CN108042801B

CN108042801B - Toluidine blue efficient photodynamic sterilization composition and application thereof

Info

Publication number: CN108042801B
Application number: CN201711167625.7A
Authority: CN
Inventors: 常丽; 李次会; 王宏
Original assignee: Beijing Dede Entrepreneurial Technology Co Ltd
Current assignee: Beijing Dede Entrepreneurial Technology Co Ltd
Priority date: 2017-11-21
Filing date: 2017-11-21
Publication date: 2020-03-27
Anticipated expiration: 2037-11-21
Also published as: CN108042801A

Abstract

The invention relates to a toluidine blue efficient photodynamic sterilization composition which comprises toluidine blue and methylene blue in a mass ratio of 1: 0.03-30. The composition provided by the invention uses the synergistic effect of toluidine blue and methylene blue, not only is better than the bactericidal effect of toluidine blue single component under the same condition, but also the obtained product is insensitive to the change of the optical wavelength range, and the bactericidal effect is not influenced by the change of the wavelength, so that the bactericidal effect is not reduced due to the change of the optical wavelength range in the application. Meanwhile, the composition provided by the invention has good stability and is convenient to store and apply.

Description

Toluidine blue efficient photodynamic sterilization composition and application thereof

Technical Field

The invention relates to the field of medicine and health, and in particular relates to a toluidine blue efficient photodynamic sterilization composition and a preparation method thereof.

Background

Photodynamic therapy is a treatment method that kills bacteria without/minimally invasive using the optical properties of a substance by combining a photosensitizer with a light wave of a certain wavelength to generate Reactive Oxygen Species (ROS). The method is widely applied to the fields of comprehensive treatment of various cancers, treatment of skin diseases, control and treatment of oral diseases and the like, and has the advantages of quick response, effect on a large number of bacteria, no immune response, no side effect and reusability. Toluidine blue gel is a photosensitizer gel with several major advantages: the gel preparation has certain viscosity and adhesiveness, and can make the medicine stay at affected part to improve therapeutic effect. Toluidine blue, on the other hand, is a nucleic acid-prone eosinophilic dye that resides in tissues with a high nuclear content. The toluidine blue has better penetration depth in tissues, can be effectively distributed in infected target tissues, but cannot penetrate normal tissues, so that the normal tissues are prevented from being killed and killed by photodynamic, and the toluidine blue has better selectivity on lesion areas and bacteria. Most importantly, the toluidine blue photosensitizer has no obvious toxic or side effect, so that the harm to human health systems is reduced, and the popularization and the application of the photodynamic therapy are facilitated.

Patent document CN106822894A discloses a photosensitive gel preparation product for photodynamic therapy of periodontitis and a preparation method thereof, wherein the gel product takes toluidine blue as a main photosensitive substance and is mainly prepared from the following components in percentage by weight: 0.001-1% of toluidine blue, 0.05-3% of hydrophilic gel, 0.01-0.4% of sodium hydroxide or 0.07-2% of triethanolamine, and the balance of water; the hydrophilic gelling agent is preferably carbomer. The patent also discloses a preparation method of the gel preparation, which comprises the following steps: dissolving carbomer in water, and stirring to swell completely; adding toluidine blue to the swollen carbomer for summarization; and adding sodium hydroxide or triethanolamine, supplementing water, fully stirring, and fully swelling to obtain the photosensitive gel preparation. The method for photodynamic therapy of periodontitis can replace antibiotic therapy, and prevent the problem of antibiotic abuse, the preparation is used for treating oral diseases caused by infection such as periodontitis, the bacteriostasis rate of one-time irradiation therapy reaches 99.99%, serious patients can be treated repeatedly at intervals, and the preparation has no toxic or side effect on normal organs and tissues, and has very wide market prospect and application value. However, the gel provided by the scheme is sensitive to the wavelength range of light when in use, and the sterilization effect of the gel is obviously reduced due to the change of the wavelength, so that the application effect of the gel is influenced; meanwhile, the long-acting stability of the product provided by the scheme is not ideal.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a toluidine blue efficient photodynamic sterilization composition.

Specifically, the composition provided by the invention comprises toluidine blue and methylene blue in a mass ratio of 1: 0.03-30.

In the scheme provided by the invention, toluidine blue and methylene blue are used as double-component photosensitive reagents, and have a synergistic effect on a sterilization effect. According to a great deal of practice, when the toluidine blue and the methylene blue are mixed and used according to the mass ratio of 1: 0.03-30, the bactericidal effect of the toluidine blue single component is better than that of the toluidine blue single component under the same condition, the obtained product is insensitive to the change of the optical wavelength range, the bactericidal effect of the product is not influenced by the change of the wavelength, and therefore the bactericidal effect cannot be reduced due to the change of the optical wavelength range in the application. In the present invention, it is further preferable that the mass ratio of toluidine blue to methylene blue is 1:0.1 to 20.

