WO2014180012A1

WO2014180012A1 - Preparation based on gold, silver, mixture of gold and silver, and glutathione/chitosan, and application of same

Info

Publication number: WO2014180012A1
Application number: PCT/CN2013/076542
Authority: WO
Inventors: 王雪梅; 高生平; 叶静; 王建玲
Original assignee: 东南大学
Priority date: 2013-05-07
Filing date: 2013-05-31
Publication date: 2014-11-13
Also published as: CN103212056B; CN103212056A

Abstract

Disclosed are a preparation based on gold, silver, a mixture of gold and silver, and glutathione/chitosan, and an application of same. The preparation can target some lesion positions such as tumor cells/tumor tissue in the living body, and effectively accelerate apoptosis of the tumor cells and ablation of the tumor tissue. Therefore, the preparation can be used in clinic as a potential drug preparation for tumor early treatment, and can implement correct targeted therapy for the tumor portion of the living body.

Description

[Name of invention by ISA according to Rule 37.2] Based on gold, silver and gold-silver mixtures with glutathione/chitosan preparations and their applications

Technical field

The invention relates to the synthesis of precious metal gold, silver ions and glutathione/chitosan preparations and the disease targeted treatment method thereof, and particularly relates to a precious metal gold, silver and gold-silver mixture with glutathione and chitosan A method for synthesizing a preparation and its use in the treatment of diseases such as living tumors.

Background technique

It is well known that early detection of cancerous sites and timely targeted therapy is one of the most effective treatments for cancer. However, because the early symptoms of cancer are not obvious, it is difficult to be discovered in time, and the current routine histopathological diagnosis method is difficult to detect the biochemical and molecular abnormalities of the diseased tissue and early stage of the cell, so it is convenient, quick and accurate for early diagnosis of cancer. The method is of great significance, so researching more advanced diagnostic methods and preparations to achieve accurate and timely early diagnosis and treatment of cancer has always been one of the efforts of people.

Nanomaterials have attracted much attention in the research of cancer treatment because of their special properties. Application in biomedical therapy: early diagnosis of diseases such as tumors, analysis or molecular imaging of tumor evolution in vivo; improvement of drug therapy targeting, avoiding biological or physiological barriers in vivo; Perform real-time monitoring.

For cancers that have already occurred, how to improve the cure rate of cancer is very important through effective means. The research on the application of nanotechnology for the treatment of malignant tumors has been carried out in the world. The present invention has found that precious metal gold, silver plasma and glutathione/chitosan synthetic preparations have a unique role in promoting tumor cell apoptosis, and provide excellent means and methods for cancer targeted therapy in biomedicine; Precious metal preparations such as gold and silver can accurately locate and target malignant tumor cells/tissue, which is an important direction for early prevention and treatment of cancer, and provides a new method and basis for the treatment of major diseases such as cancer.

technical problem

The present invention provides a formulation based on gold, silver and gold and silver with glutathione/chitosan and its use.

Technical solution

In view of the deficiencies of the prior art, the present invention provides a formulation based on gold, silver and gold and silver with glutathione/chitosan and its use.

A formulation based on gold, silver and gold and silver and a glutathione/chitosan preparation characterized by the following steps:

A, the concentration will be 0.05-5.0 A mixture of mmol/L gold reagent and glutathione/chitosan at a concentration of 0.1-100 mmol/L is added dropwise to complete the solution until the solution is just clear; glutathione/chitosan The mixed solution is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

or

B, 0.01-1.0 A mixed solution of mmol/L silver reagent and glutathione/chitosan at a concentration of 0.1-100 mmol/L is added dropwise to complete the solution until the solution is just clear; wherein glutathione/chitosan The mixed solution is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

or

C. Gold reagent with a concentration of 0.05-5.0 mmol/L and 0.01-1.0 mmol/L The silver reagent is mixed in a ratio of 4:1 to 1:4 by volume, mixed with glutathione/chitosan at a concentration of 0.1-100 mmol, and mixed by dropwise addition until the solution is just clarified; a mixed solution of glutathione/chitosan is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

The gold reagent is one of chloroauric acid, tetrakisium octachloride, gold chloride, gold trichloride, gold monochloride or a mixed solution of any two; the silver reagent is a silver ammonia solution, nitric acid One or a mixture of any two of silver, silver potassium cyanide, and silver chlorate solution.

