CN110152021B - Drug carrier system with cancer cell internal target administration capability and preparation method thereof - Google Patents
Drug carrier system with cancer cell internal target administration capability and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a drug carrier system which is obtained by taking carbon quantum dots and beta-cyclodextrin modified Prussian blue as a matrix, combining the matrix with NO donor 5-chloro-2-nitrophenyl trifluoromethane grafted polylysine and then modifying the matrix with folic acid. The drug carrier system provided by the invention can realize effective enrichment at tumor parts through folic acid targeting and tumor part EPR effects, and under near-infrared laser irradiation, prussian blue nanoparticles can ablate tumor cells with excellent photo-thermal conversion efficiency. Meanwhile, the drug carrier system can control the release of nitric oxide under the illumination of 400nm wavelength, thereby improving EPR effect, enhancing the enrichment of nano particles at tumor parts, simultaneously NO can induce tumor cell apoptosis, reverse multidrug resistance and the like, and inhibiting tumor growth.
Description
Technical Field
The invention belongs to the field of drug carriers, and particularly relates to a drug carrier system with cancer cell internal targeted drug delivery capability and a preparation method thereof.
Background
In recent years, the incidence of cancer has been increasing with the deterioration of natural environments. Traditional cancer therapies include: surgical treatment, chemotherapy and radiotherapy. Although the life of a patient can be prolonged to a certain extent, the treatment modes generally have the defects of large toxic and side effects, large damage to normal tissues, large trauma and the like, so that the treatment effect of the treatment modes on the tumor is limited. Therefore, there is increasing interest in developing new therapeutic modalities.
Chemotherapy is currently the primary treatment regimen for anti-tumor, but chemotherapy is challenged by multidrug resistance (MDR), which greatly limits the efficacy of chemotherapy. And often employ high doses and increase the frequency of administration. However, high doses and increased dosing frequency do not significantly improve the therapeutic effect, but rather often cause serious adverse side effects on vital organs (heart, liver and kidney), possibly further worsening the drug resistance. Compared with the traditional small molecule drug tumor chemotherapy, the near infrared irradiation setting is controllable, such as irradiation time, light source position, power output and the like, so the photothermal therapy (PTT) photodynamic therapy (PDT) has the characteristics of low toxicity and high specificity.
Photothermal therapy (PTT) is a new method of tumor treatment, non-invasive and spatio-temporally controllable. The basic principle of photothermal therapy is: the photothermal material is gathered at a tumor part through an EPR effect, and then under the irradiation of near infrared light, the local temperature of the tumor is raised to be higher than 42 ℃, so that tumor cells are damaged and even eliminated, and the purpose of treating cancer is achieved. Due to its special ion exchange, adsorption and mechanical trapping properties, Prussian Blue (PB) has been approved by the U.S. Food and Drug Administration (FDA) in 2003 as an antidote to thallium and cesium internal radioactive contamination, showing its good biosafety and biocompatibility. Compared with other emerging nano near-infrared absorption materials (such as polypyrrole nano particles, gold nano particles and CuS nano particles), the PB nano particles have the advantages of good biocompatibility, higher photo-thermal conversion efficiency, light stability, easiness in size control and the like.
Photodynamic therapy (PDT) is a new therapeutic approach, and has attracted more and more attention in tumor therapy due to its advantages of low invasiveness, low side effects, low drug resistance, and the like. Current photodynamic therapy induces apoptosis, necrosis and tissue destruction mostly by Reactive Oxygen Species (ROS). Unfortunately, abnormal angiogenesis and poor blood flow within solid tumors can lead to an imbalance in oxygen supply and consumption, resulting in high levels of hypoxia. The hypoxic environment of tumor tissue may reduce the concentration of available oxygen in PDT, limiting ROS production. NO production does not need to rely on oxygen in the tumor tissue, and hypoxia at the tumor site can be reduced through NO-mediated vasodilation.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a drug carrier system aiming at the defects in the prior art, compared with the traditional drug carrier, the system has more efficient cancer cell targeted drug delivery capability, can effectively improve the drug utilization rate and reduce the toxic and side effects.
The technical scheme adopted by the invention for solving the problems is as follows:
the drug carrier system provided by the invention is obtained by taking carbon quantum dots and beta-cyclodextrin modified Prussian blue as a matrix, combining the matrix with NO donor 5-chloro-2-nitrophenyl trifluoromethane grafted polylysine, and modifying the matrix with folic acid.