The composition provided by the invention is preferably an aqueous agent or a gel, and is prepared by taking a phosphate buffer solution or a Tris buffer solution with the pH value of 6.5-9 as a solvent, so that the composition has good stability and a good sterilization effect is kept.

In order to further improve the stability of the composition for convenient storage and use, the present invention preferably mixes sodium hyaluronate with the composition to form a gel. According to the invention, a large number of practices show that after the sodium hyaluronate is mixed with toluidine blue and methylene blue, the obtained gel can be ensured to be uniform and stable after being placed for a long time, and no color change or layering occurs. Preferably, the amount of the sodium hyaluronate is 0.1 to 100 times of that of toluidine blue.

The present invention further found that when the concentration of the composition is controlled within a specific range, it is more advantageous for its long-term stability and can reduce skin irritation while ensuring its bactericidal effect. Specifically, the concentration of the composition is preferably 0.0001g/ml to 0.001g/ml in terms of toluidine blue by mass volume. If the concentration of the composition is too high, particularly if the concentration of the sodium hyaluronate is too high, the viscosity of the system is increased, the system is agglomerated and delaminated under the action of ions, and the stability is reduced.

As a preferable embodiment of the present invention, when the composition is an aqueous solution, the preparation method thereof comprises the following steps: dissolving toluidine blue in phosphate buffer or Tris buffer, and adding methylene blue to disperse completely.

As a preferable scheme of the invention, when the composition is a gel, the preparation method comprises the following steps: dissolving toluidine blue in phosphate buffer or Tris buffer, adding methylene blue for dispersing, and adding sodium hyaluronate to prepare gel.

The composition provided by the invention can be applied to sterilization of wound surfaces before and after various operations, effectively kill various pathogenic bacteria, prevent postoperative infection, and is particularly suitable for infection prevention after minimally invasive operations.

In actual use, the composition is applied to wound surface and then irradiated by light. The illumination wavelength is preferably 630 nm-670 nm, and the illumination time is preferably 0.5-1.5 min.

The composition provided by the invention comprises toluidine blue and methylene blue, the synergistic effect of the toluidine blue and the methylene blue is better than the bactericidal effect of a toluidine blue single component under the same condition, the obtained product is insensitive to the change of the optical wavelength range, and the bactericidal effect is not influenced by the change of the wavelength, so that the bactericidal effect is not reduced due to the change of the optical wavelength range in the application. Meanwhile, the composition provided by the invention has good stability and is convenient to store and apply.

Detailed Description

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

Example 1

The embodiment provides a high-efficiency photodynamic sterilization composition which is a gel and is prepared by adopting the following method: taking 100mL of phosphate buffer solution with the pH value of 7.5, firstly adding 0.1g of toluidine blue to prepare a solution with the concentration of 0.001g/mL, adding 0.01g of methylene blue after full dispersion, and adding 0.05g of sodium hyaluronate after full dispersion to prepare a gel state.

Example 2

The embodiment provides a high-efficiency photodynamic sterilization composition which is a gel and is prepared by adopting the following method: taking 100mL of phosphate buffer solution with the pH value of 8.0, firstly adding 0.5g of toluidine blue to prepare 0.005g/mL solution, adding 0.05g of methylene blue after full dispersion, adding 0.1g of sodium hyaluronate after full dispersion to prepare a gel state.

Example 3

The embodiment provides a high-efficiency photodynamic sterilization composition which is a gel and is prepared by adopting the following method: taking 100mL of phosphate buffer solution with the pH value of 7.0, firstly adding 0.01g of toluidine blue to prepare a solution with the concentration of 0.0001g/mL, adding 0.01g of methylene blue after full dispersion, adding 1g of sodium hyaluronate after full dispersion to prepare a gel state.

Example 4

The embodiment provides a high-efficiency photodynamic sterilization composition which is a gel and is prepared by adopting the following method: 100mL of phosphate buffer solution with the pH value of 7.0 is taken, 0.1g of toluidine blue is firstly added to prepare 0.001g/mL solution, 0.01g of methylene blue is added after the solution is fully dispersed, and 0.01g of sodium hyaluronate is added to prepare the solution after the solution is fully dispersed.

Example 5

The embodiment provides a high-efficiency photodynamic sterilization composition which is a gel and is prepared by adopting the following method: taking 100mL of Tris buffer solution with the pH value of 8.0, firstly adding 0.1g of toluidine blue to prepare a solution with the concentration of 0.001g/mL, adding 0.1g of methylene blue after full dispersion, and adding 1g of sodium hyaluronate to prepare a gel state after full dispersion.

