CN117180491A - Recombinant collagen freeze-dried dressing for repair and preparation method thereof - Google Patents
Recombinant collagen freeze-dried dressing for repair and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a recombinant collagen freeze-dried dressing for repair and a preparation method thereof, wherein the freeze-dried dressing is prepared from the following components in percentage by weight: 0.5 to 3 percent of trehalose, 0.1 to 1 percent of poloxamer, 0 to 0.2 percent of carrageenan, 0.2 to 0.8 percent of recombinant collagen, 0.05 to 10 percent of beta-glucan, 0.02 to 5 percent of mannitol and the balance of ultrapure water. The recombinant collagen adopted by the invention is preferably recombinant humanized collagen as a core raw material, and is compounded with beta-glucan, trehalose, poloxamer, carrageenan and mannitol, and the freeze-dried hydrocolloid is prepared by a low-temperature freeze-drying technology. When in use, the water-soluble gel is fully and evenly mixed with matched solution for timely use. The recombinant humanized collagen freeze-dried dressing provided by the invention has good long-term stability at room temperature, a definite interconnected porous structure, uniform pore diameter and good cell compatibility, can enhance cell activity, and is used as a novel wound dressing for repairing skin after laser and photon treatment.
Description
Technical Field
The invention relates to the field of biotechnology preparations, in particular to a recombinant collagen freeze-dried dressing for repairing.
Technical Field
With the improvement of income and the continuous improvement of the requirements for beauty, the medical and cosmetic industry becomes a large consumption hot spot, and the gel dressing with the skin repair capability after phototherapy has great demands for the continuous increase of people with the skin repair requirement after phototherapy.
Hydrogels play an important role in the field of wound dressings because of their good permeability, biocompatibility, and ability to provide a moist environment that facilitates wound healing. Collagen is present in the outer matrix of our skin cells, and it accounts for about 80% of the outer matrix of the skin. The human body needs to update 10% of collagen every day, and the collagen has great effect of promoting skin repair. The recombinant collagen has no immunological rejection reaction to human body, no risk of carrying virus, and is very friendly to human skin. Research shows that the recombinant collagen can promote regeneration of myoglobin and promote wound healing.
Then, how to prepare the recombinant collagen into a hydrogel preparation without affecting the efficacy thereof is a great technical problem at present.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a recombinant collagen freeze-dried dressing capable of effectively promoting skin repair and regeneration, which is prepared by selecting a specific protective agent and recombinant collagen to form a hydrocolloid and adopting a special freeze-drying process, has uniform pore diameter and good cell compatibility, can improve cell viability and is stable at room temperature.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the recombinant collagen freeze-dried dressing is prepared from the following components in percentage by weight:
0.5 to 3 percent of trehalose, 0.1 to 1 percent of poloxamer, 0 to 0.2 percent of carrageenan, 0.2 to 0.8 percent of recombinant collagen, 0.05 to 10 percent of beta-glucan, 0.02 to 5 percent of mannitol and the balance of ultrapure water.
Preferably, in the recombinant collagen freeze-dried dressing, the poloxamer is poloxamer-407 or poloxamer-188.
The preparation method of the recombinant collagen freeze-dried dressing comprises the following steps:
(1) Taking trehalose, poloxamer, carrageenan and recombinant collagen, and adopting a direct dissolution method or an emulsification method to prepare liquid before freeze-drying;
(2) Adding mannitol as a freeze-drying protective agent into the liquid before freeze-drying, dissolving and mixing uniformly, standing at a low temperature of-20 ℃ for pre-freezing for 24 hours, and freeze-drying to obtain freeze-dried powder injection; then adding beta-glucan solution, and gently shaking and rapidly dispersing to form uniform hydrogel solution.
Preferably, in the above preparation method, the direct dissolution method of step (1) includes the steps of: weighing trehalose, poloxamer and carrageenan, adding water, stirring at 50deg.C for swelling, adding recombinant collagen powder, performing ultrasonic treatment under ice bath for 10min, and performing redistilling and dialysis overnight to obtain liquid A before lyophilization.