The preparation method of the drug carrier system mainly comprises the following steps:
step one, combiningBeta-cyclodextrin-forming and cystamine dihydrochloride modified Prussian blue (PB-CD-NH)2)
Dispersing Prussian Blue (PB) in a buffer solution with the pH value of 5-6, and then adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide to react for 20-30 hours under the condition of an ice-water bath; then adding beta-cyclodextrin (EDA-beta-CD) and cystamine dihydrochloride to react at room temperature for 20-30 hours, separating and washing a solid product to obtain PB-CD-NH2；
Step two, synthesizing carbon quantum dot modified Prussian blue (PB-C-dots-CD)
Dispersing carbon quantum dots (C-dots) in a buffer solution with the pH value of 5-6, and then adding EDC and N-hydroxysuccinimide to react for 20-30 hours under the condition of ice-water bath; then, PB-CD-NH is subsequently added2Reacting at 25 ℃ for 20-30 hours, separating and washing a solid product to obtain PB-C-dots-CD;
step three, synthesizing NO donor 5-chloro-2-nitrophenyl trifluoromethane grafted polylysine (PLL (NF))
Dissolving Polylysine (PLL), potassium carbonate and 5-chloro-2-nitrophenyl trifluoromethane in a solvent DMF, refluxing for 72-96 hours at 25-35 ℃ for a reflux reaction for three days, fully dialyzing, and freeze-drying to obtain PLL (NF);
step four, synthesizing Prussian blue (PB-C-dots-CD-PLL (NF)) modified by NO donor 5-chloro-2-nitrophenyl trifluoromethane graft polylysine
Dispersing PB-C-dots-CD and PLL (NF) in a buffer solution with the pH value of 7.2-7.4, stirring and reacting for 45-55 hours at normal temperature, separating a solid product, washing and drying to obtain PB-C-dots-CD-PLL (NF);
step five, synthesizing folic acid modified Prussian blue (PB-C-dots-CD-PLL (NF) -FA)
Dissolving folic acid in a buffer solution with the pH value of 5-6, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide to react for 20-30 hours under the condition of ice-water bath, then adding PB-C-dots-CD-PLL (NF) to react for 20-30 hours at room temperature, and separating a solid product, namely folic acid modified Prussian blue (PB-C-dots-CD-PLL (NF) -FA).
According to the scheme, the Prussian blue is Prussian blue nano particles modified by the citric acid surface, and the particle size is within the range of 30nm-200 nm.
According to the scheme, the excitation wavelength of the carbon quantum dots is 400nm-660 nm.
According to the scheme, in the step one, the concentration of the prussian blue in the buffer solution is in the range of 1-2.5 mg/mL.
According to the scheme, in the step one, the concentrations of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in the buffer solution are both 4-6 mg/mL; the mass ratio of EDA-beta-CD, cystamine dihydrochloride and Prussian blue is (6-9) to (1-4) to 10 respectively.
According to the scheme, in the second step, the concentration of the carbon quantum dots in the buffer solution is in the range of 1-20 mg/ml.
According to the scheme, in the second step, the concentration of EDC and N-hydroxysuccinimide in the buffer solution is 15-30 mg/ml; PB-CD-NH2The mass ratio of the carbon quantum dots to the carbon quantum dots is (1-10): 1.
According to the scheme, in the third step, the mass ratio of the potassium carbonate to the PLL is 1 (1-3); the mass ratio of PLL to 5-chloro-2-nitrophenyl trifluoromethane is 1: (0.5-4).
According to the scheme, in the fourth step, the concentration ranges of PB-C-dots-CD and PLL (NF) in the buffer solution are respectively 1-2 mg/mL.
According to the scheme, in the fifth step, the folic acid is dissolved in the buffer solution with the pH of 5-6, and the concentration is 0.1-0.7 mg/ml.
According to the scheme, in the fifth step, the concentrations of EDC and N-hydroxysuccinimide in the buffer solution are both 5-15 mg/ml; the mass ratio of PB-C-dots-CD-PLL (NF) to folic acid is 10 (0.5-3).
