TWI565478B - A manufactruring method of non-cytotoxic hyaluronic acid silver nanoparticles for skin cellulose cells - Google Patents

Description

Method for producing hyaluronic acid nano silver particles which are not toxic to skin fiber cells

The invention relates to a method for manufacturing nano silver nanoparticles of hyaluronic acid, in particular to a material for synthesizing nano silver particles with hyaluronic acid, for preparing nanometer silver hyaluronic acid which is non-toxic to skin fiber cells and capable of promoting skin fiber cell growth. particle.

The metallic element silver, although having excellent electrical and thermal conductivity, is not very chemically active. If the metal silver is subjected to nanocrystallization, the silver particle size after the treatment is less than 100 nm, which is called nano silver. At present, it is necessary to use a chemical reduction method for obtaining nano-silver particles having high uniformity, by adding a reducing agent to a silver ion solution, causing ions to obtain electrons for reduction, and adding an appropriate amount of a protective agent to inhibit aggregation of silver particles. The particle size becomes large.

Nano-materials, although they have a small volume and easy to enter the cells of the organism, but the biocompatibility and biological toxicity can not be ignored, so in the derivation of the material, the nanoparticles can be combined with biomolecules to form a complex. In order to facilitate access to cells and the use of bio-molecular characteristics to achieve biomarkers, bio-detection, gene therapy, drug delivery, drug targets, immunoassays, enzyme immobilization and bioselective separation.

Hyaluronic acid is irregular in the normal physiological conditions of the extracellular matrix (ECM), which can be freely circulated or present in tissue-associated state. Because hyaluronic acid has many biological properties and amazing viscoelastic properties, high or Low molar mass of hyaluronic acid has been widely used in pharmaceutical or cosmetic care products.

In general, conventional nano-silver particles are important in antibacterial, catalytic, and surface plasma resonance, and their biological activities are mostly carried out using chitosan as a protective agent to synthesize nano particles.

However, there is currently no medical grade of chitosan protective agent available, so direct application of conventional nano silver particles to the human body may have doubts. In view of the problems of the prior art, the present inventors have utilized a variety of bioactive properties of hyaluronic acid to greatly enhance the utility of synthetic nanoparticles, and proposed a method for producing hyaluronic acid as a material for synthesizing silver nanoparticles.

In view of the above-mentioned problems of the prior art, the object of the present invention is to provide a nano-silver nanoparticle which is non-toxic to skin fiber cells, so as to solve the problem that the nano silver particles synthesized by the lack of a protective agent are poisonous to skin fiber cells. The problem of function.

According to the object of the present invention, a method for producing a nanoparticle of hyaluronic acid nanoparticle which is not toxic to skin fibrocytes is provided, which comprises the steps of: mixing hyaluronic acid as a protective agent with silver nitrate to form a mixed solution; and adding a reducing agent In the mixed solution, a reduction reaction is carried out to obtain nano silver nanoparticles of hyaluronic acid.

Preferably, the concentration of hyaluronic acid is 0.02 to 0.12 mg/ml.

Preferably, the concentration of silver nitrate can be from 0.1 to 0.3 mg/ml.

Preferably, the reducing agent comprises sodium borohydride.

Preferably, the concentration of the reducing agent may be from 0.001 to 0.01 M.

Preferably, the particle diameter of the nanoparticle of hyaluronic acid is between 10 and 50 nm.

Preferably, hyaluronic acid and silver nitrate are mixed for 10 minutes to 30 minutes in a dark environment.

In accordance with the purpose of the present invention, a nanoparticulate silver nanoparticle which is non-toxic to skin fibrocytes is further produced by the above-described manufacturing method.

According to the present invention, the hyaluronic acid nano silver particles which are not toxic to skin fiber cells and the method for producing the same are characterized in that hyaluronic acid rich in various biologically active properties is used as a protective agent for synthetic nano silver particles. The synthesized nano-silver hyaluronic acid particles are not toxic to skin fibroblasts, thereby promoting skin fiber cell growth.

S11~S12‧‧‧Step process

Fig. 1 is a flow chart showing a method for producing a nanoparticle of hyaluronic acid nanoparticle which is not toxic to skin fibrocytes.

Fig. 2 is an analysis diagram showing the particle size of the nanoparticle of hyaluronic acid of the present invention.

Fig. 3 is a graph showing the analysis of the wavelength and absorbance of the nanoparticle silver hyaluronic acid particles of the present invention.

Figure 4 is a schematic diagram showing the cytotoxicity test of the nanoparticle silver hyaluronic acid particles of the present invention.

The technical features, contents, and advantages of the present invention, as well as the advantages thereof, will be apparent to those skilled in the art, and the present invention will be described in detail with reference to the accompanying drawings.

Method for producing hyaluronic acid nano silver particles which are not toxic to skin fiber cells of the present invention

Please refer to FIG. 1 , which is a flow chart of a method for producing hyaluronic acid nano silver particles which are not toxic to skin fibrocytes. In the flow chart, the manufacturing method may include the following steps: Step S11: mixing hyaluronic acid as a protective agent with silver nitrate to form a mixed solution. Step S12: adding a reducing agent to the mixed solution to carry out a reduction reaction, that is, preparing silver nanoparticles of hyaluronic acid. By the above steps, nanoparticulate silver nanoparticles which are not toxic to skin fibrocytes can be produced to thereby promote skin fiber cell growth.

