-PEG-VI-PBA micelles prepared according to the present invention. It can be seen that the polymer shows a negative potential when it is placed in a phosphate buffered solution at pH 7.4. When the pH of the polymer buffer solution was 6.5, there was no significant change in the Ze-Ta of PC 7A-PEG-VI-PBA. When the pH of the polymer buffer solution was 6.0, the Ze-Ta potential of the polymer changed significantly and the potential changed from-5 mv to 17 mv. Indicating that the C7A chain becomes positively charged under weakly acidic conditions. C7A changes from a hydrophobic to a hydrophilic species, thereby disrupting the micelle.
2. Preparation and performance test of drug-loaded micelle
(1) Preparation of polymeric blank micelle
The prepared CdSeTe @ PC7A-PEG-VI-PBA (10 mg) is fully dissolved in 2 mL of DMSO, the mixed solution is dripped into deionized water at the speed of 1 drop for 15 seconds and stirred for 2 ~ 3 hours, and then the micelle solution is filled into a dialysis bag (MWCO = 3000) and placed into the deionized water for dialysis for 45 ~ 50 hours, so that the polymer blank micelle is obtained.
(2) Preparation of drug-loaded micelles
CdSeTe @ PC7A-PEG-VI-PBA (10 mg) and DOX (2 mg) were dissolved in 2 mL of DMSO. Then, slowly dripping (dripping 1 drop per 15 seconds) the mixed solution into 9 mL of distilled water by using a rubber head dropper under the dark condition, stirring for 24 hours, and then putting into a dialysis bag for dialysis for 24 hours; after dialysis, the mixed solution is equally divided into two parts, and the two parts are respectively placed in 100 mL phosphate buffer solution with pH =7.4 (100 mL) and acetic acid buffer solution with pH =5.0 (100 mL) for dialysis for 24h, so that the drug-loaded micelle DOX @ CdSeTe @ PC7A-PEG-VI-PBA is obtained. FIG. 8 is a Transmission Electron Micrograph (TEM) of drug loaded micelle DOX @ CdSeTe @ PC 7A-PEG-VI-PBA. Figure 8 shows that the micelles have a uniform spherical distribution with a size of 105 nm.
During the dialysis, 3mL of dialysate was taken at regular time intervals (1, 2, 4, 8, 12, 24 h) and then placed in 3mL of phosphate buffer. The absorbance of the collected dialysate at 485 nm was measured by a SolidSpec-3700/3700DUV type ultraviolet-visible spectrophotometer (UV/Vis) from Shimadzu corporation, Japan, and was substituted into a standard curve equation to calculate the DOX loading mass. FIG. 9 is a particle size distribution diagram of drug loaded micelle DOX @ CdSeTe @ PC 7A-PEG-VI-PBA. As can be seen from FIG. 9, the micelle had a particle size of 146nm and a PDI of 0.327. The size is different compared to TEM because the sample is lyophilized and dehydrated during the preparation process of the transmission electron microscope.
(3) Measurement of CMC
The Critical Micelle Concentration (CMC) is an important parameter to demonstrate the ability to self-assemble. The CMC value of the polymer was determined by using pyrene fluorescence probe technique. Preparing 10 a pyrene fluorescent probe-6Adding the solution with mmol/L concentration into a test tube, adding a series of blank micellar solutions with different concentrations into the test tube after acetone is completely volatilized, fully shaking for 2 h, and standing for 5 h. The critical micelle concentration of the micelle can be accurately measured by utilizing the transition of the fluorescence spectrum of the pyrene fluorescent probe when the critical micelle concentration is reached. Under the conditions of an excitation wavelength of 330 nm and an entrance slit of 5nm, emission peaks of a micellar solution of pyrene at 336 nm and 339 nm were measured. Through I336/I339The ratio between the two was determined to find the micelle CMC. FIG. 10 shows336/I339The change lg ρ is-3.16, indicating that pyrene is encapsulated in a hydrophobic core and its fluorescence value changes. It can be observed that the nanomicelles have a small CMC value (6.9X 10)-4mg/mL), indicating that the micelle does not readily dissociate even under conditions of high in vivo dilution.