Compared with the prior art, the invention has the beneficial effects that:
firstly, the drug carrier system provided by the invention can realize effective enrichment at a tumor part through folic acid targeting and a tumor part EPR effect, and under near-infrared laser irradiation, prussian blue nano particles can ablate tumor cells through excellent photo-thermal conversion efficiency. Meanwhile, the drug carrier system can control the release of nitric oxide under the illumination of 400nm wavelength, thereby improving EPR effect, enhancing the enrichment of nano particles at tumor parts, simultaneously NO can induce tumor cell apoptosis, reverse multidrug resistance and the like, and inhibiting tumor growth.
Secondly, the NO donor in the drug carrier system is controlled to release by 400nm laser, so that the NO donor can not release in advance and can control the toxic and side effects of the NO donor on normal cells; and NO can mediate vasodilatation, so that the carrier is more enriched at the tumor part, and the hypoxia of the tumor part is reduced.
And thirdly, the prussian blue can form fluorescence quenching with the carbon quantum dots, and the prussian blue is modified by the carbon quantum dots grafted by the disulfide bonds, so that GSH response in tumors can be realized to cause the disappearance of the fluorescence quenching, the fixed-point monitoring of the drugs can be realized, the carrier can be well tracked in vivo, and the visual navigation is realized. Because the concentration of Glutathione (GSH) in the cells is 1000 times of that in the extracellular matrix, the fluorescence quenching disappears in the tumor cells, and the fluorescence visualization is realized.
Fourthly, polylysine has good biocompatibility and a large number of unique membrane properties, and can enhance the absorption of cells to macromolecules; folic acid has good targeting property and can enhance the absorption of tumor cells to the carrier.
Through the series of designs, the invention can construct a composite nano-drug carrier system which has the capacity of tumor initiation targeting and is based on PLL (NF) -FA/beta-CD/PB, the fluorescence is visualized in tumor cells, then exogenous stimulation is applied to release nitric oxide and photo-thermal ablation of the tumor cells, normal cells can not be injured, compared with the traditional drug-carrying carrier, the system has more efficient cancer cell targeting drug delivery capacity, the drug utilization rate can be effectively improved, and the toxic and side effects can be reduced.
Drawings
Fig. 1 is a TEM image of prussian blue nanoparticles PB used in the examples;
FIG. 2 is a TEM image of carbon quantum dots C-dots used in the examples;
FIG. 3 shows the concentration of 1W/cm used in the examples2Photo-thermal images of prussian blue nanoparticles PB with different concentrations under 808nm laser irradiation;
FIG. 4 shows the concentration of 1W/cm used in the examples2And a photo-thermal cycle chart of 0.25mg/ml prussian blue nanoparticle PB under 808nm laser irradiation;
FIG. 5 is a nuclear magnetic diagram of PLL (NF) used in the examples;
FIG. 6 shows the NO release of PLL (NF) used in the examples;
FIG. 7 is a Fourier infrared plot of the product of each step in example 1;
FIG. 8 is a thermogravimetric analysis of the product of each step in example 1;
FIG. 9 shows fluorescence before and after GSH treatment of the product obtained in example 1;
FIG. 10 is a graph of cellular uptake versus time for the nanocarriers used in example 1;
FIG. 11 shows the photothermal killing effect of the nano-carrier used in example 1 at 50ug/mL and 100 ug/mL;
FIG. 12 shows MTT toxicity assay of nanocarriers used in example 1.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the content of the present invention, but the present invention is not limited to the following examples.
The PB, β -CD-OTs, EDA- β -CD, and C-dots referred to in the following examples can be prepared by the following methods, and can also be prepared by other methods.
1. Synthesis of Prussian blue nanoparticles (PB)
The prussian blue nanoparticles (PB NPs) with citric acid surface modification were prepared in the following manner, and the specific preparation steps were as follows: a certain amount of FeCl3And K4[Fe(CN)6]Adding into 20mL deionized water containing 0.5mmol citric acid respectively to make FeCl3And K4[Fe(CN)6]The final concentration of the compounds is 1.0 mmol/L; then the FeCl is added3Solutions and K4[Fe(CN)6]The solutions were heated to 60 ℃ separately and K was added4[Fe(CN)6]The solution is added dropwise under stirring at 60 DEG CTo FeCl3In solution; the solution gradually turned blue as the addition proceeded, and the blue solution was allowed to continue stirring for 30min and then cooled to room temperature.