In this embodiment, before step S11, 8-20 ml of hyaluronic acid having a concentration of 0.02 to 0.12 mg/ml is firstly taken into a container, and a stirring magnet is added to stir at a suitable rotation speed for 10 minutes to 30 minutes. Thereafter, 0.1 to 0.45 ml of silver nitrate having a concentration of 0.1 to 0.3 mg/ml is added in a dark environment, and stirred for 10 minutes to 30 minutes. Next, in step S12, 0.075 to 0.35 ml of a sodium borohydride having a concentration of 0.001 to 0.01 M as a reducing agent is added to carry out a reduction reaction with silver nitrate, and after the reaction for 10 minutes to 30 minutes, the reducing agent is again added to oscillate. The reaction was stirred at an appropriate speed for 2 hours to obtain the nano-silver cellulose particles. The above is only a preferred embodiment, but should not be construed as limiting.

Qualitative analysis of nano-silver particles of hyaluronic acid which are not toxic to skin fibroblasts

(1) Determination of particle size

In this embodiment, a particle size analyzer is used to measure the particle size of the nanoparticulate silver hyaluronate particles obtained by the above method. According to the measurement results, the particle diameter of the nano-silver hyaluronic acid particles is between 10 and 50 nm, wherein 74.719% of the nano-silver cellulose particles have a particle size distribution of about 16.84 nm, as shown in FIG. 2 . .

(2) Determination of wavelength and absorbance

Please refer to the following list 1 and figure 3 below. Table 1 is a table of relevant data for analysis of wavelength and absorbance of nano silver and hyaluronic acid in nanoparticle silver hyaluronic acid particles which are not toxic to skin fiber cells according to the present invention. Fig. 3 is a graph showing the analysis of the wavelength and absorbance of the nanoparticle silver hyaluronic acid particles of the present invention.

In general, in the qualitative analysis of nano silver, a full-wavelength scan is performed by an ultraviolet/visible (UV-vis) spectrometer, which shows that there is a specific absorption spectrum near a wavelength of 410 nm. Therefore, in the present embodiment, the wavelength and the absorbance of the nano silver and hyaluronic acid in the sodium hyaluronic acid particles synthesized by the above method are measured by a spectrophotometer to observe the wavelength of the nano silver in the silver nanoparticle of the hyaluronic acid. Whether it falls within the normal range of 395nm~415nm. It can be seen from the results that the wavelength of nano silver measured at the beginning was 395 nm, and the wavelength was maintained at about 395 nm after 4 months of continuous stability test. Thus, it can be found that the sodium hyaluronic acid silver nanoparticles prepared by the present invention have better stability and are not easily deteriorated.

Cytotoxicity test for hyaluronic acid nano silver particles that are not toxic to skin fibroblasts

First, skin fibroblasts (NIH3T3) were seeded in 96well microplates at a cell size of 4 x 104 cells/well per well of 100 μl, and cultured in an incubator for 12 hours. Then, respectively, adding 0.25, which is obtained by the above method, 0.5, 1, 2 μg/ml of nano-silver hyaluronic acid particles were placed in an incubator for 24 hours and 48 hours, respectively. Thereafter, 10 μl/well of 0.5 mg/ml of MTT solution was added, and after incubation for 4 hours in a 37 ° C, 5% CO 2 cell incubator, 100 μl/well of 10% sodium dodecyl sulfate-hydrochloric acid ( SDS-HCl) to dissolve the formazan crystals produced by the cytotoxicity test. Finally, the absorbance of the nanoparticle silver hyaluronic acid particles was detected at an wavelength of 570 nm using an ELISA reader to calculate the cell activity, as shown in FIG.

According to the experimental results of the cytotoxicity test, the cell viability of the sodium hyaluronan particles in the incubator cultured for 24 hours can reach nearly 100%, and the cell viability of the silver hyaluronic acid silver nanoparticles can be maintained after 48 hours of culture. Up to 100%. According to the results, it is shown that the 0.25, 0.5, 1, 2 μg/ml sodium hyaluronan particles prepared by the above method are not toxic to skin fiber cells, and the growth of skin fiber cells can be effectively increased with increasing concentration.

In summary, the hyaluronic acid nano silver particles which are not toxic to skin fiber cells and the method for producing the same according to the present invention are made by using hyaluronic acid rich in various biologically active properties as a protective agent for synthetic nano silver particles. The synthetic nano-silver hyaluronic acid particles are not toxic to skin fibrocytes, and can thereby promote skin fiber cell growth. In this way, it contributes to the medical application of the nano-silver cellulose particles.

The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

S11~S12‧‧‧Step process

Claims (1)

The invention relates to a method for preparing nano-silver particles of hyaluronic acid which is not toxic to skin fiber cells, comprising the steps of: mixing hyaluronic acid and silver nitrate as a protective agent in a dark environment for 10 minutes to 30 minutes to form a mixed solution. Wherein the concentration of the hyaluronic acid is 0.01 to 0.12 mg/ml, and the concentration of the silver nitrate is 0.1 to 0.3 mg/ml; and the sodium borohydride having a concentration of 0.001 to 0.01 M as a reducing agent is added thereto. In the mixed solution, the reaction is carried out with silver nitrate, and after reacting for 10 minutes to 30 minutes, the reducing agent is added again, and the reaction is stirred at an appropriate rotation speed by an oscillator to obtain a silver nanoparticle of hyaluronic acid, of which 74.719% The particle size distribution of the silver nanoparticles of hyaluronic acid is about 16.84 nm.