The above is based on the use of a mixture of gold, silver and gold and silver and a glutathione/chitosan preparation for the preparation of an antitumor drug.

Principle: Glutathione is a thiol-containing molecule responsible for maintaining the home's redox equilibrium homeostasis. Changes in glutathione levels can be used as an important indicator for monitoring cellular oxidative stress. Cellular cancer can cause changes in glutathione concentration, while glutathione complexes, chitosan as a protective agent for gold and silver ion complexing agents can target cancerous cells, and glutathione can reduce gold Silver ions form gold and silver nanoclusters. Gold and silver nanoclusters can promote the apoptosis of cancerous cells, so the preparation of gold, silver ions and glutathione/chitosan can be used as a tumor drug.

Beneficial effect

A mixed solution of a certain concentration of gold reagent and silver reagent is combined with a mixed solution of glutathione/chitosan, and the synthetic precious metal preparation is subjected to targeted treatment on an animal model such as transplanted nude mice by an injection method. . The invention discovers and develops a preparation of noble metal gold, silver and glutathione/chitosan, and realizes accurate targeted treatment of living tumor sites. Because the method is easy to operate, The results are intuitive, sensitive, and highly targeted, and can be used for the treatment of tumors, and can also be applied to the treatment of living tumors such as deep tumors and large animals, and has potential clinical therapeutic application value.

Since precious metal gold, silver ions and glutathione/chitosan preparations were first used for tumor targeted therapy in the living body of the research group, domestic and foreign patents and other literatures did not report on the invention.

The invention can realize non-invasive, in situ and real-time dynamic tumor targeted therapy, and further combines fluorescence, Raman, ultrasound, CT and nuclear magnetic, etc., can perform multi-modal and multi-modal simultaneous diagnosis and accurate targeted positioning and treatment, Has broad medical application prospects.

DRAWINGS

1 is a graph showing the results of an experimental group MTT of Example 11 of the present invention;

Figure 2 is a graph showing the results of an experimental group MTT of Example 12 of the present invention;

Figure 3-4 is a comparison chart of the local injection of the experimental group for tumor diagnosis and treatment;

Figure 5-6 is a comparison of the diagnosis and treatment of tumors by tail vein injection in the experimental group.

Embodiments of the invention

Example 1

0.01-1.0 A mixed solution of mmol/L silver reagent and glutathione/chitosan at a concentration of 0.1-100 mmol/L is added dropwise to complete the solution until the solution is just clear; wherein glutathione/chitosan The mixed solution is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

The silver reagent is silver nitrate.

Example 2

The silver reagent is a silver ammonia solution.

Example 3

The concentration will be 0.05-5.0 A mixture of mmol/L gold reagent and glutathione/chitosan at a concentration of 0.1-100 mmol/L is added dropwise to complete the solution until the solution is just clear; glutathione/chitosan The mixed solution is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

The gold reagent is chloroauric acid.

Example 4

The gold reagent is tetrakisium octachloride.

Example 5

The gold reagent is gold chloride.

Example 6

The gold reagent is gold trichloride.

Example 7

The gold reagent is monochlorinated gold.

Example 8

Gold reagent at a concentration of 0.05-5.0 mmol/L and 0.01-1.0 mmol/L The silver reagent is mixed in a ratio of 4:1 to 1:4 by volume, mixed with glutathione/chitosan at a concentration of 0.1-100 mmol, and mixed by dropwise addition until the solution is just clarified; a mixed solution of glutathione/chitosan is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

The gold reagent is chloroauric acid; the silver reagent is a silver ammonia solution.

