CN113908286A

CN113908286A - Polyethyleneimine modified quantum dot nano particle, preparation method thereof and application of quantum dot nano particle as nano drug carrier

Info

Publication number: CN113908286A
Application number: CN202111196812.4A
Authority: CN
Inventors: 赵美霞; 任斌; 甘莹; 李景华; 张志强
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2022-01-11

Abstract

The invention belongs to the field of chemical biology, and particularly relates to a polyethyleneimine modified quantum dot particle, a preparation method thereof and application of the polyethyleneimine modified quantum dot particle as a nano drug carrier. The polyethyleneimine modified quantum dot nanoparticle drug carrier has a structure shown in the following formula:

Description

Polyethyleneimine modified quantum dot nano particle, preparation method thereof and application of quantum dot nano particle as nano drug carrier

Technical Field

The invention belongs to the technical field of chemical biology, and particularly relates to a polyethyleneimine modified quantum dot nanoparticle, a preparation method thereof and application of the nanoparticle as a nano drug carrier.

Background

Quantum Dots (QDs) play an important role in monitoring and imaging of cancer cells due to their unique photochemical properties. Meanwhile, because QDs have larger specific surface area, the QDs can be used as carriers of anticancer drugs to realize drug delivery. Weng et al designed and synthesized the targeting ligand of conjugated hydrophilic QDs and PEG phospholipid, combined on the lipid membrane layer of liposome during the liposome hydration process, loaded anticancer drug adriamycin, and obtained the targeting traceable drug delivery carrier. Although relatively simple in design, both external and internal QDs loading reduce drug loading capacity and fluorescence intensity. Therefore, there is an urgent need for a QDs drug delivery system with strong drug delivery capacity and high fluorescence intensity.

Cationic nanoparticles have high affinity and high phagocytosis rate on negatively charged cell surfaces. Various cationic polymers, such as chitosan, Polyethyleneimine (PEI) and peptides, have been found and coupled to nanoparticles, acting as carriers for gene or drug delivery. PEI is widely studied as a carrier for non-viral gene and drug delivery due to its good water solubility and abundant positive charge. Park et al reported a quantum dot-amphiphilic polyethyleneimine (AmPEI) nanocomposite that has successfully delivered silencing RNA (SiRNA) to cancer cells. However, the toxic effect of PEI on cells is large, limiting its application in the biomedical field. It has been found that decreasing the relative molecular mass reduces the toxicity of PEI. Therefore, low molecular weight PEI has great potential in the biomedical field.

10-Hydroxycamptothecin (HCPT), doxorubicin (Dox) and the like are considered as potential anticancer drugs. HCPT, Dox, etc. have been widely used to treat various cancers. However, the poor solubility and dose toxicity of these drugs in water greatly limit their clinical applications, but the nano drug delivery system can solve these problems well. Therefore, there is a need to develop a simple, economical and feasible hydrophilic modification and nano-drug delivery method for antitumor drugs.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a polyethyleneimine modified quantum dot nano particle, a preparation method thereof and application of the polyethyleneimine modified quantum dot nano particle as a nano drug carrier.

In order to achieve the purpose, the invention adopts the following technical scheme:

a polyethyleneimine modified quantum dot nanoparticle has the following structure:

；

in the formula, QDs refers to quantum dots CdSe or CdSe/ZnS. In the quantum dot nano particle, a ligand is polyethyleneimine; the quantum dots are any one of CdSe and CdSe/ZnS.

The invention provides a preparation method of the Polyethyleneimine (PEI) -modified quantum dot nanoparticles, which comprises the steps of mixing CdSe or CdSe/ZnS quantum dots 2 (QDs) with a chloroform solution of polyethyleneimine 1, adjusting the pH to 10-11, stirring at 20-28 ℃ for reaction for 12-24h, removing chloroform by rotary evaporation after the reaction is finished, purifying with ethanol, and drying in vacuum to obtain polyethyleneimine-modified quantum dot nanoparticles 3; the synthetic route is as follows:

。

in the preparation method of the polyethyleneimine-modified quantum dot nanoparticle, the mass ratio of the quantum dot CdSe or CdSe/ZnS to the polyethyleneimine is preferably 1: 10-20.

