CN110179810B

CN110179810B - A pharmaceutical composition with anti-tumor effect

Info

Publication number: CN110179810B
Application number: CN201910550220.4A
Authority: CN
Inventors: 史劲松; 李恒; 季珂; 虞海惠; 耿燕; 许正宏; 刘朋; 濮婷琳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2022-03-25
Anticipated expiration: 2039-06-24
Also published as: CN110179810A

Abstract

The invention discloses a pharmaceutical composition with chitosan oligosaccharide and doxorubicin hydrochloride as active ingredients. The administration mode of incubating the chitosan oligosaccharide and the tested cells for a certain time and then administering the adriamycin hydrochloride is adopted, so that the independent administration concentration of the adriamycin hydrochloride reaching the same tumor cell inhibition rate can be obviously reduced. The pharmaceutical composition has good anti-tumor effect on MDA-MB-231 human breast cancer cells, PATU-8988 human pancreatic cancer cells and HepG2 human liver cancer cells. The preparation method is simple, has low cost and is easy to be applied and popularized in various medicament forms.

Description

A pharmaceutical composition with anti-tumor effect

Technical Field

The invention relates to the field of medicines, in particular to an anti-tumor application of chitosan oligosaccharide as a medicinal adjuvant.

Background

Doxorubicin Hydrochloride (Doxorubicin Hydrochloride) is a broad-spectrum antitumor drug that acts on various stages of the cell cycle to induce apoptosis. The disadvantage of using adriamycin hydrochloride is that the adriamycin hydrochloride has strong cytotoxicity, is easy to cause normal cell death and can cause drug resistance after long-term use. Therefore, it is often used in combination with other drugs clinically.

Chitosan oligosaccharides (chitosanoligosaccharides) are small molecular oligosaccharides obtained by degrading chitosan derived from shrimp and crab shells, and have a polymerization degree of 2-10. It has good water solubility, easy dispersion and absorption, and wide bioactivity, such as antioxidant, antibacterial, neuroprotection, anticoagulant, antitumor, and blood lipid reducing effects. The chitosan oligosaccharide is a natural saccharide only carrying cations in nature, can directly change the permeability of a tumor cell membrane in a targeted manner through electrostatic action, and has no toxic effect on normal cells. Can also indirectly play an anti-tumor role by enhancing the function of an immune system.

Therefore, the pharmaceutical composition containing the chitosan oligosaccharide and the doxorubicin hydrochloride is designed and developed, the dosage of the doxorubicin hydrochloride used alone is reduced, the side effect of the doxorubicin hydrochloride is reduced, and the anti-tumor effect is improved.

Disclosure of Invention

The invention aims to provide a chitosan oligosaccharide-assisted anti-tumor pharmaceutical composition and a drug delivery scheme thereof, develops the extended activity of chitosan oligosaccharide as functional sugar, and provides new resources for developing natural anti-tumor preparations in the fields of medicine, food science and the like. In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

an antitumor composition containing chitosan oligosaccharide and doxorubicin hydrochloride as active components is effective on different tumor cells. The chitosan oligosaccharide and the tested cells are incubated for a certain time, and then doxorubicin hydrochloride is administered, wherein the preferable time is 0.5-18 h. The concentration of the chitosan oligosaccharide is preferably in the range of 2-128 mug/mL.

The method has the advantages that the survival rate of the tumor cells is obviously reduced, compared with the doxorubicin hydrochloride group which is singly administered with half lethal dose, the survival rate of the tumor cells is reduced by 20-30%, and the damage to normal cells is reduced.

Drawings

FIG. 1 is a graph showing the survival rate of chitosan oligosaccharide to normal cells. The test cell is MRC-5

FIG. 2 is a graph of cell viability for optimized chitosan oligosaccharide concentrations and incubation times with test cells. The tested cell is MDA-MB-231

Detailed Description

The invention will be further illustrated by the following detailed description.

Example 1 Effect of Chitosan oligosaccharides on Normal cell survival

Human embryonic lung fibroblast MRC-5 is selected and given with chitosan oligosaccharide with different concentrations for 24, 48 and 72 hours, and as can be seen from figure 1, the survival rate of the cells has no significant difference along with the prolonging of the action time, which indicates that the chitosan oligosaccharide does not inhibit the normal survival rate of the cells.

The experimental process comprises the following steps: inoculating cells: growing the cells to 90%, carrying out cell passage operation, preparing the completely digested cells into single cell suspension by using complete culture medium, and culturing at 5X 10³one/mL of the solution was plated in a 96-well plate at 200. mu.L per well, and 150. mu.L of DPBS was added to each well in the marginal wells to prevent evaporation of the solution, and the groups were set as blank and drug groups, each group having 3 replicate wells.

Culturing the cells: placing 96-well plate at 37 deg.C and 5% CO₂And performing adherent culture in a cell culture box for 24 hours.

Administration: adding chitosan oligosaccharide with different concentrations into each well of the drug group, adding 100 μ L of complete culture medium into each well of the control group, and placing into an incubator for incubation for 24, 48 and 72 hours.

And (3) detection: absorbing the stock solution, adding 100 mu L of mixed solution of CCK-8 and complete culture medium diluted by 1:10 into each hole, incubating for 1-2h, and detecting the light absorption value at 450 nm.

The cell survival rate (%) was calculated by the formula (drug OD value-blank OD value)/(control OD value-blank OD value) × 100%.

