CN108245484B

CN108245484B - Nano-medicine composition and preparation method and application thereof

Info

Publication number: CN108245484B
Application number: CN201711451072.8A
Authority: CN
Inventors: 吴雁; 王绚; 赵彩艳
Original assignee: Beijing Institute of Nanoenergy and Nanosystems
Current assignee: Beijing Institute of Nanoenergy and Nanosystems
Priority date: 2017-12-27
Filing date: 2017-12-27
Publication date: 2020-02-21
Anticipated expiration: 2037-12-27
Also published as: CN108245484A

Abstract

The invention relates to a nano-drug composition and a preparation method thereof, wherein the nano-drug composition is prepared by carrying out an emulsification reaction on an amphiphilic polymer, a hydrophobic drug and a hydrophilic drug; wherein the amphiphilic polymer is a product obtained by mixing 4/1 polyethylene glycol-poly-epsilon-caprolactone with polyethylene glycol-poly-epsilon-caprolactone modified by tumor cell penetrating peptide Cys-Arg-Gly-Asp-Lys; the hydrophobic drug is IR 780; the hydrophilic drug is perfluorooctyl bromide. The preparation method provided by the invention is simple and feasible, the provided preparation method solves the compounding problem of hydrophilic drugs and hydrophobic drugs in the nano-drug composition, and the nano-drug composition prepared by the method shows good photodynamic treatment effect through in vitro active oxygen detection, animal experiments and the like.

Description

Nano-medicine composition and preparation method and application thereof

Technical Field

The invention relates to the field of medicine preparation, in particular to a nano-medicine composition and a preparation method thereof.

Background

Photodynamic therapy is a non-invasive treatment modality. The photosensitizer can convert oxygen molecules around the tumor into toxic Reactive Oxygen Species (ROS) under laser irradiation, thereby killing tumor cells. Photodynamic therapy is widely used in the treatment of many malignant and non-malignant cancers. However, photodynamic therapy is currently limited by the depth of penetration of the photosensitizer at the tumor site. The reason for this is that the hydraulic forces between the tumor tissue and the dense tumor stroma prevent the therapeutic agent from entering the deep and oxygen-deficient cell layer, which, however, plays an important role in tumor metastasis metabolism. In addition, the active oxygen is very short in active time and diffusion limited, and it is clear that a large volume of the tumor is not damaged by toxic active oxygen. Furthermore, the internal oxygen tension in the tumor microenvironment is another determining factor affecting the efficacy of photodynamic therapy. When tumors proliferate uncontrollably, the blood vessels inside the tumor inevitably become barren far beyond the supply of oxygen, resulting in acute hypoxia of the tumor. During photodynamic therapy, the hypoxic state of the tumor is exacerbated by the consumption of oxygen and vascular damage. When the drug reaches the tumor site, the photodynamic therapy effect will be greatly limited by insufficient oxygen supply due to the characteristic of hypoxia in the deep tissue of the tumor. Therefore, the problem to be solved is how to penetrate effective concentration of photosensitizer into the tumor and to accurately distribute the active oxygen generated by the photodynamic to the whole tumor.

Disclosure of Invention

The invention aims to provide a preparation method of a nano-drug composition, the nano-drug composition prepared by the method, and application of the nano-drug composition in preparing a drug for treating tumors.

According to the principle that the amphiphilic polymer forms the water-in-oil-in-water nano micelle through a double-emulsion method, the hydrophilic drug and the hydrophobic drug are respectively encapsulated in different capsule layers of the nano capsule, so that the co-delivery of the hydrophilic drug and the hydrophobic drug is realized, and the problem of compounding of the hydrophilic drug and the hydrophobic drug in the nano drug is solved.

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

a method for preparing a nano-drug composition comprises the following steps:

emulsifying the amphiphilic polymer, the hydrophobic drug and the hydrophilic drug;

wherein the amphiphilic polymer is a product formed by mixing 4/1 polyethylene glycol-poly-epsilon-caprolactone and polyethylene glycol-poly-epsilon-caprolactone modified by tumor cell penetrating peptide Cys-Arg-Gly-Asp-Lys. Cys-Arg-Gly-Asp-Lys (CRGDK) is a tumor cell penetrating peptide and can improve the permeability of the nano-particle to the tumor. If sufficient oxygen is delivered to the hypoxic site within the tumor as needed, along with the photosensitizer, the depth of photodynamic therapy will be increased and eradication of deeper tumors will be facilitated. In view of this, a precisely targeted nanoparticle with deep tumor penetration coupled with oxygen self-sufficiency was developed. Polyethylene glycol (PEG) -Polyepsilon Caprolactone (PCL) was chosen as the carrier due to its excellent biocompatibility and biodegradability.