Preferably, in the above preparation method, the emulsification method of step (1) includes the steps of: weighing poloxamer and carrageenan, adding water, stirring at 50deg.C for swelling, dropwise adding mixed solution of collagen and trehalose, ultrasonic treating with probe under ice bath for 30min, stirring at room temperature overnight, and filtering with 0.45 μm filter membrane to obtain liquid B before lyophilization.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a recombinant collagen freeze-dried dressing for wound repair and a preparation method thereof. The recombinant collagen adopted by the invention is preferably recombinant humanized collagen as a core raw material, and is compounded with beta-glucan, trehalose, poloxamer, carrageenan and mannitol, and the freeze-dried hydrocolloid is prepared by a low-temperature freeze-drying technology. When in use, the water-soluble gel is fully and evenly mixed with matched solution for timely use. The recombinant humanized collagen freeze-dried dressing provided by the invention has good long-term stability at room temperature, a definite interconnected porous structure, uniform pore diameter and good cell compatibility, can enhance cell activity, and is used as a novel wound dressing for repairing skin after laser and photon treatment.
Drawings
FIG. 1 is a schematic view of the appearance of a lyophilized powder for injection and a hydrogel;
FIG. 2 is a surface SEM image of a lyophilized formulation of recombinant collagen;
FIG. 3 is a graph showing the results of a cell compatibility test;
FIG. 4 is a graph showing the results of cell adhesion experiments;
FIG. 5 is a graph showing the distribution of recombinant proteins at different levels of skin after photon modeling (blank control, sample group for 8 hours);
FIG. 6 is a graph showing the distribution of recombinant proteins at different levels of skin after photon modeling (12 hours in sample group, 24 hours in sample group);
(note: blue fluorescence: DAPI; green fluorescence: FITC-recombinant collagen; red dotted line:
the upper area is the epidermis layer, and the lower area is the dermis layer
FIG. 7 shows high performance liquid chromatograms of recombinant collagen in serum at various time points;
FIG. 8 is a graph of wound healing in rats;
fig. 9 is a graph comparing two healthy subjects before and after using the product.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples.
Poloxamer series (188, 407, … …) (Pasteur, germany) and trehalose (pharmaceutical grade) of the invention are purchased from Shanxi Santa Clay medical science and technology Co., ltd, sodium hydroxide (Sodium hydroxide) is purchased from Shanxi Santa Clay medical science and technology Co., ltd, recombinant collagen is purchased from Jiangsu Jiangshan Yuan Biotechnology Co., and Guangzhou and south university medical biotechnology research and development center Co., ltd, and L-929 and NIH-3T3 cells are all purchased from a national experiment cell resource sharing platform; CCK-8 kit was purchased from abcam under the product number ab228554; DMEM medium is available from Thermal-Fisher under product number 11965-118;
all data results of the present invention are expressed as mean ± standard deviation. Statistically significant measurements were performed using one-way ANOVA (one-way ANOVA), multiple comparisons were performed using LSD-t test with indicative controls, and Tamhane's t2 test if the data failed the normalization test. P value <0.05 is a statistically significant criterion for variance.
Example 1: preparation of recombinant collagen freeze-dried dressing by direct dissolution method (the following percentages are weight percent)
Weighing trehalose 1%, poloxamer 0.5%, carrageenan 0.1%, adding water 96.1%, stirring at 50deg.C for swelling, adding recombinant collagen powder 0.3% (purchased from medical biotechnology research development center Co., guangzhou and south university), performing ultrasonic treatment under ice bath for 10min, and performing redistillation and dialysis overnight to obtain liquid A before lyophilization.
And adding 1% of mannitol as a freeze-drying protective agent into the liquid A before freeze-drying, dissolving and mixing uniformly, placing in a low-temperature refrigerator at-20 ℃ for pre-freezing for 24 hours, and freeze-drying for 24 hours to obtain the freeze-dried powder injection. The appearance of the freeze-dried product is plump, no collapse, smooth surface, uniform color and fine texture; adding 1% of beta-glucan solution, and gently shaking and rapidly dispersing to form uniform hydrogel solution, namely the recombinant collagen freeze-dried dressing. The appearance forms of the freeze-dried powder injection and the hydrogel freeze-dried dressing are shown in figure 1, and the microstructure is shown in figure 2.
Example 2: recombinant collagen freeze-dried dressing (the following percentage is weight percentage) prepared by an emulsification method
Weighing poloxamer 0.8%, carrageenan 0.2%, adding water 84.4%, stirring at 50deg.C for swelling, dropwise adding mixed solution of collagen 0.6% (purchased from Jiangsu Jiangshan poly source biotechnology Co., ltd.) and trehalose 3%, ultrasonic treating with probe under ice bath for 30min, stirring at room temperature overnight, filtering with 0.45 μm filter membrane to obtain liquid B before lyophilization.