Adding acetone with the same volume as the blue solution, standing at room temperature for 30min, centrifuging at 12500r/m for 60min, collecting nanoparticles, and repeatedly washing for 3 times; and finally, placing the collected Prussian blue nano particles in a vacuum drying oven, and drying for 12 hours at the temperature of 50 ℃ for later use.
Preparation of (1) p-toluenesulfonyloxy-beta-cyclodextrin (beta-CD-OTs)
Weighing 25g of beta-cyclodextrin (beta-CD), dissolving the beta-cyclodextrin (beta-CD) in 300mL of 0.4M NaOH solution, and stirring in an ice water bath until the cyclodextrin is completely dissolved; slowly dripping 18g of p-toluenesulfonyl chloride (TsCl) into the beta-CD solution, stirring in an ice water bath for reaction for 90min, and then carrying out suction filtration; and (3) adjusting the pH of the filtrate to 8.5 by using HCl, continuously stirring at room temperature for reacting for 2 hours, placing the obtained product in a refrigerator (4 ℃) overnight, carrying out suction filtration, washing filter residues with deionized water for three times, and drying at 60 ℃ to obtain the beta-CD-OTs.
(2) Synthesis of EDA-beta-CD
Weighing 2.5g of prepared beta-CD-OTs, adding the weighed beta-CD-OTs into 15ml of redistilled ethylenediamine, dissolving the beta-CD-OTs at normal temperature, introducing nitrogen for about 30min, placing the mixture into a water bath at 80 ℃, carrying out reflux reaction for 48h under the protection of nitrogen, then precipitating the mixture by using acetone, carrying out suction filtration by using an organic filter membrane with the thickness of 450nm, finally dissolving the mixture by using water, continuously precipitating the mixture by using acetone for two times to obtain yellowish white powder, drying the yellowish white powder for 50h at normal temperature to obtain about 2.2g of a product EDA-beta-CD, wherein the yield is about 95
3. Synthesis of carbon Quantum dots (C-dots)
1.6g of citric acid and 0.6g of urea are weighed and dissolved in 10mL of DMF, then the mixed solution is poured into a 20mL of polytetrafluoroethylene high-pressure reaction kettle, the reaction is carried out for 12 hours at 200 ℃, solids are removed by centrifugation after the reaction is finished, the obtained supernatant is washed three times by using (petroleum ether: ethyl acetate ═ 4:1) mixed solution, and the carbon quantum dots can be obtained by vacuum drying for 12 hours at 70 ℃.
The Prussian blue nano particles and the carbon quantum dots are diluted by a certain multiple, and the shapes of the Prussian blue nano particles and the carbon quantum dots are observed by a transmission electron microscope, as shown in figures 1 and 2, the carrier is of a cubic structure with the particle size of about 45nm, and the particle size of the carbon quantum dots is 3-4 nm.
Fig. 3 is a photothermal test of the prussian blue nanoparticles at different concentrations, and it can be seen from fig. 3 that the prussian blue nanoparticles have good photothermal properties.
Fig. 4 is a photo-thermal stability experiment of prussian blue nanoparticles, and it can be seen from fig. 4 that prussian blue nanoparticles have good photo-thermal stability and can be heated repeatedly.
FIG. 5 is a nuclear magnetic diagram of PLL (NF), and it can be seen from FIG. 7 that the integral ratio and chemical shift of PLL (NF) are expected, indicating the success of synthesis of PLL (NF).
FIG. 6 shows the NO release experiment, which shows that NO is released in large amount under 400nm illumination, and the synthesis and grafting success of PLL (NF) is also proved.
Example 1
A preparation method of a composite nano-drug carrier system specifically comprises the following steps:
1. synthesis of PB-CD-NH2
Weighing PB 200mg, dispersing in 100ml PBS with pH 5.5, adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 2.87g and N-hydroxysuccinimide 1.7262g at 4 ℃ in an ice-water bath, reacting for 24 hours, adding EDA-beta-CD 140mg and cystamine dihydrochloride 60mg, reacting for 24 hours, and centrifugally cleaning to obtain a solid product, namely PB-CD-NH2。
2. Synthesis of PB-C-dots-CD
Weighing 60mg of carbon quantum dots, dispersing the carbon quantum dots in 100ml of PBS (phosphate buffer solution) with the pH value of 5.5, adding 1.7262g of EDC and 2.87g of N-hydroxysuccinimide in an ice-water bath at the temperature of 4 ℃, reacting for 24 hours, then adding 200mg of PB-CD-NH2, reacting for 24 hours at room temperature, and centrifugally cleaning to obtain a solid product, namely PB-C-dots-CD.