Further, the preparation method of the polyethyleneimine modified quantum dot nanoparticles can be used for adjusting the pH to 10-11 by adding ammonia water.

The invention provides application of the polyethyleneimine modified quantum dot nanoparticles as a nano-drug carrier in drug loading.

The invention also provides application of the polyethyleneimine modified quantum dot nanoparticles as a nano-drug carrier in the aspect of cell biological activity.

The quantum dot nano particle modified by the polyethyleneimine has the advantages of small particle size, good water solubility, uniform dispersion and good stability, and simultaneously has low cytotoxicity to various cells, so that the quantum dot nano particle can be used as a good nano drug carrier to be applied to the field of chemical biology, and can lay a foundation for better designing the nano drug carrier. Compared with the prior art, the invention has the beneficial effects that:

1) according to the invention, polyethyleneimine is modified on the surfaces of quantum dot nanoparticles by an effective method, so that the quantum dot nanoparticles have good water solubility, stability and biocompatibility, and are prepared into a nano material with high biocompatibility, thereby realizing the effect of serving as a nano drug carrier in the field of chemical biology;

2) the preparation method of the nano-drug carrier for modifying the polyethyleneimine to the surface of the quantum dot nanoparticle is simple and easy to implement and has low cost;

3) the quantum dot nano-drug carrier modified by the polyethyleneimine has good optical characteristics, can provide good optical signals for carrying and releasing drugs, and provides convenient conditions for tracing the drugs.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) map of quantum dots 2 (CdSe, a in the figure) and polyethyleneimine modified quantum dot nanoparticles 3 (CdSe @ PEI, b in the figure) and 10-hydroxycamptothecin (cHCPT) -loaded product 6(CdSe @ PEI-cHCPT, c in the figure) in example 1;

FIG. 2 is the UV absorption spectrum of the product 6(CdSe @ PEI-cHCPT) after the quantum dot 2 (CdSe) and the polyethyleneimine modified quantum dot nanoparticle 3 (CdSe @ PEI) and the 10-hydroxycamptothecin (cHCPT) in example 1;

FIG. 3 is the fluorescence spectra of the product 6(CdSe @ PEI-cHCPT) after loading of quantum dot 2 (CdSe) and polyethyleneimine modified quantum dot nanoparticle 3 (CdSe @ PEI) and 10-hydroxycamptothecin (cHCPT) in example 1;

FIG. 4 is the UV absorption spectrum of the product 6(CdSe/ZnS @ PEI-cHCPT) after loading of the quantum dots 2 (CdSe/ZnS) and the polyethyleneimine modified quantum dots 3 (CdSe/ZnS @ PEI) and 10-hydroxycamptothecin (cHCPT) in example 2;

FIG. 5 shows fluorescence spectra of quantum dot 2 (CdSe/ZnS), polyethyleneimine-modified quantum dot nanoparticle 3 (CdSe/ZnS @ PEI) and 10-hydroxycamptothecin-loaded product 6(CdSe/ZnS @ PEI-cHCPT) in example 2.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Name and model of the experimental instrument:

japanese JEOL JEM-200CX transmission electron microscope;

U.S. Perkin-Elmer Lambda-850 UV Spectrophotometer;

U.S. Perkin-Elmer Ls55 spectrofluorometer;

BioTek multifunctional microplate reader, USA.

In the following examples, the quantum dots CdSe or CdSe/ZnS used are prepared by techniques conventional in the art, for example, reference is made to the following documents:

Z. A. Peng; X. Peng. Formation of High-Quality CdTe, CdSe, and CdS Nanocrystals Using CdO as Precursor. J. Am. Chem. Soc.2001, 123, 183-184;

J. J. Li; Y. A; Wang; W. Guo; J. C. Keay; T. D. Mishima; M. B. Johnson; X. Peng. Large-Scale Synthesis of Nearly Monodisperse CdSe/CdS Core/Shell Nanocrystals Using Air-Stable Reagents via Successive Ion Layer Adsorption and Reaction. J. Am. Chem. Soc.2003, 125, 12567-12575。

example 1

When the quantum dot 2 is CdSe, the synthetic steps for preparing the polyethyleneimine modified quantum dot nano-drug carrier are as follows:

。

1) preparation of compound 3: 0.20 g of polyethyleneimine 1 (PEI, 1.8 kDa)) was added to 10 mL of chloroform in a 100 mL round-bottomed flask and dissolved completely by means of ultrasound. Quantum dot 2 (CdSe, 10.0 mg) was added to the flask. 0.2 mL of aqueous ammonia was added to adjust the pH to 10, the resulting solution was stirred at room temperature (25 ℃ C.) for 12 hours, and chloroform was removed from the mixture by rotary evaporation. Re-dissolving with ethanol, centrifuging and further purifying, repeating the operation for three times, and drying in vacuum to obtain the polyethyleneimine modified quantum dot nanoparticle 3 (CdSe @ PEI).

2) Preparation of compound 5: a mixture of 104.3 mg of compound 410-hydroxycamptothecin (cHCPT, 0.29 mmol), 35.0 mg (0.29 mmol) of 4-dimethylaminopyridine and 40.0 mg (0.4 mmol) of succinic anhydride is added to 4 mL of anhydrous dimethylformamide. The mixture was stirred at room temperature in the dark for 24h, then 0.5 mL of 20% aqueous methanol was added and stirred for an additional 30 min to hydrolyze excess anhydride. The mixture was concentrated by rotary evaporation, the precipitate collected by centrifugation and dried under vacuum to give crude cHCPT. The crude cHCPT was dissolved in a mixture of Dichloromethane (DCM) and methanol (MeOH) (10: 1 by volume), 2.00 g of 100 mesh silica was added and the crude was further purified by elution with a DCM/MeOH gradient (100: 1, 50:1, 30:1, 20:1, 10:1, v/v), and the 10:1 eluate was collected and rotary evaporated to give pure 5 (cHCPT) as a yellow solid.

3) Preparation of product 6: 46.8 mg of Compound 5 (cHCPT) was dissolved in 5 mL of anhydrous dimethyl sulfoxide (DMSO), 182.1 mg (0.95 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 109.3 mg (0.95 mmol) of 1-hydroxypyrrolidine-2, 5-dione (NHS) were added for activation, followed by 205.6 mL (1.43 mmol) of Triethylamine (TEA). The mixture was reacted at room temperature for 1 hour under nitrogen atmosphere and then added to an aqueous solution of the above compound 3 (CdSe @ PEI). The mixture was stirred at room temperature under nitrogen and dark conditions for 24 h. The product was collected by centrifugation and washed with dimethylsulfoxide, ethanol, respectively, to remove excess cHCPT, EDC and NHS. After drying in vacuo, a red solid 6(CdSe @ PEI-cHCPT) was obtained.

The sizes and the dispersion degrees of the quantum dot 2 (CdSe), the polyethyleneimine modified quantum dot nano particle 3 (CdSe @ PEI) and the drug-loaded product 6(CdSe @ PEI-cHCPT) are characterized by using a transmission electron microscope, and the result is shown in figure 1. As can be seen in fig. 1: the quantum dot nano particles 3 modified by the polyethyleneimine are uniformly dispersed in water and are spherical, and the particle size of the quantum dots modified by the polyethyleneimine is increased after the quantum dots serving as nano drug carriers load cHCPT.

The ultraviolet absorption spectrum is utilized to characterize the quantum dot 2 (CdSe), the polyethyleneimine modified quantum dot nano particle 3 (CdSe @ PEI) and the drug-loaded product 6(CdSe @ PEI-cHCPT), and the result is shown in figure 2. As can be seen in fig. 2: the quantum dot nano particle product 3 modified by the polyethyleneimine still has a typical absorption peak of the quantum dot, and the modified quantum dot absorption peak is subjected to red shift of different degrees relative to the original quantum dot, which indicates that the polyethyleneimine is successfully loaded on the quantum dot; due to the addition of cHCPT, the ultraviolet spectrum absorption spectrum of CdSe @ PEI-cHCPT shows a new absorption band at 375-430 nm, which indicates that the cHCPT has been successfully modified on the quantum dots.