And adding 3% of mannitol as a freeze-drying protective agent into the liquid B before freeze-drying, dissolving and mixing uniformly, placing in a low-temperature refrigerator at-20 ℃ for pre-freezing for 24 hours, and freeze-drying for 24 hours to obtain the freeze-dried powder injection. The appearance of the freeze-dried product is plump, no collapse, smooth surface, uniform color and fine texture; adding 8% of beta-glucan solution, and gently shaking and rapidly dispersing to form uniform hydrogel solution, namely the recombinant collagen freeze-dried dressing.
Example 3: stability test
The experiment results of the stability of the freeze-dried dressing in the embodiment 1 show that the freeze-dried preparation has no mildew phenomenon after being stored at the temperature of 4 ℃ and the temperature of 25 ℃ for 18 months, and the physical and chemical indexes such as appearance, pH value, water loss, uniformity and the like are not obviously changed; the freeze-dried preparation has slightly poor stability at room temperature of 25 ℃ and slightly reduced activity after 18 months of standing, but has no obvious change at 4 ℃. The stability test results of example 2 are substantially identical to those of comparative document 1.
Table 1 results of freeze-dried dressing stability experiments at different temperatures
Example 4: cell compatibility experiments
(1) Experimental group:
experimental group (recombinant collagen lyophilized dressing), blank control group (complete medium) and blank gel group.
(2) The experimental process comprises the following steps:
placing the 24-pore plate on an ultra-clean bench, and sterilizing with ultraviolet for more than 2 hours. After sterilization is completed, the seed plate is started. Enzyme digestion of P5L-929 in logarithmic phase, 500. Mu.L of cell suspension was added to each well, cell density was 5X 10 3 Culturing in cell culture incubator, taking out cell plate at 24, 48, 72 hr for MTT detection, and detecting cell absorbance with enzyme marker to analyze relative proliferation rate.
(3) Experimental results:
the MTT experimental result is shown in figure 3, and the recombinant collagen freeze-dried dressing has good biocompatibility on L-929 cells.
Example 5: cell adhesion experiments
(1) Sample preparation:
lyophilized powder for injection in example 1: 3mL of PBS was taken to dissolve the sample, the sample was diluted 25-fold to begin administration, and 6 gradients were diluted 5-fold later.
Control (animal collagen): the samples were diluted 65-fold to begin dosing and diluted 5-fold 6-fold later (4% acetic acid dissolved the highest filterable concentration).
(2) Experimental procedure
The day before the test, 24-well plates were coated with a serum-free H-DMEM medium containing 125. Mu.M recombinant collagen, while a Control group was set with a serum-free H-DMEM medium containing 125. Mu.M recombinant collagen, 3 wells per group, and overnight at 4 ℃. The supernatant was discarded before the experiment and washed 3 times with sterile PBS. NIH-3T3 cells with good growth condition are digested with pancreatin, resuspended in serum-free H-DMEM medium, and diluted to 1×10 4 Is inoculated in 24-well plates, 500. Mu.L per well, and incubated at 37℃for 3h. The culture supernatant was discarded, washed 3 times with PBS, fixed in 4% paraformaldehyde solution for 30min, and washed 3 times with PBS. Microscopic observations and photographic recordings.
(3) Experimental results
As shown in fig. 4, the cell spreading (area and number) of the collagen lyophilized powder preparation group is superior to that of the control group, which indicates that the collagen lyophilized powder preparation has remarkable adhesion promoting effect on NIH-3T3 cells.
Example 6: cytotoxicity test
Part 5 of the biological evaluation of medical devices according to GB/T16886.5-2017': in vitro cytotoxicity test "the results of the measurements conducted on the guide documents indicate that the recombinant collagen freeze-dried dressing of examples 1 and 2 of the present invention is non-cytotoxic.
Table 2 results of in vitro cytotoxicity test of recombinant collagen lyophilized dressing
Example 7: skin irritation test
Part 10 of the biological evaluation of medical instruments according to GB/T16886.10-2017': the irritation and skin sensitization test is measured according to the instruction file, in the guinea pig skin irritation test, the gel-like freeze-dried dressing provided by the invention is respectively applied to the dehairing area for single time and multiple times, and then the phenomena of redness, eruption, blister and the like of the skin are avoided, and the results show that the recombinant collagen freeze-dried dressing provided by the invention in examples 1 and 2 has no irritation reaction.
