CN109833486B

CN109833486B - Novel nano drug delivery system

Info

Publication number: CN109833486B
Application number: CN201910243102.9A
Authority: CN
Inventors: 王梅云; 高海燕; 白岩; 陈丽娟; 魏巍
Original assignee: Henan Provincial Peoples Hospital
Current assignee: Henan Provincial Peoples Hospital
Priority date: 2019-03-27
Filing date: 2019-03-27
Publication date: 2021-11-05
Anticipated expiration: 2039-03-27
Also published as: CN109833486A

Abstract

The invention provides a novel nano drug delivery system, which comprises ICG, Dox and TPGS; wherein ICG represents indocyanine green, Dox represents adriamycin, TPGS represents D-alpha-tocopherol polyethylene glycol succinate, and ICG and Dox are emulsified into the emulsified nano-drug through electrostatic interaction, pi-pi stacking and hydrophobic interaction and TPGS. The method adopts a combined treatment mode to treat, and utilizes multi-mode imaging to monitor the drug release in the treatment process, thereby having great significance for the whole treatment process and the evaluation of the treatment effect.

Description

Novel nano drug delivery system

Technical Field

The invention particularly relates to a novel nano drug delivery system.

Background

Multidrug resistance (MDR) is a common mode of drug resistance in tumor cells and is also a major cause of failure in tumor chemotherapy. Multidrug resistance in tumor cells is caused by the overexpression of proteins, such as P-glycoprotein, of efflux antitumor drugs on the cell membrane. Cancer treatment is performed in a variety of ways, including surgery, chemotherapy, radiation therapy, immunotherapy, and the like. Among them, chemotherapy still dominates due to its high efficiency. In addition, photothermal therapy has recently been receiving attention as an emerging therapeutic modality because of its non-invasive nature, and the principle thereof is that cancer cells are killed by heat generated by absorbing near infrared light with a photothermal material. The single treatment mode is difficult to completely eliminate the tumor, and two or more treatment modes are combined, so that the limitation of the single treatment mode can be effectively solved. Furthermore, during the course of treatment, it is extremely important to track the course of drug delivery and to evaluate the effect of the treatment. Researchers have focused more and more on imaging modalities. The single-mode imaging is still limited by tissue penetration depth, low contrast, low sensitivity and the like, and the multi-mode imaging is a new imaging mode, is expected to make up the inherent limitation of the single-mode imaging, and has potential guiding significance for treating tumors.

Disclosure of Invention

The invention provides a novel nano drug-loading system, which adopts a combined treatment mode to treat, and utilizes multi-modal imaging to monitor drug release in the treatment process, thereby having great significance for the whole treatment process and the evaluation of treatment effect.

The invention adopts the following technical scheme for realizing the aim:

a novel nano drug delivery system comprises ICG, Dox and TPGS; wherein ICG represents indocyanine green, Dox represents adriamycin, and TPGS represents D-alpha-tocopherol polyethylene glycol succinate.

A new type of nano medicine carrying system, its ICG and Dox are emulsified into nano emulsified medicine through electrostatic interaction, pi-pi accumulation and hydrophobic interaction and through TPGS.

The near-infrared fluorescent dye ICG is negatively charged, the chemotherapeutic drug doxorubicin Dox is positively charged, the two are combined through the positive charge and the negative charge, and meanwhile, the ICG and the Dox are combined through pi stacking effect and hydrophobic effect due to the fact that the ICG and the Dox both have large pi bonds and hydrophobic groups, and are further emulsified into the uniform emulsified nano-drug through TPGS.

A novel nano drug delivery system comprises the following preparation processes:

1) solutions were prepared separately:

dissolving adriamycin in water to prepare a solution of 1 mg/mL; dissolving indocyanine green in water to prepare a solution of 1 mg/mL; adding D-alpha-tocopherol polyethylene glycol succinate into pure water, and oscillating by a vortex oscillator at room temperature to uniformly dissolve the D-alpha-tocopherol polyethylene glycol succinate, wherein the concentration of the solution is 0.01-0.025 mg/mL.

2) Solution mixing, shaking and emulsifying:

mixing the three solutions in the step 1 according to the mass ratio of 1:1:1, and then carrying out constant-temperature oscillation for 12 hours in a dark state, wherein the working parameters of a constant-temperature oscillator are set to be 37 ℃ and 700 r/h.