The hydrophobic drug can be selected from drugs with good photosensitive effect and good water solubility, and preferably IR780 is selected.

The hydrophilic drug can be selected from drugs with higher oxygen storage capacity and good biocompatibility, preferably perfluorooctyl bromide (PFOB), and the PFOB is a drug which is insoluble in water and organic solvents, has higher oxygen storage capacity and good biocompatibility.

In a whole, the technical scheme is that PFOB and IR780 are encapsulated and enter the PEG-PCL nanoparticles, and the nanoparticles can enter deeper layers of tumors and are uniformly distributed in gaps of tumor tissues under the modification of CRGDK on the surfaces of the nanoparticles. In the hypoxic region, PFOB in the nanoparticles continuously provide oxygen to meet oxygen consumption during photodynamic therapy, thereby achieving the best therapeutic effect of depleting the entire tumor tissue.

The method comprises the following specific steps:

1) mixing and emulsifying the solution of the amphiphilic polymer and the hydrophobic drug and the hydrophilic drug to obtain a first emulsion;

2) mixing the first emulsion and the emulsifier emulsion, emulsifying to obtain a second emulsion, and separating to obtain the nanometer medicinal composition.

Step 1) of the present invention,

the content of the amphiphilic polymer in the solution of the amphiphilic polymer and the hydrophobic drug is not particularly required, but in order to further improve the use effect of the nano-drug composition, the concentration of the amphiphilic polymer is preferably 10 to 50mg/ml, more preferably 20 mg/ml.

The content of the hydrophobic drug in the solution of the amphiphilic polymer and the hydrophobic drug is not particularly required, but in order to further improve the use effect of the nano-drug composition, the concentration of the hydrophobic drug is preferably 0.01 to 50mg/mL, more preferably 0.015 to 0.05mg/mL, and still more preferably 0.025 mg/mL.

As a preferable technical scheme, in order to further improve the use effect of the nano-drug composition, in the preparation method, the mass ratio of the amphiphilic polymer to the hydrophobic drug in the step 1) is 1000:1-2: 1. When the mass ratio is within the range, the generated nano-drug composition has better activity and stability.

The hydrophilic drug is in liquid state, and in order to further improve the using effect of the nano-drug composition, the amount of the hydrophilic drug is preferably 100-300 microliters.

The volume ratio of the amphiphilic polymer and hydrophobic drug solution to the hydrophilic drug solution is not particularly required, but in order to further improve the use effect of the nano-drug composition, as a preferred technical scheme, in the preparation method, the volume ratio of the amphiphilic polymer and hydrophobic drug solution to the hydrophilic drug in step 1) is 100:1-2:1, preferably 5-10:1 (with 5:1 as the best option). When the volume ratio is in the range, the generated nano-drug composition can be ensured to have better stability.

The amphiphilic polymer and hydrophobic drug are dissolved in an organic solvent to form a solution, the organic solvent being capable of dissolving the amphiphilic polymer but being insoluble in water and not chemically reacting with the amphiphilic polymer, the hydrophobic drug and the hydrophilic drug under emulsification conditions; preferably selected from one or more of dichloromethane, trichloromethane or methyl butanone, more preferably dichloromethane and/or trichloromethane.

Step 2) of the present invention,

the emulsifier can be selected conventionally in the field of preparation of general pharmaceutical compositions, and as a preferred technical scheme, in the preparation method, the emulsifier in the step 2) is selected from one or more of polyvinyl alcohol, F68, Tween 80 or sodium dodecyl sulfate, and is preferably polyvinyl alcohol and/or F68. The emulsifier is selected, so that a better emulsifying effect can be achieved by aiming at the hydrophilic drug, the hydrophobic drug and the amphiphilic polymer, and the activity of the pharmaceutical composition is further improved.

The emulsifier emulsion is an aqueous solution of an emulsifier, the content of the emulsifier is not particularly required, and in order to further improve the using effect of the nano-drug composition, the concentration of the emulsifier in the emulsifier emulsion is preferably 10-100ug/mL, and more preferably 50 ug/mL.

The volume ratio of the first emulsion to the emulsifier emulsion is not particularly required, and can be a conventional choice in the field of preparation of general pharmaceutical compositions, and as a preferred technical scheme, in the preparation method, the volume ratio of the first emulsion to the emulsifier emulsion in the step 2) is 1:2-1:20, preferably 1:2-5 (with 1:3 as the best option).

According to the invention, in step 1) and step 2), as a preferred technical scheme, the emulsification method adopts one or more of an ultrasonic cell crusher emulsification method, a high-speed shearing machine emulsification method or a high-pressure homogenizer emulsification method, and further preferably adopts ultrasonic emulsification or high-speed shearing machine emulsification.