Table 3 results of the skin irritation test on lyophilized recombinant collagen dressing
Example 8: percutaneous permeation experiments
1. Animal model preparation
The guinea pigs are anesthetized by injecting 1% pentobarbital sodium, the back hair of the guinea pigs is removed by using a professional shaving tool, the skin of the guinea pigs is not scratched in the removing process, and the integrity of the tested skin is ensured. Physiological saline is uniformly smeared on the skin removing surface of guinea pigs, an intelligent photon skin tendering instrument is started, a fixed use mode and time are set, the skin removing surface is gently and slowly slid at a constant speed, and uniform irradiation of light on skin is ensured.
2. Transdermal test
Method for simulating human epidermis application in a guinea pig molding region (about 2×2cm in area) 2 ) The samples were smeared and the blank was smeared with the same volume of vehicle (without recombinant collagen). After the solution was completely absorbed, the guinea pigs were returned to the feeder cages and kept in the dark, and fed on a normal diet, with 4 replicates per group.
3. Drawing materials
After application of the samples at the 8h, 12h and 24h time points, respectively, guinea pigs were anesthetized and then:
(1) the guinea pigs were fixed supine and dehaired and disinfected at the left chest heart. Vertically penetrating the injection needle into the heart at the most obvious heartbeat position of 3mm at the left edge of the intercostal sternum of the 3 rd step, extracting part of blood, placing the blood in a collecting tube containing a proper amount of anticoagulant, repeatedly inverting the blood collecting tube to ensure that the anticoagulant and the blood are fully and uniformly mixed, and centrifuging at 3000rpm and 4 ℃ for 10min to obtain supernatant, namely blood plasma; and (3) placing the extracted part of blood in a collecting tube without anticoagulant, and centrifuging at 3000rpm and 4 ℃ for 10min after the blood is coagulated to obtain supernatant, namely serum. The collected plasma and serum were stored at-80 ℃.
(2) The skin surface of the test area was cleaned using a cotton swab dipped in normal saline to remove residual unabsorbed sample. Carefully peeling the effective diffusion area to obtain skin, washing with normal saline, removing water with gauze, and embedding with OTC embedding agent.
4. Blood sample analysis
And (3) detecting the recombinant collagen content of the collected plasma and serum samples by adopting a high performance liquid chromatography.
5. Preparation and observation of skin sections
Skin tissue was cut longitudinally into 10 μm thick sections using a cryostat, adhered to the treated slide, the sections were fixed with ice acetone for 5min, dried at room temperature for 30min, washed twice with PBS, the skin sections were mounted using an anti-fluorescence quench capper (DAPI-containing) and fluorescent images of the skin longitudinal sections were taken under a fluorescent microscope, as shown in FIGS. 5 and 6.
6. Blood sample analysis results
6.1 high Performance liquid chromatography of recombinant collagen in blood samples
As shown in fig. 7.
6.2 analysis of content
The recombinant collagen content in blood samples at different time points was calculated according to a linear correlation equation, and the results are shown in table 4.
TABLE 4 results of recombinant collagen transdermal experiments
7. Conclusion(s)
Transdermal experiments were performed by photon skin rejuvenation post-operative skin model, during the test period:
1) The main component in the recombinant collagen freeze-dried dressing is that the recombinant collagen in the blood is not detected.
2) Skin sections show that the major component of the recombinant collagen lyophilized dressing, recombinant collagen, is not transmitted through the skin or is locally absorbed by the skin.
Example 9: wound repair test of rat skin model
1. Experimental procedure
Animals were anesthetized with 1% sodium pentobarbital solution (Sigma, ostode am Harz, germany) by intraperitoneal injection. After debridement of the back skin, 4 circular (12 mm diameter) abrasion scald wounds were formed by mechanical friction and scald at 70 ℃ for 15 seconds. After creating the wound surface, rats were randomly divided into four groups (blank, blank gel, control, recombinant collagen lyophilized dressing). Wound changes were measured periodically and the time to scab, debridement, redness, and swelling subsidence was recorded. Semi-quantitative analysis of the wound area was performed using ImageJ software. Rats were euthanized on days 3, 7 and 14 post-surgery. Skin tissue surrounding the wound was isolated and fixed in 4% neutral buffered paraformaldehyde, paraffin embedded, cut into 8.0 μm thick sections, and trichromatically stained with H & E and Masson.