Example 9

The gold reagent is tetrakisium octachloride; the silver reagent is silver nitrate.

Example 10

Gold reagent at a concentration of 0.05-5.0 mmol/L and 0.01-1.0 mmol/L The silver reagent is mixed in a ratio of 1:1 by volume, mixed with glutathione/chitosan at a concentration of 0.1-100 mmol, and mixed by dropwise addition until the solution is just clarified; wherein glutathione a mixed solution of chitosan obtained by mixing glutathione and chitosan by a volume ratio of 1:1;

The gold reagent is chloroauric acid; the silver reagent is silver nitrate.

Example 11

MTT toxicity concentration test

1) Collect log phase Hela cells (purchased from Shanghai Institute of Biochemistry and Cell Research, Chinese Academy of Sciences), adjust the cell suspension concentration, add 100 ul per well, and plate the cells to adjust the density to 1000-10000 wells. Bacteria filled with PBS).

2) 5% CO2, incubate at 37 °C, until the cell monolayer is covered with the bottom of the well (96-well flat bottom plate), each concentration gradient is 1 ug/mL, 4 Ug/mL, 7 ug/mL, 10 ug/mL, 13 ug/mL, 16 Ug/ml silver nitrate, silver nanoclusters, silver nitrate obtained in Example 1 and glutathione/chitosan complex solution were each provided with 5 duplicate wells.

3.) 5% CO2, incubate at 37 ° C for 24 hours, add 20ul per well MTT solution (5 mg/ml, ie 0.5% MTT) was continued for 4 h. The culture was terminated and the culture medium in the well was carefully aspirated.

4) 150 ul of dimethyl sulfoxide was added to each well, and shaken at a low speed for 10 min on a shaker to fully dissolve the crystals. In the enzyme-linked immunosorbent assay OD The absorbance of each well was measured at 490 nm.

The experimental results are shown in Fig. 1. The nanocluster and related precious metal ion solution have great toxicity, and the complex solution of glutathione and chitosan and silver nitrate is low in toxicity and can be used for intravenous injection.

The preparation method of the silver nanocluster is prepared according to the application No. 201310015357.2, as follows: the culture solution containing the 0.1-100 μmol/L silver-containing solution is incubated with the cells cultured for 24 hours in a cell culture incubator for 10-72 hours, and the culture solution is prepared. Silver nanoclusters can be formed in the medium, and the silver nanoclusters in the culture solution are ultrasonically dispersed by an ultrasonic disperser, and then separated and purified to obtain silver nanoclusters.

Example 12

The MTT toxicity time parameter test includes the following steps:

1) Collect log phase Hela cells, adjust the cell suspension concentration, add 100 ul per well, and plate the cells to adjust the density to 1000-10000 wells (the edge wells are filled with sterile PBS).

2) 5% CO2, incubate at 37 °C, until the cell monolayer is covered with the bottom of the well (96-well flat bottom plate), and the concentration gradient is 16 for each well. Ug/ml silver nitrate, silver nanoclusters, and Example 1 obtained silver nitrate and glutathione/chitosan complex solution, each with 5 duplicate wells.

3.) 5% CO2, incubate at 37 °C for 12, 24, 36, 48, 72 hours, add 20ul per well MTT solution (5 mg/ml, ie 0.5% MTT) was continued for 4 h. The culture was terminated and the culture medium in the well was carefully aspirated.

The experimental results are shown in Fig. 2. In Example 1, the solution of silver nitrate and glutathione/chitosan complex solution is low in 24 hours, and after 24 hours, glutathione reduces silver ions to form silver nanoclusters, which can promote Tumor cell apoptosis.