In the preparation method of the present invention, the separation in the step 2) is a process of removing the organic solvent and separating, and the process and method are not particularly required, but as a preferable embodiment, the method of removing the organic solvent in the step 2) is a method of removing the organic solvent by reduced pressure rotary evaporation; in the step 2), in the process of separating to obtain the nano-drug composition, the method for separating is not particularly required, and preferably, the separation is to remove the supernatant by centrifugation to obtain the drug composition.

The preparation method is simple and feasible, solves the problem of compounding of hydrophilic drugs and hydrophobic drugs in the nano-drug composition, and the nano-drug composition prepared by the method shows good photodynamic treatment effect through in vitro active oxygen detection, animal experiments and the like.

The invention also provides the nano-drug composition prepared by the method.

The invention also provides the application of the nano-drug composition in preparing drugs for treating tumors.

The nano-drug composition is used for treating tumors and can achieve good photodynamic therapy effect.

Drawings

FIG. 1 is a transmission electron microscope image of the nano-drug composition obtained in the example;

FIG. 2 is a graph of the particle size of the example nano-drug composition measured by a laser particle sizer;

FIG. 3 is a diagram of an experiment for measuring in vitro active oxygen of the nano-drug composition;

FIG. 4 is a histological section view for observing the distribution of the nano-drug composition in tumor tissue;

FIG. 5 shows the saline-free CRGDK-unmodified and coated IR780 nm drug group; the CRGDK-modified nano-drug composition-free laser group and a growth curve chart of a tumor treated by the CRGDK-modified nano-drug composition laser group.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Amphiphilic polymers (where mPEG-PLGA/CRGDK-PEG-PLGA. 4/1) were obtained from Sienna Rexi Biotech Ltd and had a molecular weight of 2.5X 10⁴；

Polyvinyl alcohol, available from Xiong chemical reagents GmbH, model number PVA-124, with a saponification degree of 85 mol%; a viscosity at 25 ℃ of 700 mPas;

transmission electron microscopy FEI, Tecnai G220S-TWIN, 200 kV;

zeta potentiometer Zetasizer NanoZS.

Example 1

The present embodiment provides a nano-drug composition and a preparation method thereof.

1) Dissolving 20mg of an amphiphilic polymer and 25. mu.g of IR780 in 1mL of methylene chloride solution to obtain a first solution; the second solution is 200 mu L PFOB; adding 200 mu L of the second solution into the first solution, and emulsifying by using ultrasonic waves at the power of 70W for 3 minutes to obtain a first emulsion.

2) Preparing polyvinyl alcohol into a 1% aqueous solution to obtain a third solution; adding the first emulsion into 4ml of the third solution, and emulsifying by ultrasonic waves at the power of 200W for 5 minutes to obtain a second emulsion.

3) And (4) at room temperature, carrying out rotary evaporation to remove the organic solvent to obtain a rotary evaporated product. Centrifuging the rotary evaporated product at 13000r/min for 10 min, and collecting precipitate to obtain the nanometer medicinal composition.

The nano-drug composition obtained in this example (see fig. 1) is typically spherical and observed under a transmission electron microscope, and the particle size is about 200 nm.

The particle size of the nano-drug composition obtained in this example was measured to be 201.5. + -. 4.7nm using Zeta potentiometer (see FIG. 2).

Example 2

1) Dissolving 30mg of an amphiphilic polymer and 30. mu.g of IR780 in 1mL of methylene chloride solution to obtain a first solution; the second solution is 220 mu L PFOB; and adding the second solution into the first solution, and emulsifying by using ultrasonic waves at the power of 70W for 3 minutes to obtain a first emulsion.

The characterization information is the same as in example 1.

Test example 1

The experimental example provides the detection and data of active oxygen generated during the photodynamic therapy of tumor by the nano-drug composition prepared in example 1.

By measuring the singlet oxygen production of the nano-drug composition under aerobic conditions (normal atmospheric conditions) and under hypoxic conditions (which is realized by putting a reagent capable of reducing the oxygen content, Anaerocult, which is a reagent for producing an anaerobic culture medium in an anaerobic tank into a closed container), after 30s, observation shows that the nano-drug composition can still produce more singlet oxygen under the hypoxic environment, which indicates that the oxygen self-sufficient nano-drug composition can adapt to the hypoxic environment (see figure 3).

Test example 2

This test example provides data on the distribution of the nano-drug composition prepared in example 1 at the tumor site.