(4) In vitro drug release properties of micelles
To determine the pH-dependent drug release profile, the in vitro release profile of DOX was recorded in PBS at pH7.4 (physiological conditions) and 5.0 (intracellular environment). As shown in FIG. 11, the release rate of micelles was significantly accelerated in the buffer solution at pH5.0, and the release was slow in the buffer solution at pH 7.4. This is due to the protonation of 2- (hexamethyleneimino) ethylmethacrylate in 5.0 PBS resulting in the release of DOX. Furthermore, in the buffer solution at ph7.4, DOX release was only 22.3% within 24 hours, whereas in the buffer solution at ph5.0, DOX release reached about 53.5% within the first 8 hours and 83.7% after 24 hours. This indicates that the drug carrier can circulate at the normal pH of the human body and release the drug under the acidic conditions of cancer cells.
(5) Endocytosis and drug release
Logarithmic phase B16F10 cells were harvested at 5X 10 cells per well3Individual cells were seeded in 96-well plates and incubated for 24h (37 ℃, 5% CO by volume fraction)2). The medium was removed and supplemented with 180. mu.L of fresh DMEM medium containing 10% FBS by volume, and 20. mu.L of DOX @ PC7A-PEG-VI-PBA and DOX @ CdSeTe @ PC7A-PEG-VI-PBA, respectively, were added and the cells were incubated for 4h, 10 h. The culture solution was removed, Hoechst 33342 was added, and photographed by observing with an inverted fluorescence microscope of the Japanese OLYMPUS IX71 type.
FIG. 12 shows fluorescence images of B16F10 cells incubated with DOX and DOX @ CdSeTe @ PC7A-PEG-VI-PBA micelles for 4 hours and 10 hours, respectively. After 4 hours DOX incubation, weak fluorescence was observed in the nuclei of B16F10 cells of DOX @ CdSeTe @ PC7A-PEG-VI-PBA group. This is weaker than the fluorescence of DOX. hcl group, probably because DOX easily enters cancer cells by direct diffusion, whereas DOX @ CdSeTe @ PC7A-PEG-VI-PBA micelles enter cells by endocytosis, requiring a longer time to release DOX. After 10 hours of culture, fluorescence of DOX in the nucleus of cells was significantly enhanced, since the release of DOX encapsulated in micelles increased with time. In addition, DOX is released in large amounts under acidic conditions, which also increases the drug concentration in the B16F10 cell nucleus. The result shows that the CdSeTe @ PC7A-PEG-VI-PBA nano micelle can wrap DOX and effectively deliver the DOX to the nucleus, and release the DOX in the weak acid environment of tumor cells.
(6) In vitro cytotoxicity
B16F10 cells are adopted to research the cytotoxicity of the empty carrier, DOX & HCl, DOX @ PC7A-PEG-VI-PBA, DOX @ CdSeTe @ PC7A-PEG-VI-PBA micelle through 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT). Cells were seeded in 96-well plates (5X 10 per well) in 100. mu.L of Darber modified eagle Medium (DMEM Medium) containing 15% Fetal Bovine Serum (FBS) by volume fraction3Individual cells) and maintained at 37 ℃ and volume fraction of5% CO2Culturing in a humid atmosphere for 24 h. The medium was removed and supplemented with 180. mu.L of fresh DMEM medium containing 10% FBS by volume, and 20. mu.L of empty vector (0.0312, 0.0625, 0.125, 0.25, 0.5, 1 mg/mL), DOX. HCl, DOX @ PC7A-PEG-VI-PBA, DOX @ CdSeTe @ PC7A-PEG-VI-PBA (all at 0.25, 0.05, 0.1, 0.5, 1, 5 ug/mL) were added to different wells and incubated for 48 h, respectively. Then, 10. mu.L of MTT reagent was added to each well, and the cells were further incubated for 2 h, and the Optical Density (OD) was measured as absorbance at 490 nm using a BioRad X-mark type microplate reader in the U.S.A. All experiments were repeated 3 times and cell viability was calculated according to the following formula:
FIG. 13 shows the cytotoxicity of PC7A-PEG-VI-PBA micelles. The free micelle shows low cytotoxicity in B16F10 cells, and the cell survival rate is 83% even after 48 hours of incubation at the highest concentration (1.0 mg/mL), which indicates that the PC7A-PEG-VI-PBA micelle is substantially nontoxic, has good biocompatibility and can be used as a safe nano-drug carrier.
