CN115317656A - Medical dressing containing collagen and preparation method thereof - Google Patents

Abstract

The invention discloses a medical dressing containing collagen and a preparation method thereof, wherein the dressing comprises the following components: sulfonamide zwitter-ion modified silicone oil, type III recombinant human collagen, phosphotungstic acid, glycerol, triethanolamine and water. The modified polysiloxane elastomer is used as a dressing substrate, and is crosslinked by a physical method, so that the dressing substrate has good mechanical property and self-healing property, and cracks are repaired after water absorption and drying. The function of slowly releasing the protein is achieved by utilizing the interaction of phosphotungstic acid and recombinant human III type collagen, the time of acting on the wound is as long as 48 hours, and the collagen can be continuously and stably released.

Description

Medical dressing containing collagen and preparation method thereof

Technical Field

The invention belongs to the technical field of synthetic biomedical materials, and particularly relates to a medical dressing containing collagen and a preparation method thereof.

Background

Scars on the skin caused by various reasons such as operation, infection, burn and scald affect the beauty of the human body, and can seriously affect the daily life and social activities of patients. With the development of times and the improvement of living standard, the requirements of people on scar beautification are higher and higher, so scar removing preparations with various components such as silicone gel, animal collagen gel, human-like collagen gel and the like are developed successively. The recombinant human III-type collagen functional gel is a novel collagen, can be exogenously supplied to skin to supplement the collagen, is connected with skin fiber fracture caused by wound and infection, inhibits melanin generation, inhibits scars and accelerates skin healing. However, the gel as the recombinant human type III collagen has the following disadvantages: (1) The gel has low mechanical strength after film forming, is easy to run off under the action of external friction or water scouring, has short time of acting on the wound, and is easy to cause the dryness, infection and secondary injury of the wound if not supplemented in time; (2) The moisture retention is poor, and according to the 'moisture wound healing theory', the wound heals better under the moisture sterile environment; (3) The sustained release property is not provided, the recombinant human III-type collagen is continuously and stably released, the excessive deposition or disorganization of the I-type collagen can be avoided, and the pathological scar generation can be avoided.

Disclosure of Invention

In order to solve the problems, the invention provides a medical dressing containing collagen, which is prepared from a modified polysiloxane elastomer, has good mechanical property, antibacterial property and air permeability, has self-healing property, can act on wounds for 48 hours, and can continuously and stably release type III collagen.

The technical scheme of the invention is as follows:

a medical dressing containing collagen comprises the following components in percentage by mass: sulfonamide zwitter-ion modified silicone oil, type III recombinant human collagen, phosphotungstic acid, glycerol, triethanolamine and water.

The invention also provides a preparation method of the medical dressing containing the collagen, which comprises the following steps:

(1) Preparation of sulfonamide zwitter-ion modified silicone oil

Hydrolyzing N, N-diethylamine propyl methyl dimethoxy silane under alkaline condition by using anhydrous ether as solvent to prepare full-grafted tertiary amino silicone oil; further, N, N-diethylamine propyl methyl dimethoxy silane is mixed with octamethylcyclotetrasiloxane D according to the metering ratio ₄ Mixed with hexamethyldisiloxane MM in tetramethylolhydroxideUnder the catalysis of ammonium silicon alkoxide, heating for reaction to prepare tertiary amino silicone oil;

tetrahydrofuran is used as a solvent, tertiary amino silicone oil and 1,3-Propane Sultone (PS) are heated and reacted for 12 hours at the temperature of 60 ℃, and then the sulfonamide zwitter ion modified silicone oil is prepared after further acidification.

(2) Preparation of modified Silicone Elastomers

Respectively dissolving sulfanilamide zwitterion modified silicone oil and phosphotungstic acid by using a solvent, mixing the sulfanilamide zwitterion modified silicone oil and the phosphotungstic acid under a heating condition, uniformly stirring, casting the obtained solution into a polytetrafluoroethylene mold, evaporating the solvent at room temperature, and performing vacuum drying at 80 ℃ for 48 hours to obtain the modified polysiloxane elastomer.

(3) Preparation of medical dressing containing collagen

And (3) soaking the modified polysiloxane elastomer obtained in the step (2) in a liquid medicine containing collagen for 24 hours, taking out, and drying by blowing at the temperature of 20-30 ℃ to obtain the medical dressing containing the collagen.

Further, the grafting ratio of the tertiary amino silicone oil in the step (1) is 20 to 50 percent.