3. Synthesis of PLL (NF)
200mg of PLL, 100mg of potassium carbonate and 200mg of 5-chloro-2-nitrophenyltrifluoromethane are weighed out and refluxed in 25mL of DMF for three days, dialyzed at room temperature for three days, deionized water is replaced every 6 hours, and lyophilized to obtain PLL (NF).
4. Synthesis of PB-C-dots-CD-PLL (NF)
Weighing 100mg of PB-C-dots-CD and 100mg of PLL (NF), dispersing in 60ml of PBS buffer solution (pH7.4), stirring at normal temperature for reaction for two days, centrifuging the solid product, washing with deionized water for several times, and vacuum drying to obtain the product PB-C-dots-CD-PLL (NF).
5. Synthesis of PB-C-dots-CD-PLL (NF) -FA
Weighing 20mg of folic acid, dissolving the folic acid in 200ml of PBS with the pH value of 5.5, adding 1.7262g of EDC and 2.87g of N-hydroxysuccinimide in ice-water bath at 4 ℃ for reaction for 24 hours, then adding 200mg of PB-C-dots-CD-PLL (NF), reacting at room temperature for 24 hours, and centrifugally cleaning the obtained solid product, namely PB-C-dots-CD-PLL (NF) -FA.
As can be seen from FIG. 7, in the above steps, a stretching vibration peak of the specific group grafted at each step was observed, indicating that the reaction at each step was successful. As can be seen from fig. 8, TG of the grafted carbon quantum dots increases, TG of the grafted carbon quantum dots decreases after each other step, indicating that the grafting of the nanoparticles is successful.
Application testing
1. Effect of the Presence or absence of GSH on PB-C-dots-CD-PLL (NF) -FA: appropriate amounts of PB-CD-C-dots-pll (nf) -FA were dispersed in a pH7.4 PBs buffer solution and a pH7.4 PBs buffer solution containing 10mM GSH, respectively. Placing the mixture in a shaker at 37 ℃ for 24 hours, centrifuging, washing and detecting fluorescence.
As can be seen from FIG. 9, PB-C-dots-CD-PLL (NF) -FA did not fluoresce in the absence of GSH, and fluorescence appeared in the presence of GSH, indicating successful grafting of the nanoparticles.
2. To investigate the relationship between cell uptake and co-culture time, HeLa cells were cultured at 1.0X 104The concentrations of cells/well were seeded into 6-well plates. After 24h of culture, the original culture solution in each well plate was replaced with 1mL of fresh DMEM containing PB-CD-C-dots-PLL (NF) -FA, PB-CD-C-dots-PLL (NF) and PB-CD-C-dots-PLL-FA, respectively, in terms of the concentration of PB contained in each well plate, wherein the concentration of PB was 100. mu.g/mL-1. After incubation of the cells for different times, the cells were washed three times with PBS, trypsinized, collected by centrifugation at 1100rpm for 5min and finally treated with concentrated nitric acid solution. The iron content was measured by ICP-MS (PerkinElmer, USA).
As can be seen from FIG. 10, in the eighth hour, the absorption of the nanocarriers by the cells is the highest, and both PB-CD-C-dots-PLL (NF) -FA and PB-CD-C-dots-PLL-FA are higher than PB-CD-C-dots-PLL (NF), which can effectively prove the targeting property of folic acid.
3. The photothermal cytotoxicity test adopts HeLa cells to evaluate the photothermal effect. HeLa cells were cultured at a temperature of 1.0X 104The concentrations of cells/well were seeded into 6-well plates, and 1mL of DMEM medium containing 10% FBS and 1% antibiotic was added to each plate. After 24h incubation, the culture medium in each well plate was replaced with fresh 1mL DMEM containing PBS (control), PB-CD-C-dots-PLL (NF) -FA, PB-CD-C-dots-PLL (NF) and PB-CD-C-dots-PLL-FA, respectively, and the net PB concentration was maintained at 100. mu.g/mL, based on the concentration of PB contained therein. After 24 hours of culture, the power density is 1w/cm-2After 808nm laser irradiation for 5min, the culture was continued for 24 h. The cells were washed three times with PBS and the viability of the cells was determined by MTT method.