FIG. 14 is an IC50 of free DOX, DOX @ PC7A-PEG-VI-PBA and DOX @ CdSeTe @ PC 7A-PEG-VI-PBA. As can be seen in FIG. 14, the IC50 value (1.43) for DOX @ PC7A-PEG-VI-PBA is lower than the IC50 (1.89) for free DOX, since the binding of PBA to sialic acid at the tumor site favors its entry into the cell and action on the nucleus. The IC50 value (1.25) of DOX @ CdSeTe @ PC7A-PEG-VI-PBA was similar to the IC50 value of DOX @ PC7A-PEG-VI-PBA, indicating that CdSeTe QDs were non-toxic.
In conclusion, the C7A is used as a base material, a hydrophobic macromolecule PC7A polymer is prepared through RAFT polymerization, then the PC7A polymer is subjected to chain extension at 60 ℃ by using PEG to obtain a PC7A-PEG polymer, then the PC7A-PEG is subjected to chain extension by using VI and PBA monomers to obtain a block compound PC7A-PEG-VI-PBA, and finally the block compound PC7A-PEG-VI-PBA is coordinated with quantum dots CdSeTe QDs to obtain a CdSeTe @ PC7A-PEG-VI-PBA polymer. Due to protonation of 2- (hexamethyleneimino) ethyl methacrylate (C7A), PC7A-PEG-VI-PBA nanomicelles at physiological pH are converted to positive charges under weakly acidic conditions, leading to micelle disruption and DOX release. In addition, in vitro cytotoxicity research shows that the PC7A-PEG-VI-PBA nano micelle has good biocompatibility. The drug-loaded micelle can effectively inhibit cell proliferation and promote apoptosis of B16F10 cells. Tumor targeting characteristics were assessed using fluorescence imaging, indicating that DOX @ CdSeTe @ PC7A-PEG-VI-PBA can be efficiently accumulated at the tumor site. Therefore, the compound can be used as a novel pH-responsive antitumor drug carrier in tumor cells.
Drawings
FIG. 1 shows NMR hydrogen and carbon spectra of RAFT reagents prepared according to the invention.
FIG. 2 shows NMR hydrogen spectra and NMR carbon spectra of C7A monomer prepared according to the present invention.
FIG. 3 shows the NMR spectra of PC7A polymer prepared according to the invention.
FIG. 4 shows the NMR spectrum of PC7A-PEG polymer prepared by the present invention.
FIG. 5 is a NMR spectrum of a PC7A-PEG-VI-PBA polymer prepared according to the present invention.
FIG. 6 is a UV absorption spectrum of CdSeTe @ PC7A-PEG-VI-PBA prepared by the present invention.
FIG. 7 is a graph of the pH responsiveness of PC7A-PEG-VI-PBA micelles prepared according to the present invention.
FIG. 8 is a transmission electron micrograph of the micelle carrier of the present invention.
FIG. 9 is a graph showing the distribution of the particle size of the micelle carrier of the present invention.
FIG. 10 is a graph of the critical micelle concentration of the micelle carriers of the present invention.
Fig. 11 is an in vitro simulated profile of doxorubicin release at pH =5.0 and pH =7.4 for drug-loaded micelles of the invention.
FIG. 12 is a fluorescence image of B16F10 cells incubated with DOX and DOX @ CdSeTe @ PC7A-PEG-VI-PBA micelles for 4 hours and 13 hours, respectively, for drug-loaded micelles of the present invention.
FIG. 13 shows the cytotoxicity of PC7A-PEG-VI-PBA micelles of the present invention.
FIG. 14 shows the antitumor activity of free DOX, DOX @ PC7A-PEG-VI-PBA and DOX @ CdSeTe @ PC 7A-PEG-VI-PBA.
Detailed Description
The pH responsive polymer nano-micelle and the preparation and structural characterization thereof of the present invention are further illustrated by the following specific examples.