Example 13

The nude mice were subcutaneously inoculated with a cervical cancer tumor model. The nude mice were directly injected with the local injection method to obtain silver nitrate and glutathione/chitosan complex solution for 96 hours. It was found that the tumor was reduced and gradually disappeared. The results are shown in Fig. 3-4. The results show that the silver nitrate and glutathione/chitosan complex solution obtained in Example 1 have a good targeted therapeutic effect on the tumor.

Example 14

The nude mice were subcutaneously inoculated with a cervical cancer tumor model, and the nude mice were injected with the silver nitrate and glutathione/chitosan complex solution by the tail vein injection method for 96 hours, and the tumor parts of the nude mice were observed and photographed. It was found that the tumor was reduced and gradually disappeared. The results are shown in Fig. 5-6. The solution of silver nitrate and glutathione/chitosan complex solution in Example 1 has accurate targeting and good therapeutic effect on the tumor.

Example 15

The nude mice were subcutaneously inoculated with a cervical cancer tumor model, and the complex obtained in Example 10 was injected into the nude mice by the tail vein injection method. After 96 hours, the tumor sites of the nude mice were observed and photographed, and the tumor was found to be reduced and gradually disappeared. The results show that the mixed preparation of chloroauric acid and silver nitrate obtained in Example 10 and the complex preparation of glutathione and chitosan have accurate targeting effect and good therapeutic effect on the tumor.

Example 16

The lung cancer model was subcutaneously inoculated into nude mice, and the complex obtained in Example 10 was injected into the nude mice by the tail vein injection method for 96 hours. The tumor sites of the nude mice were observed and photographed, and the tumor was found to be reduced and gradually disappeared. The results show that the complex preparation of chloroauric acid and silver nitrate mixed with glutathione and chitosan has accurate targeting and good therapeutic effect on tumor.

The results of the above specific examples show that the preparations prepared by complexing gold, silver and gold-silver mixed solution with glutathione/chitosan have good targeting and therapeutic effects on different tumors.

The invention relates to a preparation of a noble metal gold, silver and gold-silver mixture, a preparation of glutathione and chitosan and a nano-cluster biosynthesis method thereof and the application thereof in the targeted therapy of living tumors. The preparation is the first application in tumor treatment. The nano-cluster prepared by in situ biosynthesis of the lesion site can have strong fluorescence in the near-infrared region, so it can be made into a highly sensitive near-infrared fluorescence imaging contrast agent and can be used as a tumor treatment. Targeted formulation.

The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It is considered as the scope of protection of the present invention.

Claims

A formulation based on gold, silver and gold and silver and a glutathione/chitosan preparation characterized by the following steps:

A, the concentration will be 0.05-5.0 A mixture of mmol/L gold reagent and glutathione/chitosan at a concentration of 0.1-100 mmol/L is added dropwise to complete the solution until the solution is just clear; glutathione/chitosan The mixed solution is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

or

B, 0.01-1.0 A mixed solution of mmol/L silver reagent and glutathione/chitosan at a concentration of 0.1-100 mmol/L is added dropwise to complete the solution until the solution is just clear; wherein glutathione/chitosan The mixed solution is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

or

C. Gold reagent with a concentration of 0.05-5.0 mmol/L and 0.01-1.0 mmol/L The silver reagent is mixed in a ratio of 4:1 to 1:4 by volume, mixed with glutathione/chitosan at a concentration of 0.1-100 mmol, and mixed by dropwise addition until the solution is just clarified; a mixed solution of glutathione/chitosan is obtained by mixing glutathione and chitosan at a volume ratio of 1:1;

The gold reagent is one of chloroauric acid, tetrakisium octachloride, gold chloride, gold trichloride, gold monochloride or a mixed solution of any two; the silver reagent is a silver ammonia solution, nitric acid One or a mixture of two in a silver solution.
The use of a mixture of gold, silver and gold and silver and a glutathione/chitosan preparation according to claim 1 for the preparation of an antitumor drug.