As can be seen from FIG. 11, the control group used 1W/cm2And no obvious killing is caused under the laser irradiation of 808nm, which proves that the laser irradiation does not have great harm to cells. And the killing effect of PB-CD-C-dots-PLL (NF) -FA is the best, the killing effect of PB-CD-C-dots-PLL (NF) is the second, the killing effect of PB-CD-C-dots-PLL-FA is the worst, and the killing effect of high concentration is higher than that of low concentration. The combination of fig. 10 and fig. 12 shows that the drug has good targeting property and no major toxic and side effects. But the medicine NO can be effectively released and kill cells under the irradiation of light, and has good photo-thermal killing effect. The system is proved to have more efficient cancer cell targeting drug delivery capability, can effectively improve the utilization rate of the drug and reduce the toxic and side effects of the drug.
4. HeLa cells were seeded in a 96-well plate at a density of 8000 cells/well, cultured for 24 hours, and PB-CD-C-dots-PLL (NF) -FA, PB-CD-C-dots-PLL (NF) and PB-CD-C-dots-PLL-FA at different concentrations were added to co-culture the cells for 48 hours, after which the culture medium was changed to 200. mu.L of fresh DMEM medium and 20. mu.L of MTT solution (5mg/mL PBS). After an additional 4h incubation, the MTT solution was carefully removed, 150 μ L DMSO was added to the wells, and the media tray was shaken at room temperature and mixed well. The absorbance at 570nm of each well was recorded on a microplate reader (Model 550, Bio-Rad, USA) and the cell viability was calculated as follows:
wherein OD570(control)Is the absorbance, OD, measured without addition of material570(treated)Is the absorbance measured after addition of the material.
As can be seen from FIG. 12, the cell survival rate of the vector system is higher than 90%, and the vector system has good biocompatibility and no obvious toxic or side effect.
Example 2
A preparation method of a composite nano-drug carrier system specifically comprises the following steps:
1. synthesis of PB-CD-NH2
Weighing 200mg of PB, dispersing in 100ml of deionized water, adjusting the pH to 5-6 by hydrochloric acid (if the pH is adjusted to be lower than 5, a small amount of sodium hydroxide can be added for adjustment), adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 2.87g and N-hydroxysuccinimide 1.7262g in an ice-water bath at the temperature of 4 ℃, reacting for 24 hours, then adding 140mg of EDA-beta-CD and 60mg of cystamine dihydrochloride for reacting for 24 hours, and centrifugally cleaning to obtain a solid product, namely PB-CD-NH 2.
2. Synthesis of PB-C-dots-CD
Weighing 60mg of carbon quantum dots, dispersing in water, adjusting the pH to 5-6 by hydrochloric acid, adding EDC1.7262g and N-hydroxysuccinimide 2.87g at the temperature of ice-water bath 4 ℃, reacting for 24 hours, then adding 200mg of PB-CD-NH2, reacting for 24 hours at room temperature, and centrifugally cleaning to obtain a solid product, namely PB-C-dots-CD.
3. Synthesis of PLL (NF)
200mg of PLL, 100mg of potassium carbonate and 200mg of 5-chloro-2-nitrophenyltrifluoromethane are weighed out and refluxed in 25mL of DMF for three days, dialyzed at room temperature for three days, and lyophilized to obtain PLL (NF).
4. Synthesis of PB-C-dots-CD-PLL (NF)
Weighing 100mg of PB-C-dots-CD and 100mg of PLL (NF), dispersing in 60ml of PBS buffer solution (pH7.4), stirring at normal temperature for reaction for two days, centrifuging the solid product, washing with deionized water for several times, and vacuum drying to obtain the product PB-C-dots-CD-PLL (NF).
5. Synthesis of PB-C-dots-CD-PLL (NF) -FA
Weighing 20mg of folic acid, dissolving in 200ml of water, adjusting the pH to 5-6 by using hydrochloric acid, adding 1.7262g of EDC and 2.87g of N-hydroxysuccinimide in an ice-water bath at 4 ℃ for reaction for 24 hours, then adding 200mg of PB-C-dots-CD-PLL (NF) in the mixture for reaction at room temperature for 48 hours, and centrifugally cleaning the mixture to obtain a solid product, namely PB-C-dots-CD-PLL (NF) -FA.