(1) Synthesis of RAFT reagent DMP
Acetone (46.7 g, 0.8 mmol), dodecanethiol (20.3 g, 0.1 mol) and methyltrioctylammonium chloride (1.6 g, 0.004 mol) were weighed out and added to a 250 mL three-necked flask with nitrogen and stirred in succession. NaOH solution (80 g, 2mol) was added dropwise to the above reaction mixture, and CS was added dropwise after 20 min2Acetone solution (15.21 mL, 0.2 mol). When the solution turned red, it was stirred for 15 min. Chloroform (35.625 mL, 0.30 mol) was then added and the dropwise addition of NaOH solution (40 g, 1 mol) was continued and after the addition was complete, stirring was continued overnight. 600 mL of water was added, acidified with 100 mL of concentrated HCl and stirred vigorously. Standing, vacuum-filtering with Buchner funnel, dissolving the solid in 1L isopropanol, and recrystallizing with n-hexane to obtain yellow crystal compound DMP (0.2 g).1H NMR (600 MHz, CDCl3): 3.28 (t, J = 7.4 Hz, 2H, -SCH 2CH2C9H18CH3), 1.72 (d, J= 16.4 Hz, 6H, COOHC(CH 3)2-), 1.67 (dt, J = 10.1, 5.0 Hz, 2H, -SCH2CH 2C9H18CH3),1.25 (m, 18H, -SCH2CH2C9 H 18CH3), 0.87 (t, J = 7.0 Hz, 3H, -SCH2CH2C9H18CH 3)。
(2) Preparation of C7A monomer compound: 1.4 g C7 (7A) (10 mmol) of the monomer was weighed out and dissolved in dichloromethane (20 mL) under ice-bath conditions, and triethylamine (0.6 g, 10 mmol) was added dropwise for 20 min. Methacryloyl chloride (1.08 g, 10 mmol) was weighed out from another container and dissolved in 5 mL of dichloromethane, after which the mixture was added dropwise to the above solution using a constant pressure dropping funnel and reacted for 24 h. And filtering the solid product, washing the solid product for three times by using anhydrous sodium carbonate, removing water by using anhydrous magnesium sulfate, and distilling under reduced pressure to obtain 0.87 g of the C7A monomer compound.1HNMR (600 MHz, CDCl3, ppm): 5.93 (br, 1H,CHH=C(CH3)-),5.39 (br, 1H, CHH=C(CH3)-), 4.07 (t, J=6.5HZ, 2H, -OCH 2CH2N-),2.67 (t, J = 6.5 HZ, 2H, -OCH2CH 2N-), 2.56 (m, 4H, -N(CH 2CH2CH2)2), 1.78 (s,3H, CH2=C(CH 3)-), 1.49 (m, 8H, -N(CH2CH 2CH 2)2).13C NMR (150MHz, CDCl3, ppm):49.7 (2C, -N(CH2CH2CH2)2), 29.5 (2C, -N(CH2 CH2CH2)2), 26.8 (2C, -N(CH2CH2 CH2)2),167.2 (1C, -C=O), 62.5 (1C, -OCH2CH2N-), 54.9 (1C, -OCH2 CH2N-), 136.0 (1C, CH2=C(CH3)-), 125.2 (1C,CH2=C(CH3)-), 17.9 (1C, CH2=C(CH3)-)。
(3) Synthesis of PC7A polymer: under the protection of nitrogen, DMP (20 mg, 0.055 mmol), AIBN (0.02 g, 0.12 mmol) and C7A monomer compound (0.4 g, 0.002 mol) are weighed and added into a sheck bottle containing 4 mL of dimethyl sulfoxide solution, polymerization reaction is carried out for 24h at 60 ℃, dialysis is carried out by using a dialysis bag with the molecular weight cutoff of 3000 to remove impurities after polymerization is completed, and reduced pressure distillation is carried out, thus obtaining 0.32 g of the PC7A compound.1H NMR (600 MHz , CDCl3, ppm): 4.0 (2H,-OCH 2CH2N-), 2.98, 2.76, 2.66 (6H, -OCH2CH 2N(CH 2CH2CH2)2), 1.61, 1.57 (8H, -N(CH2CH 2CH 2)2), 1.23 (18H, CH3(CH 2)9-)。
(4) Synthesis of PC7A-PEG Polymer: PEG (0.3 g, 0.63 mmol), AIBN (0.02 g, 0.12 mmol) and PC7A polymer (0.3 g) were weighed out under nitrogen and added4 mL of dimethyl sulfoxide solution in a sheck flask. The polymerization was carried out at 60 ℃ for 24 h. After the polymerization was completed, dialysis was performed using a dialysis bag having a molecular weight cut-off of 3000 to remove impurities, and distillation under reduced pressure was performed to obtain 0.48 g of PC7A-PEG polymer.1H NMR (600 MHz, CDCl3, ppm): 4.03 (2H,-OCH 2CH2N-), 2.73, 2.69 (6H, -OCH2CH 2N(CH 2CH2CH2)2), 1.60, 1.56 (8H, -N(CH2CH 2CH 2)2), 1.21 (18H, CH3(CH 2)9-), 3.61 (4H, CH3OCH 2CH 2O-)。