The quantum dot 2 (CdSe), the polyethyleneimine modified quantum dot nanoparticle 3 (CdSe @ PEI) and the drug-loaded product 6(CdSe @ PEI-cHCPT) are characterized by fluorescence spectroscopy, and the result is shown in FIG. 3. As can be seen in fig. 3: after the cHCPT is adsorbed, the fluorescence intensity is reduced, the emission spectrum appears a little blue shift, compared with the original quantum dot, the emission spectrum band of the product 6 is wider, and the difference of the fluorescence among the quantum dots is caused by the compounding of polyethyleneimine and cHCPT on the surface.

Example 2

When the quantum dot 2 is CdSe/ZnSe, the synthetic steps for preparing the polyethyleneimine modified quantum dot nano-drug carrier are as follows:

。

1) preparation of compound 3: 0.20 g of polyethyleneimine 1 (PEI, 1.8 kDa)) was added to 10 mL of chloroform in a 100 mL round-bottomed flask and dissolved completely by means of ultrasound. Quantum dots 2 (CdSe/ZnS, 10.0 mg) were added to the flask. 0.2 mL of ammonia was added to adjust the pH to 10, and the resulting solution was stirred at room temperature for 12 hours. The chloroform was removed from the mixture by rotary evaporation. Re-dissolving with ethanol, centrifuging and further purifying, repeating the operation for three times, and drying in vacuum to obtain the polyethyleneimine modified quantum dot nanoparticle 3 (CdSe/ZnS @ PEI).

2) Preparation of compound 5: refer to example 1.

3) Preparation of product 6: 46.8 mg of Compound 5 (cHCPT) was dissolved in 5 mL of anhydrous dimethyl sulfoxide (DMSO), 182.1 mg (0.95 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 109.3 mg (0.95 mmol) of 1-hydroxypyrrolidine-2, 5-dione (NHS) were added for activation, followed by 205.6 mL (1.43 mmol) of Triethylamine (TEA). The reaction was carried out at room temperature for 1 h under nitrogen, and then added to an aqueous solution of the above compound 3 (CdSe/ZnS @ PEI). The mixture was stirred at room temperature under nitrogen and dark conditions for 24 h. The product was collected by centrifugation and washed with dimethylsulfoxide, ethanol, respectively, to remove excess cHCPT, EDC and NHS. After vacuum drying, a red solid 6(CdSe/ZnS @ PEI-cHCPT) was obtained.

The quantum dot 2 (CdSe/ZnS), the polyethyleneimine modified quantum dot nanoparticle 3 (CdSe/ZnS @ PEI) and the drug-loaded product 6(CdSe/ZnS @ PEI-cHCPT) are characterized by ultraviolet absorption spectroscopy, and the result is shown in FIG. 4. As can be seen in fig. 4: the quantum dot nano particle product 3 modified by the polyethyleneimine still has a typical absorption peak of the quantum dot, and the modified quantum dot absorption peak is subjected to red shift of different degrees relative to the original quantum dot, which indicates that the polyethyleneimine is successfully loaded on the quantum dot.

The quantum dot 2 (CdSe/ZnS) and the polyethyleneimine modified quantum dot nanoparticle 3 (CdSe/ZnS @ PEI) and the drug-loaded product 6(CdSe/ZnS @ PEI-cHCPT) are characterized by fluorescence spectroscopy, and the result is shown in FIG. 5. As can be seen in fig. 5: after the cHCPT is adsorbed, the fluorescence intensity is reduced, the emission spectrum appears a little blue shift, compared with the original quantum dot, the emission spectrum band of the product 6 is wider, and the difference of the fluorescence among the quantum dots is caused by the compounding of polyethyleneimine and cHCPT on the surface.