The nano-drug composition was injected into the tail vein of tumor-bearing nude mice (injection amount is 200. mu.l, measured as IR780, IR780 concentration is 100. mu.g/ml), and after 24 hours, tumor tissues were extracted and tissue sections were analyzed, and it was observed that the nano-drug composition was not only distributed at the tissue edges but also penetrated into the tissues (see FIG. 4).

Test example 3

The experimental example provides experimental detection and data of the nano-drug composition prepared in example 1 in photodynamic therapy of tumor in vivo.

Tumor-bearing mice were divided into 4 groups, namely a normal saline group, a CRGDK-free modified nano-drug composition-free laser group (NPs/IR780+ NIR), a CRGDK-free modified nano-drug composition laser group (NPs/IR780/PFOB + NIR), and a CRGDK-modified nano-drug composition laser group (CRGDK-NPs/IR780/PFOB + NIR). Respectively injecting the nano-drug into a tumor-bearing nude mouse tail vein (the injection amount is 200 mul, calculated by IR780, the IR780 concentration is 100 mug/ml), and after 24 hours, the laser group uses the laser with the wavelength of 808nm and the power of 2W/cm²The tumor site was irradiated with the laser light for 20 s. The size of the tumor in each group of tumor-bearing nude mice was recorded daily over 14 days. (see fig. 5) it can be found that the nano-drug composition of example 1 provided by the present invention has an excellent effect in inhibiting tumor.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method of preparing a nano-drug composition, comprising:

1) mixing and emulsifying a solution containing an amphiphilic polymer and a hydrophobic drug and a hydrophilic drug to obtain a first emulsion;

2) adding an emulsifier emulsion into the first emulsion, and performing secondary emulsification to obtain the nano-drug composition;

wherein the amphiphilic polymer is a product of mixing 4/1 polyethylene glycol-polyepsilon caprolactone with a tumor cell penetrating peptide Cys-Arg-Gly-Asp-Lys modified polyethylene glycol-polyepsilon caprolactone;

the hydrophobic drug is IR 780;

the hydrophilic drug is perfluorooctyl bromide;

the solution is formed by dissolving the amphiphilic polymer and the hydrophobic drug in an organic solvent, wherein the organic solvent is dichloromethane and/or trichloromethane.

2. The method of claim 1, wherein the emulsification in step 1) and step 2) is independently selected from one of ultrasonic cell disruptor emulsification, high speed shearing emulsification, and high pressure homogenizer emulsification.

3. The method of claim 2, wherein the emulsifying is ultrasonic emulsification or high speed shearer emulsification.

4. The production method according to any one of claims 1 to 3, wherein the concentration of the amphiphilic polymer in the solution is 10 to 50 mg/ml.

5. The method of claim 4, wherein the concentration of the amphiphilic polymer in the solution is 20 mg/ml.

6. The method according to any one of claims 1 to 3, wherein the concentration of the hydrophobic drug in the solution is 0.01 to 50 mg/mL.

7. The method of claim 6, wherein the concentration of the hydrophobic drug in the solution is 5-20 mg/mL.

8. The method of claim 7, wherein the concentration of the hydrophobic drug in the solution is 10 mg/mL.

9. The production method according to any one of claims 1 to 3, wherein the mass ratio of the amphiphilic polymer to the hydrophobic drug in step 1) is 1000:1 to 2: 1.

10. The production method according to any one of claims 1 to 3, wherein the volume ratio of the solution of the amphiphilic polymer and the hydrophobic drug to the hydrophilic drug in step 1) is 100:1 to 2: 1.

11. The method for preparing a hydrophobic drug according to claim 10, wherein the volume ratio of the solution of the amphiphilic polymer and the hydrophobic drug to the hydrophilic drug in step 1) is 5-10: 1.

12. The method according to any one of claims 1 to 3, wherein in step 2), the emulsifier is selected from one or more of polyvinyl alcohol, F68, Tween 80 or sodium dodecyl sulfate;

the concentration of the emulsifier in the emulsifier emulsion is 10-100 ug/mL.

13. The preparation method according to claim 12, wherein in step 2), the emulsifier is polyvinyl alcohol and/or F68; the concentration of the emulsifier in the emulsifier emulsion was 50 ug/mL.

14. The method according to any one of claims 1 to 3, wherein the volume ratio of the first emulsion to the emulsifier emulsion in step 2) is from 1:2 to 1: 20.

15. The method according to claim 14, wherein the volume ratio of the first emulsion to the emulsifier emulsion in step 2) is 1: 2-5.

16. A nano-drug composition prepared by the method of any one of claims 1 to 15.

17. Use of the nano-pharmaceutical composition of claim 16 in the preparation of a medicament for the treatment of a tumor.