(5) Synthesis of PC 7A-PEG-VI-PBA: PC7A-PEG (0.3 g), AIBN (0.02 g, 0.12 mmol), VI (0.07 g, 0.74 mmol) and PBA (0.05 g, 0.34 mmol) were weighed out under nitrogen and added to a sheck flask containing 4 mL of dimethylsulfoxide solution. The polymerization was carried out at 60 ℃ for 24 h. After the polymerization was completed, dialysis was performed using a dialysis bag having a molecular weight cut-off of 3000 to remove impurities, and distillation under reduced pressure was performed to obtain 0.421 g of PC7A-PEG-VI-PBA polymer.1H NMR (600 MHz,DMSO, ppm): 3.98 (2H, -OCH 2CH2N-), 1.60, 1.53(8H, -N(CH2CH 2CH 2)2), 3.52 (4H,CH3OCH 2CH 2O-), 6.85 (2H, -Ar-CH 2- of VI), 7.89 (2H, -Ar-CH- of PBA)。
(6) Synthesis of CdSeTe QDs
Te (0.0127 g, 1 mmol) and Se (0.0078 g, 1 mmol) were placed in two different 5 mL round-bottomed flasks, to which equal amounts of NaBH were added4(0.074 g, 2 mmol) of solid and then sealed. Then, 3mL of ultrapure water (except for O) was added to each flask under a nitrogen blanket2). Then, the reaction is carried out in a water bath at the temperature of 50 ℃ for 1.5 h to generate NaHTe and NaHSe.
Adding CdCl2• 2H2O (0.18 g, 0.8 mmol) was charged into a three-necked flask, and after 40 mL of ultrapure water was added, the flask was allowed to stand forIt is dissolved completely. 160 uL of thioglycolic acid was added thereto and adjusted to pH = 11.0 with 1mL of NaOH (2M). Introduction of N2To remove O2. After 30 min, adding NaHTe and NaHSe, heating to 120 ℃, and reacting for 10 h. Precipitating with isopropanol, and vacuum drying to obtain CdSeTe QDs (with red fluorescence under ultraviolet lamp).
(7) Preparation of CdSeTe @ PC7A-PEG-VI-PBA Polymer:
PC7A-PEG-VI-PBA (20 mg) was dissolved in 2 mL of DMSO, then CdSeTe QDs (5 mg) was dissolved in 1mL of water and added dropwise to the above solution after stirring for 24 hours, the new solution was dialyzed in distilled water (MWCO = 3000) through a dialysis bag for 20 ~ hours to remove uncomplexed CdSeTe QDs.
Claims (9)
1. A pH-responsive polymer nano-micelle has a structural formula as follows:
wherein a =22, b =10, c =5, d = 2; number average molecular weight Mn =9664.06 g/mol;
representing CdSeTeQDs.
2. The method for preparing pH-responsive polymer nanomicelle according to claim 1, comprising the steps of:
(1) the preparation of the C7A monomer compound comprises the steps of reacting 2- (hexamethylene imino) -ethyl methacrylate with triethylamine and methacryloyl chloride in dichloromethane at room temperature for 20 ~ 24 hours, washing a reaction product with anhydrous sodium carbonate, and removing water with anhydrous magnesium sulfate to obtain the C7A monomer compound;
(2) synthesizing a PC7A polymer, namely under the protection of nitrogen, in dimethyl sulfoxide, taking azobisisobutyronitrile as an initiator, and carrying out polymerization reaction on a C7A monomer compound and a RAFT reagent DMP at 50 ~ ℃ for 20 ~ hours;
(3) synthesizing a PC7A-PEG polymer, namely under the protection of nitrogen, in dimethyl sulfoxide solution, taking azobisisobutyronitrile as an initiator, and carrying out polymerization reaction on PC7A and polyethylene glycol methyl methacrylate at 50 ~ ℃ for 20 ~ hours;
(4) synthesizing a compound PC7A-PEG-VI-PBA, namely reacting PC7A-PEG with 1-vinyl imidazole and 4-vinyl phenylboronic acid in dimethyl sulfoxide solution under the protection of nitrogen for 20 ~ 24 hours at 50 ~ 60 ℃ by using azobisisobutyronitrile as an initiator, dialyzing by using a dialysis bag with the molecular weight cutoff of 3000 after polymerization is finished to remove impurities, and distilling under reduced pressure to obtain the PC7A-PEG-VI-PBA polymer;
(5) the preparation of the CdSeTe @ PC7A-PEG-VI-PBA polymer comprises the steps of dissolving PC7A-PEG-VI-PBA in DMSO, adding an aqueous solution of CdSeTe QDs, stirring for 20 ~ 24 hours, dialyzing in distilled water for 20 ~ 24 hours by a dialysis bag with the molecular weight cutoff of 3000 to remove the uncoordinated CdSeQDs, and carrying out vacuum distillation to obtain the CdSeTe @ PC 7A-PEG-VI-PBA.