3) Centrifugal cleaning and filtering:

collecting the TPGS emulsified nano-drug in the step 2, repeatedly centrifuging and cleaning for three times by adopting a 10KD ultracentrifuge tube, and removing free molecules; the operating parameters of the ultracentrifuge were set as follows: the temperature is 37 ℃, the rotating speed is 4500 rpm, the time is 20 minutes, then filtration is carried out by using a filter membrane to obtain the purified sterile TPGS emulsified nano-drug, and the purified sterile TPGS emulsified nano-drug is stored in a dark place, wherein the size of the filter membrane is 0.22 mu m.

The invention adopts the technical scheme and has the following beneficial effects:

the nano-drug self-delivery system disclosed by the invention is simple in preparation method, simple and safe in components, stable in structure and capable of showing enhanced fluorescence effect and good biological safety, and meanwhile, the influence of the nano-drug emulsified by TPGS on the drug-resistant cell efflux microenvironment is utilized. Therefore, the delivery process of the drug is tracked under the guidance of the image, and the synergistic killing effect on the drug-resistant tumor is realized.

Drawings

Fig. 1A is a TEM image of a nano-drug self-delivery system prepared according to the ICG to Dox mass ratio of 1: 2.

Fig. 1B is a TEM image of a nano-drug self-delivery system prepared according to the ICG to Dox mass ratio of 1: 1.

Fig. 1C is a TEM image of a nano-drug self-delivery system prepared according to the ICG to Dox to TPGS mass ratio 1:1: 1.

Fig. 1D is a particle size diagram of a nano-drug self-delivery system prepared according to the present invention.

Fig. 2 is a graph showing the absorption profile of the nano-drug self-delivery system prepared according to the present invention.

Fig. 3A is a fluorescent image of a nano-drug self-delivery system prepared according to the present invention.

Fig. 3B is a fluorescence image of the nano-drug self-delivery system prepared according to the present invention.

Fig. 4 shows the cellular effect of the nano-drug self-delivery system prepared according to the present invention.

Detailed Description

1. solutions were prepared separately:

2. Solution mixing, shaking and emulsifying:

3. Centrifugal cleaning and filtering:

The ICG and FDA approved near infrared fluorescent dye is suitable for noninvasive biological imaging, and can convert near infrared absorbed light into heat for tumor thermal treatment. Dox is a broad spectrum anticancer drug that exerts its effects by disrupting the tertiary structure of DNA.

TPGS is a derivative of vitamin E, has the function of serving as an emulsifier in the preparation of nano-drugs, and is a novel emulsifier which is safe, effective and excellent in physicochemical property. The TPGS emulsified nano-drug can show more excellent performance. In addition, researches show that the TPGS emulsified nano-drug can effectively avoid the recognition of P-glycoprotein and influence the discharge microenvironment of drug-resistant cells, so that the accumulation of chemotherapeutic drugs in cells is increased, and tumor cells are killed.

Examples

The gastric cancer drug-resistant strain SCG7901/VCR is selected by the cells.

(1) Firstly, the mass of the indocyanine green is 10mg, the volume of the ultrapure water is 10mL, and the indocyanine green is uniformly dissolved by oscillating for 5min by a vortex oscillator at room temperature to obtain an indocyanine green aqueous solution for later use.

(2) Then, adriamycin was dissolved uniformly in an amount of 10mg and ultrapure water was dissolved in a volume of 10mL to obtain an adriamycin aqueous solution for use.

(3) Finally, the TPGS had a mass of 0.1-0.25mg, in this example 0.2mg, and the volume of ultrapure water was 10mL, and was uniformly dissolved by vortex oscillation for 10min at room temperature to give an aqueous TPGS solution for use.

(4) Mixing the (1) and the (2) in a mass ratio of 1:1 and 1:2, and mixing the (1) and the (2) and the (3) reagents in a mass ratio of 1:1:1, and keeping away from light and carrying out constant-temperature oscillation for 12h, wherein the working parameters of the constant-temperature oscillator of the embodiment are set to be 37 ℃ and 700 revolutions per hour. And packaging with tinfoil paper for shading.

(5) And (5) collecting the TPGS emulsified nano-drug prepared in the step (4), and repeatedly centrifuging and cleaning for three times by adopting a 10KD ultracentrifuge tube to remove free molecules. The working parameters of the ultracentrifuge are set as follows; the temperature was 37 deg.C, the rotation speed was 4500 rpm, and the time was 20 minutes. Then filtering with a filter membrane to obtain the purified sterile TPGS emulsified nano-drug, and storing the purified sterile TPGS emulsified nano-drug in the dark.

The size of the filter in this example was 0.22 μm.