Example 6

The embodiment provides a high-efficiency photodynamic sterilization composition which is a gel and is prepared by adopting the following method: taking 100mL of Tris buffer solution with the pH value of 8.5, firstly adding 0.3g of toluidine blue to prepare a solution with the concentration of 0.003g/mL, adding 0.5g of methylene blue after full dispersion, and adding 0.5g of sodium hyaluronate to prepare a gel state after full dispersion.

Example 7

The embodiment provides a high-efficiency photodynamic sterilization composition which is a gel and is prepared by adopting the following method: taking 100mL of Tris buffer solution with the pH value of 8.5, firstly adding 1g of toluidine blue to prepare a solution with the concentration of 0.01g/mL, adding 0.1g of methylene blue after full dispersion, adding 2.5g of sodium hyaluronate after full dispersion to prepare a gel state.

Comparative example 1

Compared with example 1, the difference is only that: carbomer was used in place of the sodium hyaluronate.

Comparative example 2

Compared with example 1, the difference is only that: the phosphate buffer was replaced with 0.1% sodium hydroxide solution.

Comparative example 3

Toluidine blue gel with a concentration of 0.005g/ml was formulated with carbomer gel with a concentration of 0.05 g/ml.

Comparative example 4

Toluidine blue in water at a concentration of 0.002 g/ml.

Comparative example 5

An aqueous methylene blue solution at a concentration of 0.02 g/ml.

Experimental example 1: sterilizing effect

Inoculating strains incubated for 16-24h into nutrient broth culture medium test tubes respectively, and adjusting the concentration of bacteria liquid to 1.0 × 10⁸CFU/mL. Taking 0.5ml of bacterial liquid by a pipette, flatly paving the bacterial liquid in the middle of a stainless steel sheet with the diameter of 8mm, and controlling the tiled area to be not more than 5mm by using a bacteriostatic ring. Taking 0.5ml of liquid or 0.5g of gel of each example or comparative example respectively, placing the liquid or gel on the bacterial liquid, fully spreading the liquid so as to enable the sample to cover the bacterial liquid, and detecting the sterilization effect of each example or comparative example on staphylococcus aureus and escherichia coli under the conditions of irradiating 635nm, 650nm, 660nm and 670nm for 1 min. Then, the stainless steel sheet is washed by 10ml of sterile water and ultrasonically treated to remove bacteria, the bacteria liquid is obtained through centrifugal separation, the obtained bacteria liquid is cultured for 24 hours at 37 ℃, the growth condition of the bacteria is observed, and the sterilization effect is calculated through CFU (computational fluid dynamics) to obtain the result shown in Table 1.

Table 1: rate of sterilization

The results in table 1 show that the two-component photosensitive reagent provided by the invention has a synergistic effect on the sterilization effect, and is superior to the sterilization effect of a single component under the same conditions. Meanwhile, the single-component reagent is sensitive to the wavelength range of light, and the sterilization effect of the single-component reagent is obviously reduced due to the change of the wavelength. The double components are insensitive to the change of the optical wavelength range, and the sterilization effect of the double components is not influenced, so that the sterilization effect of the double components is not reduced due to the change of the optical wavelength range in the application.

Experimental example 2: stability test

100ml of each of the gels obtained in examples and comparative examples was placed in a sealed transparent glass bottle, and stored in a constant temperature and humidity chamber in the dark, an accelerated aging test was carried out at 55 ℃ and 95% humidity, and the state of the gel was observed periodically.

The results are shown in Table 2.

Table 2: gel stability

As can be seen from the results in Table 2, the gel provided by the invention has good stability, is uniform and stable after being placed for a long time, and does not have layering or discoloration.

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. The toluidine blue efficient photodynamic sterilization composition is characterized by comprising toluidine blue, methylene blue, sodium hyaluronate and a solvent in a mass ratio of 1: 0.1-1; the composition takes phosphate buffer solution or Tris buffer solution with pH of 6.5-9 as the solvent; the dosage of the sodium hyaluronate is 0.1-100 times of that of toluidine blue.

2. The composition of claim 1, wherein the composition is an aqueous or gel formulation.

3. The composition according to claim 1 or 2, wherein when the composition is an aqueous agent or a gel, the mass volume concentration of toluidine blue is 0.0001g/ml to 0.001 g/ml.

4. The composition according to claim 1 or 2, wherein the composition is an aqueous preparation, and the preparation method comprises the following steps: fully dissolving toluidine blue in phosphate buffer solution or Tris buffer solution, and adding methylene blue for full dispersion;

when the composition is a gel, the preparation method comprises the following steps: dissolving toluidine blue in phosphate buffer or Tris buffer, adding methylene blue for dispersing, and adding sodium hyaluronate to prepare gel.