3. The method for preparing pH-responsive polymer nanomicelle according to claim 2, wherein: in the step (1), the molar ratio of 2- (hexamethylene imino) -ethyl methacrylate to triethylamine is 1: 1; the molar ratio of 2- (hexamethyleneimino) -ethyl methacrylate to methacryloyl chloride is 1: 1.
4. The method for preparing pH-responsive polymer nanomicelle according to claim 2, wherein: in the step (2), the structural formula of the RAFT reagent 2- (dodecylthio thiocarbonylthio) -2-methylpropanoic acid (DMP) is as follows:
。
5. the method for preparing pH-responsive polymer nanomicelle according to claim 2, wherein in step (2), the molar ratio of the C7A monomer compound to the RAFT reagent DMAP is 30: 1, and the amount of AIBN used is 0.3 ~ 0.4.4% of the molar amount of the C7A monomer compound.
6. The method for preparing pH responsive polymer nanomicelle according to claim 2, wherein in step (3), the molar ratio of PC7A polymer to polyethylene glycol methyl methacrylate is 1:15, and the amount of AIBN used is 20 ~ 25% of the molar amount of PC7A polymer.
7. The method for preparing pH-responsive polymer nanomicelle according to claim 2, wherein: in the step (4), the molar ratio of the 1-vinyl imidazole to the PC7A-PEG is 10: 1; the molar ratio of 4-vinylphenylboronic acid to PC7A-PEG was 5: 1.
8. The method for preparing pH-responsive polymer nanomicelle according to claim 2, wherein: in the step (5), the mass ratio of the PC7A-PEG-VI-PBA to the CdSeTe QDs is 4: 1.
9. The use of the pH-responsive polymeric nanomicelle according to claim 1 as a tumor-targeted drug delivery vehicle.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111991563A (en) * | 2020-09-03 | 2020-11-27 | 西北师范大学 | PH response type nano-drug delivery system and preparation method thereof |
| CN112107542A (en) * | 2020-09-03 | 2020-12-22 | 西北师范大学 | Has tumor pH and H2O2Multifunctional polymer micelle with specific activated antitumor activity and preparation method thereof |
| CN114276557A (en) * | 2021-12-31 | 2022-04-05 | 福州大学 | Acid-responsive hyperbranched poly-prodrug nano-micelle and preparation method and application thereof |
| CN115304977A (en) * | 2021-05-07 | 2022-11-08 | 香港大学 | pH/CO for controlling, rejecting and/or inactivating viruses and bacteria 2 Responsive smart anti-pathogen coatings |
| CN116333190A (en) * | 2023-03-06 | 2023-06-27 | 郑州大学 | Multiple-response prodrug polymer, preparation method and application |
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2019
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Non-Patent Citations (1)
| Title |
|---|
| QIANQIAN QI等: "Tumor-targeting and imaging micelles for pH triggered anticancer drug release and Combined Photodynamic Therapy", 《JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111991563A (en) * | 2020-09-03 | 2020-11-27 | 西北师范大学 | PH response type nano-drug delivery system and preparation method thereof |
| CN112107542A (en) * | 2020-09-03 | 2020-12-22 | 西北师范大学 | Has tumor pH and H2O2Multifunctional polymer micelle with specific activated antitumor activity and preparation method thereof |
| CN115304977A (en) * | 2021-05-07 | 2022-11-08 | 香港大学 | pH/CO for controlling, rejecting and/or inactivating viruses and bacteria 2 Responsive smart anti-pathogen coatings |
| CN114276557A (en) * | 2021-12-31 | 2022-04-05 | 福州大学 | Acid-responsive hyperbranched poly-prodrug nano-micelle and preparation method and application thereof |
| CN116333190A (en) * | 2023-03-06 | 2023-06-27 | 郑州大学 | Multiple-response prodrug polymer, preparation method and application |
| CN116333190B (en) * | 2023-03-06 | 2024-05-24 | 郑州大学 | Multiple-response prodrug polymer, preparation method and application |
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