Further, the ratio of the molar weight of the tertiary amino group of the tertiary amino silicone oil in the step (1) to the molar weight of 1,3-propane sultone is 1:1~2.

Further, the mass ratio of the sulfonamide zwitterion modified silicone oil to the phosphotungstic acid in the step (2) is 1 to 10-30%.

Further, the solvent in step (2) is selected from: one or a combination of water, methanol, ethanol, isopropanol and acetonitrile.

Further, the collagen-containing liquid medicine in the step (3) comprises the following specific components: 0.5 to 1 percent of III type recombinant human collagen, 3~4 percent of glycerin, 4~7 percent of triethanolamine and the balance of water.

Has the advantages that:

(1) The modified polysiloxane elastomer is used as a dressing substrate, and is crosslinked by a physical method, so that the dressing substrate has good mechanical property and self-healing property, and cracks are repaired after water absorption and drying. On the other hand, the dressing substrate has good water absorption and transparency, can absorb exudates and keep a wound wet when being applied to the wound, and can observe the wound.

(2) According to the invention, quaternary ammonium salt on the sulfonamide zwitterion modified silicone oil is combined with phosphotungstic acid through electrostatic acting force, and the synergistic antibacterial effect of the quaternary ammonium salt and the phosphotungstic acid is exerted, so that wound bacterial and fungal infection is prevented.

(3) The function of slowly releasing the protein is achieved by utilizing the interaction of phosphotungstic acid and recombinant human III type collagen, the time of acting on the wound is as long as 48 hours, and the collagen can be continuously and stably released.

(4) Biological safety experiments show that the dressing has less skin irritation and cytotoxicity.

Drawings

FIG. 1 shows the preparation of sulfonamide zwitterion modified silicone oil ¹ H-NMR。

FIG. 2 is a graph showing the change in collagen release rate with time in test example 4.

FIG. 3 is a circular dichroism map of test example 4.

Detailed Description

The present invention will be described in further detail with reference to specific examples, which are not intended to limit the present invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Example 1

Preparing the sulfanilamide zwitter ion modified silicone oil.

(1) Preparation of full-grafted tertiary amine silicone oil

Dissolving 200g of N, N-diethylamine propyl methyl dimethoxy silane in 400mL of anhydrous ether, stirring and heating to 40 ℃, slowly dropwise adding 20wt% of NaOH aqueous solution 40 g, carrying out reflux reaction for 4h, pouring the reaction solution into a separating funnel, washing with water until the pH is less than or equal to 8, taking an organic layer, and using anhydrous MgSO (MgSO) as a solvent ₄ Drying, filtering and volatilizing the solvent to obtain the full-grafted tertiary amino silicone oil.

(2) Preparation of partially grafted tertiary amino silicone oil

Adding the fully grafted tertiary amino silicone oil D into a reaction kettle ₄ MM and anteriorHeating tetramethylammonium hydroxide accounting for 1wt% of the total mass of the raw materials to 90 ℃ for reaction for 9 hours, then heating to 150 ℃, continuing stirring for 30 minutes, obtaining a crude product, and performing rotary evaporation for 5 hours at 120 ℃ to remove small-molecule low-boiling-point substances, thereby obtaining part of grafted tertiary amino silicone oil.

The grafting rate and molecular weight of tertiary amino silicone oil at different feeding amounts are shown in the table below.

TABLE 1

Sample (I)	Fully grafted tertiary amino silicone oil, g	D 4 ，g	MM，g	Mn，g/mol	A graft ratio of 1%
						PAMS-1	58.34	55.22	2.62	6216	22.34%
PAMS-2	74.26	23.17	2.37	5428	48.23%

The number average molecular weight (Mn) was determined by means of a Waters model 515-2414 gel permeation chromatograph with THF as the mobile phase.

Utilization of grafting Rate ¹ H-NMR calculation is carried out according to the following method:

the chemical expression of the tertiary amino silicone oil is as follows:

Si(CH ₃ ) ₃ O[Si(CH ₃ ) ₂ O] _x [Si(CH ₃ )(CH ₂ CH ₂ CH ₂ N(C ₂ H ₅ ) ₂ ] _y Si(CH ₃ ) ₃

graft ratio 1=

×100%

x and y correspond to the number of repeating units, S _Me Corresponding to Si-CH ₃ The peak value is 0 to 0.22ppm, S _CH2 Corresponding to Si-CH ₂ The peak appeared at 0.54 ppm.