Activity assay

Cell culture: a549 (human non-small cell lung cancer cell), HeLa (cervical cancer cell), HCT-116 (human colon cancer cell) and QSG-7701 (human hepatocyte). The cells were incubated with 10% (v/v) fetal bovine serum and with 1% (v/hr)v) 1640 culture of the mixed solution of penicillin and streptomycin was carried out in a culture flask. The culture flask is placed at 37 ℃ and contains 5% CO₂And the cultivation was carried out in an incubator with a humidity of 90%.

Cytotoxicity test: cytotoxicity of the polyethyleneimine modified quantum dot nanoparticle drug carrier 3 and the drug-loaded product 6 prepared in examples 1 to 2 in a549, HeLa, HCT-116 and QSG-7701 cells was determined by the MTT method: after subculturing the cells for 3 or 4 times, when the cells grow to logarithmic phase, digesting the cells with 0.25% trypsin to form single cell suspension, counting the viable cells by adopting a blood counting chamber, and adjusting the concentration of the viable cells to 5 × 10⁴Perml/mL in 96-well plates, 100. mu.L per well, 5% CO at 37 ℃₂And cultured in an incubator with a humidity of 90% for 24 hours, the old medium was aspirated, and products 3 and 6 prepared in examples 1 to 2 were added at different concentrations diluted with the medium, respectively. The 96-well plate after the addition of the sample was placed at 37 ℃ with 5% CO₂The culture box is incubated for 48 hours, then MTT 20 mu L/well (2.5 mg/mL) is added, the supernatant is discarded after 4 hours, DMSO 100 mu L/well is added, the oscillation is carried out for about 10 min, the OD value is measured by an M200 microplate reader, and the wavelength is set to be 570 nm and 690 nm. The cell viability of the wells to which no sample was added was set as a control to 100%, and the cell viability was calculated to evaluate the cytotoxicity of the samples. The results are shown in Table 1 below.

TABLE 1 cytotoxicity of the products and intermediates prepared in examples 1 to 2

Table 1 shows the in vitro growth inhibition activity of the polyethyleneimine modified quantum dot nanoparticle drug carrier 3 and the drug-loaded product 6 prepared in examples 1 to 2 on A549, HeLa, HCT-116 and QSG-7701 cells. IC (integrated circuit)₅₀The value is the sample concentration value (. mu.g/mL), IC, at which the number of cells capable of normal division growth is suppressed to a level of 50%₅₀A larger value indicates a less cytotoxic sample. The experimental results show that: the toxicity of the polyethyleneimine modified quantum dot nanoparticle drug carrier 3 on cellsThe quantum dot after drug loading has smaller ratio, the product 6 after drug loading can more effectively inhibit the growth of various cancer cells, has smaller damage to normal cells, and has higher antiproliferative activity to HeLa cells compared with HCT-116, QSG-7701 and A549 cells. That is, the low-toxicity effect can be achieved by selecting low-molecular-weight PEI as a quantum dot ligand.

To sum up, the following steps are carried out: the quantum dot nanoparticle drug carrier 3 modified by polyethyleneimine can be used as a nano drug carrier, is applied to the field of chemical biology, and can lay a foundation for better designing the nano drug carrier.

Claims

1. A polyethyleneimine modified quantum dot nanoparticle is characterized by having the following structure:

；

in the formula, QDs refers to quantum dots CdSe or CdSe/ZnS.

2. The preparation method of the polyethyleneimine-modified quantum dot nanoparticle as claimed in claim 1, wherein the preparation method comprises the steps of mixing quantum dot CdSe or CdSe/ZnS with a chloroform solution of polyethyleneimine, adjusting pH to 10-11, stirring at 20-28 ℃ for reaction for 12-24h, removing chloroform by rotary evaporation after the reaction is finished, purifying with ethanol, and drying in vacuum.

3. The method of claim 2, wherein the mass ratio of the quantum dot CdSe or CdSe/ZnS to the polyethyleneimine is 1: 10-20.

4. The method of claim 3, wherein ammonia is added to adjust the pH to 10-11.

5. The use of the polyethyleneimine modified quantum dot nanoparticles of claim 1 as a nano-drug carrier in drug delivery.

6. The use of the polyethyleneimine modified quantum dot nanoparticles as claimed in claim 1 as a nano-drug carrier in cellular biological activity.