Example 3
A preparation method of a composite nano-drug carrier system specifically comprises the following steps:
1. synthesis of PB-CD-NH2
Weighing PB 200mg, dispersing in 100ml PBS with pH 5.5, adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride 2.87g and N-hydroxysuccinimide 1.7262g at 4 ℃ in an ice-water bath, reacting for 24 hours, adding EDA-beta-CD 180mg and cystamine dihydrochloride 20mg, reacting for 24 hours, and centrifugally cleaning to obtain a solid product, namely PB-CD-NH2。
2. Synthesis of PB-C-dots-CD
Weighing 60mg of carbon quantum dots, dispersing the carbon quantum dots in 100ml of PBS (phosphate buffer solution) with the pH value of 5.5, adding 1.7262g of EDC and 2.87g of N-hydroxysuccinimide in an ice-water bath at the temperature of 4 ℃, reacting for 24 hours, then adding 200mg of PB-CD-NH2, reacting for 24 hours at room temperature, and centrifugally cleaning to obtain a solid product, namely PB-C-dots-CD.
3. Synthesis of PLL (NF)
200mg of PLL, 100mg of potassium carbonate and 600mg of 5-chloro-2-nitrophenyltrifluoromethane are weighed out and refluxed in 25mL of DMF for three days, dialyzed at room temperature for three days, and lyophilized to obtain PLL (NF).
4.PB-C-dots-CD-PLL(NF)
Weighing 100mg of PB-C-dots-CD and 100mg of PLL (NF), dispersing in 60ml of PBS buffer solution (pH7.4), stirring at normal temperature for reaction for two days, centrifuging the solid product, washing with deionized water for several times, and vacuum drying to obtain the product PB-C-dots-CD-PLL (NF).
5. Synthesis of PB-C-dots-CD-PLL (NF) -FA
Weighing 20mg of folic acid, dissolving the folic acid in 200ml of PBS with the pH value of 5.5, adding 1.7262g of EDC and 2.87g of N-hydroxysuccinimide in ice-water bath at 4 ℃ for reaction for 24 hours, then adding 200mg of PB-C-dots-CD-PLL (NF), reacting at room temperature for 24 hours, and centrifugally cleaning the obtained solid product, namely PB-C-dots-CD-PLL (NF) -FA.
The detection methods of the intermediate products and the final products obtained in the examples 2-3 are the same as those in the example 1, and the drug carrier system is successfully prepared through verification, has good targeting property and NO large toxic or side effect, can effectively release and kill cells under illumination, and has good photo-thermal killing effect. The drug carrier system provided by the invention is proved to have higher efficient cancer cell targeting drug delivery capability, and can effectively improve the drug utilization rate and reduce the toxic and side effects.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many modifications and changes can be made without departing from the inventive concept of the present invention, and these modifications and changes are within the protection scope of the present invention.
Claims (10)
1. A drug carrier system with the capability of targeted drug delivery in cancer cells is characterized by being prepared by the following main steps:
(1) synthesizing prussian blue modified by beta-cyclodextrin and cystamine dihydrochloride by taking the prussian blue nano particles modified by citric acid as raw materials;
(2) synthesizing carbon quantum dot modified Prussian blue by taking the beta cyclodextrin obtained in the step (1) and the Prussian blue modified by cystamine dihydrochloride as raw materials;
(3) and (3) combining the carbon quantum dot modified Prussian blue obtained in the step (2) with NO donor 5-chloro-2-nitrophenyl trifluoromethane grafted polylysine, and modifying with folic acid to obtain the drug carrier system with the cancer cell internal targeting drug delivery capability.