(3) Preparation of sulfonamide zwitterion modified silicone oil

Dissolving part of grafted tertiary amino silicone oil and 1,3-propane sultone in tetrahydrofuran, then placing the mixture into a round-bottom flask, heating the mixture in a water bath at 60 ℃ for 12 hours for reaction, precipitating the reaction solution with excessive petroleum ether after the reaction is finished, volatilizing the solvent after repeating the reaction for three times, dissolving the solvent again with deionized water or ethanol, dialyzing the mixture for 3 days, dissolving the dried product again with ethanol, adding 35wt% hydrochloric acid to adjust the pH =4, heating, refluxing and acidifying the mixture (40 ℃ and 6 hours), and finally volatilizing the solvent to obtain the sulfonamide zwitterion modified silicone oil.

The grafting ratio of the sulfonamide zwitterionic modified silicone oil under different feeding amounts is shown in the table below.

TABLE 2

Utilization of grafting Rate ¹ H-NMR calculation is carried out according to the following method:

the molecular structure expression of the sulfanilamide zwitter ion modified silicone oil is as follows:

graft ratio 2=

S _SCH2 Corresponding to S-CH ₂ At 2.52ppm, S _SiCH2 Corresponding to Si-CH ₂ The peak appeared at 0.54 ppm.

Example 2

Preparing the modified polysiloxane elastomer.

Dissolving sulfanilamide zwitterion modified silicone oil and phosphotungstic acid with ethanol as a solvent, wherein the concentrations of the sulfanilamide zwitterion modified silicone oil and the phosphotungstic acid are 0.5g/mL and 0.2g/mL respectively, mixing the sulfanilamide zwitterion modified silicone oil and the phosphotungstic acid at 50 ℃, uniformly stirring, casting the obtained solution into a polytetrafluoroethylene mold, volatilizing the solvent at room temperature, and performing vacuum drying at 80 ℃ for 48 hours to obtain the modified polysiloxane elastomer.

The charge amounts of the modified polysiloxane elastomer were as follows:

TABLE 3

Test example 1

The modified polysiloxane elastomer is subjected to a water absorption (WU) test under the conditions of normal temperature, deionized water and 8 hours of soaking time.

In the formula, m _w And m _d The wet and dry weights of the sample to be tested, respectively.

Test example 2

And (3) carrying out mechanical property test on the modified polysiloxane elastomer.

The elastomer is made into strips by hot pressing, specifically hot pressing at 100 ℃ for 10min and then cold pressing for 5min. Spline size: 5cm × 1cm × 0.1-0.2cm. Stretching rate: 500mm/min.

Test example 3

The modified silicone elastomer was tested for self-healing properties.

Testing self-healing performance: scratching the surface of a sample by a constant acting force of 1N and a constant distance of-0.1 mu m, then dropwise adding 200 mu L of ethanol on the surface of the scratch, spreading the scratch, drying the scratch in a normal-temperature blast box, and then carrying out a tensile test to evaluate the self-repairing rate by the ratio of the repaired tensile strength to the original tensile strength.

The results of test example 1~3 are shown in table 4.

TABLE 4

Sample (I)	WU，%	Tensile strength, MPa	Elongation at break,%	Self-repairing rate%
					R1	31.2	5.48	128.5	84.3
R2	30.1	5.12	142.3	89.5
					R3	47.5	7.21	89.7	77.1
R4	48.2	5.88	136.5	93.3

Example 3

Preparing the modified polysiloxane elastomer and soaking the liquid medicine to obtain the dressing, and meanwhile, calculating the protein loading capacity.

S1 preparation of modified polysiloxane elastomer, the formulation is shown in Table 5 below.

TABLE 5

S2, establishing a collagen content standard curve (pH7.4 PBS) by using a BCA method, and measuring A =0.45C +0.0071 and r =0.9921.

S3, accurately preparing 5mL of the following formula liquid medicine by adopting a volumetric flask: 0.5 percent of III type recombinant human collagen, 3 percent of glycerin, 4 percent of triethanolamine and the balance of water, 1mL is put into a quartz cuvette, and the measured absorbance OD1 and the corresponding concentration c thereof are measured ₁ (mg/mL)。

S4, weighing the elastomer dried to constant weight (specification of 0.5 multiplied by 0.12 to 0.15cm) to be m _d Immersing the cuvette containing the liquid medicine in the S2, taking out the cuvette after 24h by using a pair of tweezers, adding deionized water to supplement the solution in the cuvette to 1mL, and measuring the absorbance OD2 and the corresponding concentration c ₂ (mg/mL)。

S5, calculating the protein load (mg/g) = of the dressing

Table 6: protein loading

As can be seen from the data in Table 6, as the proportion of phosphotungstic acid in the elastomer is increased, the protein loading capacity of the dressing is increased after being reduced, because the crosslinking density of the elastomer is increased along with the increase of the dosage of the phosphotungstic acid, so that the water absorption capacity is reduced, the molecular chain connection is tighter, and the protein is difficult to diffuse into the dressing, but as the phosphotungstic acid content is further increased, a large number of hydrogen bond sites are arranged on the surface of the phosphotungstic acid, and the interaction with the hydroxyl groups of the protein occurs, so that the protein diffusion is facilitated.