2. Test results
Table 5 treatment of recombinant collagen lyophilized dressing damaged area of skin model wound surface of microneedle damaged rat (%)
3. Conclusion of the test
As shown in fig. 8, the rate and quality of healing of the wound surface of rats in the group using the recombinant collagen lyophilized dressing were superior to those of the other groups.
Example 10: comparison of healthy volunteers before and after use
The repair effect of the recombinant collagen freeze-dried dressing for laser and photon treatment postoperative skin is verified through a real case.
(1) Instrument and equipment
The instrument was an Oluantum SRTM photon skin tendering instrument (Wuhan Qiyi Co.) and a long pulse emerald 755mm laser (U.S. Kang Ao Co.).
(2) Method of
The volunteers were treated for general topical treatment, except for the eyelid and male beard. According to different skin conditions, different treatment parameters are selected. Laser and photon alternation is generally adopted to treat 755nm laser energy parameters: 16-26J/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the A power cooling tank: pulse width is 30ms, and delay is 20ms. Photon is directed against freckle and mottle with skin injury, and the energy of double pulse is 2.0 and 4.0J/cm 2 While for telangiectasis and coarse pores, the multipurpose 3 pulse energy is 4.0, 7.0 and 6.6J/cm 2 . The photon is treated 1 time every 3-4 weeks, the laser is treated 1 time every 6-7 weeks, and the treatment 5 times are 1 treatment course. The recombinant collagen freeze-dried dressing is adopted for coating after treatment, the application is carried out once every day in the previous week, the application is carried out every 3 weeks later, 4 weeks is a treatment course, the treatment effect is evaluated by experienced doctors after photographing before and after the use.
And (3) observing curative effect: the main symptoms of photoaging are divided into 5 indicators of skin color, telangiectasia, enlarged pores, pigmentation and skin texture. The above-mentioned indexes are objectively evaluated by experienced doctors and volunteers before and after treatment, and the satisfaction score satisfaction is mild (0-3), and the satisfaction score satisfaction is moderate (4-6) and severe (7-9).
(2) The effect of use is shown in fig. 9.
Table 6 comparison of scores before and after photo-aging of recombinant collagen lyophilized dressing in combination with laser and photon therapy skin
(3) Conclusion(s)
The skin color, telangiectasia and skin texture are improved most obviously, and the recombinant collagen freeze-dried dressing is matched with laser, so that the method is an effective method for treating the skin photoaging symptoms.
Claims (5)
1. The recombinant collagen freeze-dried dressing is characterized by being prepared from the following components in percentage by weight:
0.5 to 3 percent of trehalose, 0.1 to 1 percent of poloxamer, 0 to 0.2 percent of carrageenan, 0.2 to 0.8 percent of recombinant collagen, 0.05 to 10 percent of beta-glucan, 0.02 to 5 percent of mannitol and the balance of ultrapure water.
2. The recombinant collagen freeze-dried dressing according to claim 1, wherein the poloxamer is poloxamer-407 or poloxamer-188.
3. The method for preparing the recombinant collagen freeze-dried dressing according to claim 1, which is characterized by comprising the following steps:
(1) Taking trehalose, poloxamer, carrageenan and recombinant collagen, and adopting a direct dissolution method or an emulsification method to prepare liquid before freeze-drying;
(2) Adding mannitol as a freeze-drying protective agent into the liquid before freeze-drying, dissolving and mixing uniformly, standing at a low temperature of-20 ℃ for pre-freezing for 24 hours, and freeze-drying to obtain freeze-dried powder injection; then adding beta-glucan solution, and gently shaking and rapidly dispersing to form uniform hydrogel solution.
4. A method of preparing a recombinant collagen freeze-dried dressing according to claim 3 wherein the direct dissolution method of step (1) comprises the steps of: weighing trehalose, poloxamer and carrageenan, adding water, stirring at 50deg.C for swelling, adding recombinant collagen powder, performing ultrasonic treatment under ice bath for 10min, and performing redistilling and dialysis overnight to obtain liquid A before lyophilization.
5. A method of preparing a recombinant collagen freeze-dried dressing according to claim 3 wherein the emulsification method of step (1) comprises the steps of: weighing poloxamer, carrageenan, adding water, stirring at 50deg.C for swelling, adding dropwise mixed solution of collagen and trehalose,
ultrasonic treating with probe under ice bath for 30min, stirring overnight at room temperature, filtering with 0.45 μm filter membrane,
liquid B was obtained before lyophilization.
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