Example 2 examination of the mode of administration

The dosage regimen was determined by comparing the mode of administration of the test cell chitosan oligosaccharide or doxorubicin hydrochloride alone with the mode of administration of doxorubicin hydrochloride in combination.

Detection of IC of doxorubicin hydrochloride on different test cells₅₀As is clear from the results in Table 1, doxorubicin hydrochloride acts on the IC of MDA-MB-231 cells₅₀The lowest, 2.5. mu.g/mL.

IC on each test cell in Adriamycin hydrochloride₅₀Under the concentration, chitosan oligosaccharide is compounded, different administration modes are changed,the inhibitory effect on each tumor cell was evaluated. The results are shown in table 2, the chitosan oligosaccharide alone administration and the doxorubicin hydrochloride and chitosan oligosaccharide simultaneous incubation did not produce significant inhibition effect on the survival rate of each tested tumor cell, when the chitosan oligosaccharide was pre-incubated and then the doxorubicin hydrochloride was administered, the survival rate of each tumor cell was significantly reduced by more than 50%, wherein the cell survival rate of MDA-MB-231 was reduced by 83.9%.

The cell culture and detection and calculation methods were the same as in example 1. When the drug is administered and evaluated, 100 μ L of culture medium containing chitosan oligosaccharide or doxorubicin hydrochloride with different concentrations is added into each well of the drug group, 100 μ L of complete culture medium is added into each well of the control group, and the control group is placed into an incubator for incubation. The simultaneous incubation is to add chitosan oligosaccharide and doxorubicin hydrochloride with certain concentration into the tested cells at the same time for incubation. The chitosan oligosaccharide is pre-incubated by incubating the chitosan oligosaccharide and the tested cells for a certain time and then adding the doxorubicin hydrochloride. The dosage concentration of the doxorubicin hydrochloride is consistent with the final concentration of other groups. The adriamycin hydrochloride dosage concentration of different cell strains is IC₅₀The value is obtained.

TABLE 1 Effect of doxorubicin hydrochloride on IC of each test cell₅₀

TABLE 2 Effect of different administration modes on tumor cell survival

Note: and each cell line IC₅₀Compared with the positive medicine group, the medicine composition has the advantages that,^*p<0.05，^**p<0.01，^***p<0.001。

example 3 Chitosan oligosaccharide Pre-incubation time and concentration optimization

The MDA-MB-231 cells having the best anti-tumor effect in example 2 were used as test cells, and chitosan oligosaccharide incubation time and concentration were optimized to evaluate cell survival rate. As can be seen from FIG. 2, the survival rate decreased in a dose-dependent manner, and the concentration of the effective drug was 2. mu.g/mL, whereas the survival rate of 128. mu.g/mL or more was kept equal, and the concentration was estimated to be saturated. The time period with significant difference in pre-incubation time is 0.5-24 h, and the survival rates of 24h and 18h are equal, so 18-24 h is not selected. Therefore, the optimized incubation time is 0.5-18 h, and the concentration range is 2-128 mug/mL.

The experiment and detection procedure were the same as in example 1. For administration, the drug group is added with medium containing chitosan oligosaccharide at concentration of 0.5, 2, 8, 32, 128, 256 μ g/mL per well, 100 μ L per well, and doxorubicin hydrochloride (final concentration of IC on MDA-MB-231 cells) at intervals of 0.2, 0.5, 3, 18, 24h₅₀Value). Control group was incubated with 100. mu.L of complete medium per well in an incubator.

Example 4 doxorubicin hydrochloride concentration optimization

IC of different tested tumor cells₅₀On the basis, the concentration of the fixed chitosan oligosaccharide is 2 mug/mL, the dosage concentration of the doxorubicin hydrochloride is decreased in turn, and the concentration of the doxorubicin hydrochloride required by the cell survival rate of 50 percent is recorded. As shown in Table 3, the survival rate of each tumor cell tested was 50% and the doxorubicin hydrochloride concentration was lower than that of the single drug administration, in which the doxorubicin hydrochloride concentration of MDA-MB-231 cells was 40%.

The experimental procedure was as in example 1.

TABLE 3 cell survival 50% (IC) for different modes of administration₅₀) Corresponding adriamycin hydrochloride administration concentration

Claims

1. The application of chitosan oligosaccharide as a medicinal adjuvant in improving the in-vitro inhibition effect of doxorubicin hydrochloride on tumor cells is characterized in that the chitosan oligosaccharide and the tumor cells are incubated for a period of time, and then the doxorubicin hydrochloride is added, wherein the incubation time is 0.5-18 h; the concentration of the chitosan oligosaccharide is 2 mug/mL; and when the tumor cell is MDA-MB-231 human breast cancer cell, the concentration of the doxorubicin hydrochloride is 1.5 mug/mL, when the tumor cell is PATU-8988 human pancreatic cancer cell, the concentration of the doxorubicin hydrochloride is 3.5 mug/mL, and when the tumor cell is HepG2 human liver cancer cell, the concentration of the doxorubicin hydrochloride is 5.5 mug/mL.

2. The application of the chitosan oligosaccharide as a medicinal adjuvant in improving the in-vitro inhibition effect of doxorubicin hydrochloride on tumor cells, as claimed in claim 1, wherein the polymerization degree of the chitosan oligosaccharide is 2-10.