Test example 4

The dressing prepared in example 3 (taken out and blown dry in a 30 ℃ blast box) was subjected to in vitro release and protein activity measurements.

The dressing obtained in example 3 was put into a PBS solution of pH =7.4, stirred at 37 ℃ at a speed of 100rpm, taken at regular intervals, and the absorbance thereof was measured using the BCA method while supplementing equal amounts of the PBS solution, and the in vitro release degree was calculated.

The release of collagen as a function of time is shown in FIG. 2.

The activity of the protein solution in the color dish is identified by the leachate by adopting circular dichroism, and the specific method comprises the following steps: different samples are tested by adopting leachate with similar concentration (judged by absorbance), and meanwhile, a recombinant collagen PBS solution with similar concentration is prepared as a control (the concentration of the tested protein is 0.22 +/-0.17 mg/mL); and performing far ultraviolet CD scanning on the leachate or the control group, and analyzing the chromatogram by using dicroprot software to obtain the information of the secondary structure.

Circular dichroism chromatogram referring to fig. 3, the protein secondary structure content of the leachate is shown in table 7.

TABLE 7

Sample (I)	-helix	-folding	-turn angle + random crimp
				Recombinant collagen	0.74	0.02	0.24
R4	0.62	0.11	0.27
				R5	0.68	0.09	0.23
R6	0.54	0.29	0.17

At 192nm, 208 corresponds to

Conformation of the helical segments, corresponding at 195, 216

-conformation of the folded segments. A weaker negative absorption peak at 200nm is a random coil fragment; 205nm is the conformation of the beta-turn fragment. The collagen is accompanied with the content change of each conformation during the denaturation process, and the collagen in an undenatured collagen polypeptide chain

-a helical structure content above 50%.

As can be seen from the data in the table, the collagen in the leachate has less change in each conformation, and

the content of the helical structure is more than 50 percent, and the activity is still higher.

Test example 5

And (3) performing bacteriostatic activity test on the dressing: the elastomer was sterilized and cut into disks 15mm in diameter, which were placed in the center of a petri dish containing a solid medium. 20 μ L of the bacterial liquid (Escherichia coli, staphylococcus albus and Candida albicans, respectively, all at a concentration of 1X 10) was aspirated by a pipette ⁹ CFU/mL) were coated on the wafers. Culturing the culture medium in an incubator at 37 ℃ for 24h, taking out, removing the wafer under aseptic condition, taking out the solid culture medium with the diameter of 10mm under the wafer by using a puncher, placing the solid culture medium in a centrifuge tube, adding 2000 mu L of PBS buffer solution, and performing ultrasonic treatment for 4min to obtain the original bacterial liquid. Diluting the original bacteria liquid 10 respectively ⁴ The dilutions were obtained and 150. Mu.L of each dilution was applied to a petri dish containing solid medium. Placing the mixture in an incubator at 37 ℃ for culture 2Taking out after 4h, observing and counting the colony number N to obtain the number of bacteria or fungi in the corresponding diluent, and calculating the number N of the bacteria or fungi in the original bacterial liquid (2000 mu L) according to the dilution multiple D, wherein the calculation formula is as follows:

test example 6

The dressing was tested for biocompatibility: including hemolysis experiments and cytotoxicity experiments.