2. The method for preparing a drug carrier system with cancer cell targeting drug delivery capability of claim 1, characterized in that it essentially comprises the following steps:
step one, synthesizing beta-cyclodextrin and cystamine dihydrochloride modified Prussian blue PB-CD-NH2
Dispersing Prussian blue PB in an aqueous solution with the pH value of 5-6, and then adding EDC and N-hydroxysuccinimide to react for 20-30 hours under the condition of an ice-water bath; then adding p-toluenesulfonyloxy-beta-cyclodextrin and cystamine dihydrochloride to react at room temperature for 20-30 hours, separating and washing a solid product to obtain PB-CD-NH2；
Step two, synthesizing carbon quantum dot modified Prussian blue PB-C-dots-CD
Dispersing carbon quantum dots C-dots in an aqueous solution with the pH value of 5-6, and then adding EDC and N-hydroxysuccinimide to react for 20-30 hours under the condition of ice-water bath; then, PB-CD-NH is subsequently added2Reacting at 25 ℃ for 20-30 hours, separating and washing a solid product to obtain PB-C-dots-CD;
step three, synthesizing NO donor 5-chloro-2-nitrophenyl trifluoromethane grafted polylysine PLL (NF)
Dissolving polylysine PLL, potassium carbonate and 5-chloro-2-nitrophenyl trifluoromethane in a solvent DMF, carrying out reflux reaction at 25-35 ℃ for 72-96 hours, fully dialyzing, and freeze-drying to obtain PLL (NF);
step four, synthesizing Prussian blue PB-C-dots-CD-PLL (NF) modified by NO donor 5-chloro-2-nitrophenyl trifluoromethane grafted polylysine
Dispersing PB-C-dots-CD and PLL (NF) in a buffer solution with the pH value of 7.2-7.4, stirring and reacting for 45-55 hours at normal temperature, separating a solid product, washing and drying to obtain PB-C-dots-CD-PLL (NF);
step five, synthesizing folic acid modified Prussian blue PB-C-dots-CD-PLL (NF) -FA
Dissolving folic acid in an aqueous solution with the pH value of 5-6, adding EDC and N-hydroxysuccinimide to react for 20-30 hours under the condition of ice-water bath, then adding PB-C-dots-CD-PLL (NF) to react for 20-30 hours at room temperature, and separating out a solid product, namely PB-C-dots-CD-PLL (NF) -FA.
3. The method for preparing a drug carrier system with cancer cell targeting drug delivery capability according to claim 2, wherein in the first step, prussian blue is prussian blue nanoparticle surface-modified with citric acid, and the particle size is within the range of 30nm-200 nm; in the second step, the excitation wavelength of the carbon quantum dots is 400nm-660 nm.
4. The method for preparing a drug carrier system with cancer cell targeting drug delivery capability according to claim 2, wherein in the first step, the concentration of prussian blue in the aqueous solution is in the range of 1-2.5 mg/mL; the concentrations of EDC and N-hydroxysuccinimide in the water solution are both 15-30 mg/mL; the mass ratio of EDA-beta-CD, cystamine dihydrochloride and Prussian blue is (6-9) to (1-4) to 10 respectively.
5. The method for preparing a drug carrier system with cancer cell targeting drug delivery capability of claim 2, wherein in the second step, the concentration of the carbon quantum dots in the aqueous solution is in the range of 1-20 mg/ml; the concentration of EDC and N-hydroxysuccinimide in the buffer solution is 15-30 mg/ml; PB-CD-NH2The mass ratio of the carbon quantum dots to the carbon quantum dots is (1-10): 1.
6. The preparation method of the drug carrier system with cancer cell targeting drug delivery capability of claim 2, characterized in that in the third step, the mass ratio of potassium carbonate to PLL is 1 (1-3); the mass ratio of PLL to 5-chloro-2-nitrophenyl trifluoromethane is 1: (0.5-4).
7. The method for preparing a drug carrier system with targeting drug delivery capability in cancer cells according to claim 2, wherein in step four, the concentration ranges of PB-C-dots-CD and PLL (NF) in the buffer solution are 1-2mg/mL respectively.
8. The method for preparing a drug carrier system with cancer cell targeting drug delivery capability of claim 2, wherein in step five, folic acid is dissolved in an aqueous solution with pH of 5-6 to a concentration of 0.1-0.7 mg/ml.
9. The method for preparing a drug carrier system with cancer cell targeting drug delivery capability of claim 2, wherein in step five, the concentrations of EDC and N-hydroxysuccinimide in the buffer solution are both 5-15 g/ml; the mass ratio of PB-C-dots-CD-PLL (NF) to folic acid is 10 (0.5-3).
10. A pharmaceutical carrier system with cancer cell targeting drug delivery capability prepared by the method of any one of claims 2-9.
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