CN116115816A

CN116115816A - Preparation method of visual diagnosis and differential drug release nanofiber membrane

Info

Publication number: CN116115816A
Application number: CN202310202279.0A
Authority: CN
Inventors: 吴金丹; 吴昌迷; 高玉洁
Original assignee: Zhejiang Sci Tech University ZSTU
Current assignee: Zhejiang Sci Tech University ZSTU
Priority date: 2023-03-06
Filing date: 2023-03-06
Publication date: 2023-05-16

Abstract

The invention relates to the field of medical materials, and discloses a preparation method of a visual diagnosis and differential drug release nanofiber membrane, which comprises the following steps: (1) synthesizing modified cellulose acetate and preparing spinning solution 1; (2) preparing spinning solution 2; (3) preparing spinning solution 3; (4) spraying the spinning solution 1 on paper; and (5) coaxial electrostatic spinning. The nanofiber wound dressing prepared by the invention has pH responsiveness, fluorescent dye is fixed on the nanofiber, the infection condition of a wound is monitored by changing the fluorescence intensity according to the pH change, and the infection condition of the wound is monitored by changing the macroscopic color according to the pH change; the antibacterial agent is loaded into a coaxial structure with pH response, so that wound infection, pH rise and antibacterial agent release are realized, the property of the nanofiber dressing that the antibacterial agent is released according to the need is further realized, and the visual diagnosis and controllable treatment of chronic wounds are realized.

Description

Preparation method of visual diagnosis and differential drug release nanofiber membrane

Technical Field

The invention relates to the field of medical materials, in particular to a preparation method of a visual diagnosis and differential drug release nanofiber membrane.

Background

The electrostatic spinning technology is a technology for preparing the nanofiber membrane with large specific surface area, high porosity, adjustable structure, low cost and wide application range. The nanofiber membrane prepared by electrostatic spinning becomes an ideal drug delivery dressing due to high encapsulation efficiency, durable drug release and adjustable characteristics. Many of the prior nanofiber dressings carry antibacterial agents, but excessive use of antibacterial agents can lead to bacterial resistance, and thus achieving on-demand administration is highly desirable. By on-demand administration is meant that the material releases a certain amount of antimicrobial agent on demand at the designated wound site, which can significantly reduce drug exposure time and prevent premature release, reducing the risk of developing bacterial resistance. Generally, when subjected to external stimuli (temperature, light, pH, enzyme or ion appearance, magnetic or electric field triggered stimuli), such smart materials undergo the usual physical-chemical changes to achieve drug release. Such stimuli are classified into endogenous stimuli and exogenous stimuli, and exogenous stimuli have time limitations, cannot spontaneously release drugs according to wound environment changes, and miss optimal release time, so that it is very necessary to achieve on-demand administration of endogenous stimuli (pH).

In addition, for chronic wound infections, frequent changes of wound dressing can cause secondary tears and infections, so monitoring of wound status is becoming increasingly important in modern health care systems, early discovery of early treatments being an important step in improving wound management and reducing antibiotic treatment. Often commercial pH papers and meters are used to measure the pH of the wound, however, these invasive assays require manual removal of the dressing from the wound to detect the wound pH, which can lead to shedding of newly formed skin tissue, disrupting the wound healing process, and increasing the risk of infection, thereby causing re-injury. Thus, there is a great need to construct intelligent non-invasive sensor materials that are easily identifiable by the naked eye in personal care and everyday use. Current wound pH monitoring systems rely on visual color differentiation and measurement of electrical signals, however, these methods have problems such as visual color identification being prone to error, electrode contamination, etc. Therefore, in order to not only satisfy the real situation that the wound can be easily observed with naked eyes, but also avoid the error caused by the pollution of the dressing by blood or tissue fluid, we need to further design a dual-color monitoring system with pH-dependent visible and fluorescence intensity variation to improve the sensitivity and accuracy of the detection system. In addition, most dyes are currently supported in the polymer matrix of the dressing by a physical blending method, which inevitably results in release of the dye from the dressing, which not only reduces the sensitivity of the sensor but may cause side effects, and thus achieving dye immobilization is also a major challenge in the current construction of monitoring type dressings.

In view of the foregoing, there is a need for further development of novel wound nanofiber membranes with dye immobilized pH responsive diagnostic integration.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a visual diagnosis and differential drug release nanofiber membrane. Firstly, esterifying and grafting cellulose acetate with hydroxyl and 5 (6) -carboxyl naphthyl fluorescein with carboxyl through esterification reaction; spraying the modified cellulose acetate on paper; then coaxially spinning acrylic resin and polycaprolactone by using an electrostatic spinning technology, and receiving nanofiber filaments by using paper with a modified cellulose acetate coating to obtain a nanofiber wound dressing with pH response for stimulating drug release and monitoring wound healing; the nanofiber wound dressing prepared by the invention has pH responsiveness, and visual diagnosis and controllable treatment of chronic wounds can be realized through the change of the pH of the wounds.

The specific technical scheme of the invention is as follows: a preparation method of a visual diagnosis and differential drug release nanofiber membrane comprises the following steps:

(1) Adding cellulose acetate into a reaction container filled with a mixed solvent of dichloromethane and N, N-dimethylacetamide, and sequentially adding 4-dimethylaminopyridine, 5 (6) -carboxynaphthalene fluorescein and dicyclohexylcarbodiimide; stirring for reaction under ice bath condition, and dialyzing and filtering the reaction product in a mixed solvent of dichloromethane and N, N-dimethylacetamide to obtain a modified cellulose acetate polymer solution which is named as spinning solution 1.

(2) Dissolving antibacterial agent and polycaprolactone in mixed solvent of tetrahydrofuran and N, N-dimethylformamide to obtain spinning solution 2.

(3) Acrylic resin was dissolved in absolute ethanol to obtain a dope 3.

(4) And uniformly spraying the spinning solution 1 on paper, and airing for standby.

(5) And (3) taking the spinning solution 2 as a coaxial core layer and the spinning solution 3 as a coaxial shell layer for coaxial electrostatic spinning, receiving the paper obtained in the step (4), and stripping the nanofiber membrane from the baked paper after spinning for 4 hours to obtain the nanofiber membrane with a two-layer composite structure.

As described in the background section of the present application, at present, the abuse of antibiotics and the fixation of wound dyes are lacking in the current research, and the invention regulates the fluorescence intensity of nanofibers and the color change of nanofibers through the pH change by esterifying and grafting cellulose acetate with hydroxyl and 5 (6) -carboxynaphthalene fluorescein with carboxyl; the antibacterial agent is loaded into a coaxial structure with pH response, so that the property of the controllable drug release nanofiber dressing is realized, and the nanofiber membrane with two functions realizes visual diagnosis and controllable treatment of chronic wounds.

The pH corresponding principle of the invention is as follows: the shell layer is pH sensitive high polymer acrylic resin, when the pH value of the solution is less than or equal to 4.5, the acrylic resin chain becomes insoluble due to the protonation of COO-groups, so that the solubility of the whole nanofiber in the solution is poor, the drug embedded in the core layer is difficult to release, when the pH value of the solution is more than or equal to 7.4, the acrylic resin chain is weakened due to the protonation degree of COO-groups, the solubility of the acrylic resin chain in water is increased, the structure of the nanofiber gradually collapses, and the drug carrier embedded in the core layer is separated from the fiber, so that the property of the controllable drug-release nanofiber dressing is realized.

Preferably, in the step (1), the mass ratio of the cellulose acetate to the 5 (6) -carboxynaphthalene fluorescein to the 4-dimethylaminopyridine to the dicyclohexylcarbodiimide is (90-100): (1-3): (0.5-1).

The 5 (6) -carboxyl naphthyl fluorescein has high sensitivity and high price, and the grafting rate can achieve the function only by about 1 percent when grafting, so the mass ratio of the cellulose acetate to the 5 (6) -carboxyl naphthyl fluorescein is (90-100) to (1-5).

Preferably, in the step (1), the volume ratio of the dichloromethane to the N, N-dimethylacetamide is 2-3:6-7.

Preferably, in the step (1), the concentration of the spinning solution 1 is 5 to 10wt%.

Preferably, in step (2), the antibacterial agent is a hydrophilic agent and/or a lipophilic agent.

Preferably, in step (2), the hydrophilic drug is tetracycline; the hydrophobic drug is roxithromycin.

Preferably, in the step (2), the concentration of polycaprolactone in the spinning solution 2 is 10-15wt% and the concentration of the antibacterial agent is 5-10wt%.

Preferably, in the step (3), the concentration of the spinning solution 3 is 15-20wt%.

Preferably, in the step (4), the spraying distance is 10-20cm, and the air pressure is 2-4kPa.

Preferably, in the step (5), the electrospinning parameters of the core layer and the shell layer are as follows: the needle is 20-22G, the distance is 10-15cm, the voltage is 15-20kv, the temperature is 25-35 ℃, and the humidity is 30-50%; the spinning speeds of the core layer and the shell layer are respectively 0.003-0.006mm/s and 0.004-0.005mm/s.

Compared with the prior art, the invention has the following technical effects: the nanofiber wound dressing prepared by the invention has pH responsiveness, and fluorescent dye is fixed on the nanofiber through esterification grafting of cellulose acetate with hydroxyl and 5 (6) -carboxyl naphthyl fluorescein with carboxyl, so that the dye is prevented from falling; a pH-responsive fluorescent probe that provides a clear visible color change from purple to blue and a significant change in fluorescence over the range of pH (about 6-8) of the infected wound, i.e., a bi-color monitoring system with pH-dependent changes in both visible and fluorescence intensity; the antibacterial is loaded into a coaxial structure with pH response, so that wound infection, pH rise and antibacterial release are realized, the property of the nanofiber dressing that the antibacterial is released according to the need is further realized, and the visual diagnosis and controllable treatment of chronic wounds are realized.

Detailed Description

The invention is further described below with reference to examples.

Example 1

(1) Cellulose acetate (0.7 g) was added to a reaction vessel containing a mixed solvent of methylene chloride (3 ml) and N, N-dimethylacetamide (7 ml), followed by the addition of 4-dimethylaminopyridine (3 mg), 5 (6) -carboxynaphthalene fluorescein (3 mg) and dicyclohexylcarbodiimide (0.5 mg); stirring for reaction under ice bath condition, and dialyzing and filtering the reaction product for 72h to obtain 7%wt modified cellulose acetate polymer solution which is named as spinning solution 1 for standby;

(2) Taking N, N-dimethylformamide and tetrahydrofuran (1:1) as solvents, adding 10wt% of polycaprolactone (1.4 g) for stirring and dissolving, and then adding 5wt% of hydrophilic medicine tetracycline (0.75 g) to obtain a medicine-carrying polycaprolactone spinning solution, and naming the medicine-carrying polycaprolactone spinning solution as spinning solution 2;

(3) Preparing an acrylic resin spinning solution with 15wt% by taking absolute ethyl alcohol as a solvent, and naming the spinning solution as spinning solution 3;

(4) Uniformly spraying the spinning solution 1 (5 ml) on baking paper by using a spray gun, airing for standby, wherein the spraying distance is 15cm, and the air pressure is 3kpa;

(5) And (3) coaxially and electrostatically spinning the spinning solution 2 and the spinning solution 3, receiving the spinning solution by using the baking paper in the step (4), and stripping the nanofiber membrane from the baking paper after spinning for 4 hours to obtain the nanofiber membrane with a two-layer structure. The core layer is polycaprolactone, the shell layer is acrylic resin, and the electrostatic spinning parameters are as follows: needle 21G, distance 10cm, voltage 18kv, temperature 30 ℃, humidity 40%, spinning speeds of core layer and shell layer are 0.0007mm/s, 0.0042mm/s respectively;

(6) Weighing 60mg of the composite nanofiber membrane obtained in the step (5), putting the composite nanofiber membrane into a centrifuge tube filled with 45ml of PBS (pH 7.4) solution, releasing medicine on a shaking table at 37 ℃, and regularly sucking 3ml of solution for ultraviolet measurement;

(7) The nanofiber membrane obtained by cutting 1cm by 1cm (5) was placed in PBS having pH of 4.5/5/5.5/6/6.5/7/7.4/8 to observe the color change of the membrane, and the change of fluorescence intensity was measured by a fluorescence spectrophotometer.

Comparative examples 1 to 1

(2) Taking N, N-dimethylformamide and tetrahydrofuran (1:1) as solvents, adding 10wt% of polycaprolactone (1.4 g) for stirring and dissolving, and then adding 1wt% of hydrophilic medicine tetracycline (0.14 g) to obtain a medicine-carrying polycaprolactone spinning solution, and naming the medicine-carrying polycaprolactone spinning solution as spinning solution 2;

(4) The spinning solution 1 (5 ml) was uniformly sprayed on the baking paper with a spray gun, dried in the air for standby, the spraying distance was 15cm, and the air pressure was 3kpa.

(5) And (3) coaxially and electrostatically spinning the spinning solution 2 and the spinning solution 3, receiving the spinning solution by using the baking paper in the step (4), and stripping the nanofiber membrane from the baking paper after spinning for 4 hours to obtain the nanofiber membrane with a two-layer structure. The core layer is polycaprolactone, the shell layer is acrylic resin, and the electrostatic spinning parameters are as follows: needle 21G, 10cm apart, voltage 18kv, temperature 30℃humidity 40%, spinning speeds of core and sheath were 0.0007mm/s, 0.0042mm/s, respectively.

(6) Weighing 60mg of the composite nanofiber membrane obtained in the step (5), putting the composite nanofiber membrane into a centrifuge tube filled with 45ml of PBS (pH 4.5) solution, releasing medicine on a shaking table at 37 ℃, and regularly sucking 3ml of solution for ultraviolet measurement;

Comparative examples 1 to 2

Comparative examples 1 to 3

(4) Uniformly spraying the spinning solution 1 on baking paper by using a spray gun, airing for standby, wherein the spraying distance is 15cm, and the air pressure is 3kpa;

(5) And (3) coaxially and electrostatically spinning the spinning solution 2 and the spinning solution 3, receiving the spinning solution by using the baking paper in the step (4), and stripping the nanofiber membrane from the baking paper after spinning for 4 hours to obtain the nanofiber membrane with a two-layer structure. The core layer is polycaprolactone, the shell layer is acrylic resin, and the electrostatic spinning parameters are as follows: needle 21G, distance 10cm, voltage 18kv, temperature 30 ℃, humidity 40%, spinning speeds of core layer and shell layer are 0.0007mm/s, 0.0007mm/s respectively;

Comparative examples 1 to 4

(1) Cellulose acetate (0.7 g) is added into a mixed solvent containing dichloromethane (3 ml) and N, N-dimethylacetamide (7 ml) to obtain 7%wt cellulose acetate polymer solution which is named as spinning solution 1 for standby;

Example 2

(2) Taking N, N-dimethylformamide and tetrahydrofuran (1:1) as solvents, adding 10wt% of polycaprolactone (1.4 g) for stirring and dissolving, and then adding 5wt% of hydrophobic medicine roxithromycin (0.75 g) to obtain a medicine-carrying polycaprolactone spinning solution, and naming the medicine-carrying polycaprolactone spinning solution as spinning solution 2;

Comparative example 2-1

(2) Taking N, N-dimethylformamide and tetrahydrofuran (1:1) as solvents, adding 10wt% of polycaprolactone (1.4 g), stirring and dissolving, and then adding 5wt% of hydrophobic medicine roxithromycin (0.75 g) and 1wt% of hydrophobic medicine roxithromycin (0.14 g) to obtain medicine-carrying polycaprolactone spinning solution, and naming the medicine-carrying polycaprolactone spinning solution as spinning solution 2;

Comparative examples 2 to 2

Comparative examples 2 to 3

Comparative examples 2 to 4

Nanofiber wound dressing performance test for visual diagnosis and controllable treatment

The visible diagnosis nanofiber membrane is measured by a fluorescence spectrophotometer according to the change of fluorescence intensity along with the pH value; the absorbance of the controllable therapeutic nanofiber is measured by an ultraviolet spectrophotometer according to different drug release rates of pH; the bacteriostasis rate of the dressing is determined by the bacteriostasis rate of e bacteria and s bacteria. The results are as follows:

from the above results, it is known that the kind of antibacterial agent, the drug loading concentration of the antibacterial agent, the core-shell ratio of the coaxial nanofiber, whether fluorescein is grafted or not, and the like have a certain influence on the performance of the nanofiber wound dressing for visual diagnosis and controllable treatment. Hydrophilic and hydrophobic properties of the antimicrobial affect the drug release rate (examples 1, 2); if the drug-carrying concentration is too low (comparative example 1-1, comparative example 2-1), the nanofiber dressing does not have an antibacterial effect; in addition, if the shell of the coaxial nanofiber is too thin (comparative examples 1-3 and 2-1), the antibacterial drug release duration is shortened; if fluorescein is not grafted (comparative examples 1-4, comparative examples 2-4), the nanofiber dressing will not have a monitoring function.

The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A preparation method of a visual diagnosis and differential drug release nanofiber membrane is characterized by comprising the following steps of: the method comprises the following steps:

(1) Adding cellulose acetate into a reaction container filled with a mixed solvent of dichloromethane and N, N-dimethylacetamide, and sequentially adding 4-dimethylaminopyridine, 5 (6) -carboxynaphthalene fluorescein and dicyclohexylcarbodiimide; stirring for reaction under ice bath condition, dialyzing and filtering the reaction product in a mixed solvent of dichloromethane and N, N-dimethylacetamide to obtain a modified cellulose acetate polymer solution which is named as spinning solution 1;

(2) Dissolving antibacterial agent and polycaprolactone in a mixed solvent of tetrahydrofuran and N, N-dimethylformamide to obtain spinning solution 2;

(3) Dissolving acrylic resin in absolute ethyl alcohol to obtain spinning solution 3;

(4) Uniformly spraying the spinning solution 1 on baking paper, and airing for later use;

(5) And (3) taking the spinning solution 2 as a coaxial core layer and the spinning solution 3 as a coaxial shell layer for coaxial electrostatic spinning, receiving the baked paper obtained in the step (4), and stripping the nanofiber membrane from the baked paper after spinning for 4 hours to obtain the nanofiber membrane with a two-layer composite structure.

2. The method of manufacturing according to claim 1, wherein: in the step (1), the mass ratio of the cellulose acetate to the 5 (6) -carboxynaphthalene fluorescein to the 4-dimethylaminopyridine to the dicyclohexylcarbodiimide is (90-100): (1-5): (1-3): (0.5-1).

3. The preparation method according to claim 1 or 2, characterized in that: in the step (1), the volume ratio of the dichloromethane to the N, N-dimethylacetamide is 2-3:6-7.

4. The preparation method according to claim 1 or 2, characterized in that: in the step (1), the concentration of the spinning solution 1 is 5-10wt%.

5. The method of claim 1, wherein in step (2), the antibacterial agent is a hydrophilic agent and/or a lipophilic agent.

6. The method of claim 5, wherein in step (2), the hydrophilic drug is tetracycline and/or oxytetracycline; the lipophilic medicine is roxithromycin and/or ritonamine.

7. The method according to claim 1, wherein in the step (2), the concentration of polycaprolactone in the dope 2 is 10 to 15wt% and the concentration of the antibacterial agent is 5 to 10wt%.

8. The method of manufacturing according to claim 1, wherein: in the step (3), the concentration of the spinning solution 3 is 15-20wt%.

9. The method of manufacturing according to claim 1, wherein: in the step (4), the spraying distance is 10-20cm, and the air pressure is 2-4kPa.

10. The method of manufacturing according to claim 1, wherein: in the step (5), the electrostatic spinning parameters of the core layer and the shell layer are as follows: the needle is 20-22G, the distance is 10-15cm, the voltage is 15-20kv, the temperature is 25-35 ℃, and the humidity is 30-50%; the spinning speeds of the core layer and the shell layer are respectively 0.003-0.006mm/s and 0.004-0.005mm/s.