Hemolysis experiment: and (3) taking fresh human blood containing the sodium citrate anticoagulant, and diluting according to the ratio of human blood/normal saline =5/4 (w/w) to obtain the fresh diluted anticoagulant human blood. The elastomer was cut into strips of about 0.5X 2X 0.13cm, sterilized and placed in test tubes, and 2mL saline was added thereto. One test tube was taken as a negative control, 2mL saline was added thereto, and the other test tube was taken as a positive control, and 2mL of distilled water was added thereto. And then placing all the test tubes into a constant-temperature water bath at 37 ℃ for heat preservation for 30 min, adding the uniformly mixed fresh diluted anticoagulant human blood of 2mL into the test tubes, gently mixing, and placing the test tubes into the water bath at 37 ℃ for continuous heat preservation for 60 min. Transferring the liquid in the test tube into a centrifuge tube, centrifuging for 5min at the speed of 3000rpm, sucking the supernatant, testing the absorbance of the supernatant at 545 nm by using an SMA5000 type micro ultraviolet photometer, and calculating the hemolysis rate according to the following formula:

×100%

in the formula, D _S To test the absorbance value of the sample, D _pc Absorbance of a positive control, D _nc Absorbance of negative control.

Cytotoxicity test: the sterilized elastomer (1 g) was immersed in 10mlpmmi 1640 medium 24h and diluted to 50% of the original concentration with RPMI 1640 medium. L929 cells (murine aneuploid fibrosarcoma cells) were plated at 1.0X 10 in 96-well plates ⁵ cells/mL were cultured in the above-mentioned RPMI 1640 medium. The cells are cultured at 37 ℃,CO ₂ The cells were incubated overnight in a 5% concentration incubator. After 24 hours of cell culture, 5.0 mg/mL MTT (PBS solution) was added to the wells, followed by culture at 37 ℃ for 4 h. The media was then removed, 150 μ l of LDMSO was added, and then shaken vigorously to dissolve the purple formazan crystals that formed. The absorbance was measured with a Wellscan Mk 3 microplate reader at a wavelength of 570 nm.

Table 8: test example 5~6 results

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (7)

1. A medical dressing containing collagen is characterized by comprising the following components: sulfonamide zwitter-ion modified silicone oil, type III recombinant human collagen, phosphotungstic acid, glycerol, triethanolamine and water.

2. The method for preparing a medical dressing containing collagen according to claim 1, comprising the following steps:

(1) Preparation of sulfonamide zwitterion modified silicone oil

Using anhydrous ether as a solvent, and hydrolyzing N, N-diethylamine propyl methyl dimethoxy silane under an alkaline condition to prepare fully grafted tertiary amino silicone oil; further, N, N-diethylamine propyl methyl dimethoxy silane is mixed with octamethylcyclotetrasiloxane D according to the metering ratio ₄ Mixing with hexamethyldisiloxane MM, and heating to react under the catalysis of tetramethylammonium hydroxide silicon alkoxide to prepare tertiary amino silicone oil;

taking tetrahydrofuran as a solvent, heating and reacting tertiary amino silicone oil and 1,3-propane sultone at 60 ℃ for 12 hours, and then further acidifying to prepare sulfonamide zwitter ion modified silicone oil;

(2) Preparation of modified Silicone Elastomers

Respectively dissolving sulfonamide zwitter-ion modified silicone oil and phosphotungstic acid by using a solvent, mixing the sulfonamide zwitter-ion modified silicone oil and the phosphotungstic acid under a heating condition, uniformly stirring, casting the obtained solution into a polytetrafluoroethylene mold, evaporating the solvent at room temperature, and performing vacuum drying for 48 hours at 80 ℃ to obtain a modified polysiloxane elastomer;

(3) Preparation of medical dressing containing collagen

And (3) soaking the modified polysiloxane elastomer obtained in the step (2) in a liquid medicine containing collagen for 24 hours, then fishing out, and drying by blowing at the temperature of 20-30 ℃ to obtain the medical dressing containing the collagen.

3. The method for preparing a medical dressing containing collagen according to claim 2, wherein the grafting ratio of the tertiary amino silicone oil in step (1) is 20 to 50%.

4. The method for preparing a medical dressing containing collagen according to claim 2, wherein the ratio of the molar weight of tertiary amino groups of tertiary amino silicone oil to the molar weight of 1,3-propane sultone in step (1) is 1:1~2.

5. The preparation method of the medical dressing containing the collagen according to claim 2, wherein the mass ratio of the sulfa zwitterionic modified silicone oil to the phosphotungstic acid in the step (2) is 1 to 10-30%.

6. The method for preparing a medical dressing containing collagen according to claim 2, wherein said solvent in step (2) is selected from the group consisting of: one or a combination of water, methanol, ethanol, isopropanol and acetonitrile.

7. The method for preparing a medical dressing containing collagen according to claim 2, wherein the collagen-containing liquid medicine in the step (3) comprises the following specific components: 0.5 to 1 percent of III type recombinant human collagen, 3~4 percent of glycerin, 4~7 percent of triethanolamine and the balance of water.

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