(6) In this example, the final indocyanine green and doxorubicin contents were determined separately. The method comprises the following steps: the indocyanine green and adriamycin solutions were prepared at 0, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 75. mu.g/mL and 100. mu.g/mL, respectively, and their absorbance values at 780nm and 480nm wavelengths were measured, respectively, to obtain standard curves of the indocyanine green and adriamycin solutions. The same method obtains the absorption values of the TPGS emulsified nano-drug obtained in the step (5) at the wavelengths of 780nm and 480nm, and the absorption values are respectively substituted into a standard curve to obtain the concentrations of indocyanine green and adriamycin.

It should be noted that the above listed specific experimental parameters are only one of the disclosure

Examples are given, and in practical experiments, the oscillation time, the centrifugation time, and the like should be reasonably changed according to practical needs.

Fig. 1A, B, C is a TEM image of the TPGS emulsified nano drug prepared according to the embodiment of the present disclosure, and fig. 1D is a particle size analysis diagram of the TPGS emulsified nano drug prepared according to the embodiment of the present disclosure.

As can be seen from the combination of FIG. 1A, B, C and FIG. 1D, ICG and Dox can be self-assembled into a complete ICG-Dox nano-drug structure, and the nano-drug particle size can be changed by changing the ratio of ICG to Dox, as can be seen from TEM and particle size, when the ICG and Dox form nano-drug with a mass ratio of 1:1, the average diameter in aqueous solution is 27.45nm, after TPGS is added, as can be seen from TEM and particle size, the TPGS emulsified nano-drug is more uniformly dispersed, the form is more regular, the average particle size in aqueous solution is 19.03nm, and the more uniform TPGS emulsified nano-drug provides guarantee for further biological application.

FIG. 2 is a graph showing the UV absorption curves of ICG-Dox nano-drug and TPGS emulsified nano-drug in different proportions, and it can be seen that the UV absorption of the TPGS emulsified nano-drug is obviously enhanced at 780 nm.

Fig. 3A, 3B are fluorescence and photoacoustic images of TPGS emulsified nano-drug prepared according to the embodiments of the present disclosure. According to the figure, compared with a pure ICG-Dox nano-drug, the nano-drug emulsified by TPGS has very obvious fluorescence and photoacoustic enhancement effects, which shows that the nano-drug emulsified by TPGS has more optimized performance, and has significant meaning for the application of near-infrared fluorescence imaging in organisms.

FIG. 4 is the uptake of the TPGS emulsified nano-drug prepared according to the present disclosure by SCG7901/VCR cells. As can be seen from the figure, SCG7901/VCR cells have good uptake of TPGS emulsified nano-drugs. Compared with an ICG-Dox nano-drug, after the TPGS emulsified nano-drug and the SCG7901/VCR cells are incubated together, the SCG7901/VCR cells take up the drug better, and more adriamycin enters cell nucleus, so that the TPGS emulsified nano-drug changes the discharge microenvironment of drug-resistant cells, and provides a good prospect for treatment of drug-resistant tumors.

The TPGS nano emulsified medicine prepared by the method is simple in components, safe and nontoxic in carrier and high in medicine loading rate. Compared with the traditional single treatment mode, the combined treatment has more effective and wider application prospect. In addition, in the treatment process, the multi-modal imaging is utilized to monitor the drug release, so that the method has great significance for the whole treatment process and the evaluation of the treatment effect.

Claims

1. A novel nano drug delivery system comprises ICG, Dox and TPGS; wherein ICG represents indocyanine green, Dox represents adriamycin, TPGS represents D-alpha-tocopherol polyethylene glycol succinate, ICG and Dox of the nano-emulsion are emulsified into the emulsified nano-drug through electrostatic interaction, pi-pi accumulation and hydrophobic interaction and through TPGS, and the nano-emulsion is characterized in that: the preparation method comprises the following steps:

1) solutions were prepared separately:

dissolving adriamycin in water to prepare a solution of 1 mg/mL; dissolving indocyanine green in water to prepare a solution of 1 mg/mL; adding D-alpha-tocopherol polyethylene glycol succinate into pure water, and oscillating by a vortex oscillator at room temperature to uniformly dissolve the D-alpha-tocopherol polyethylene glycol succinate, wherein the concentration of the solution is 0.01-0.025 mg/mL;

2) solution mixing, shaking and emulsifying:

mixing the three solutions in the step 1 according to a mass ratio of 1:1:1, and then carrying out constant-temperature oscillation for 12 hours in a dark state, wherein working parameters of a constant-temperature oscillator are set to be 37 ℃ and 700 revolutions per hour;

3) centrifugal cleaning and filtering: