Hydrophilic organic copolymer and application thereof in skin protection field

Abstract

The invention discloses a hydrophilic organic copolymer and application thereof in the field of skin protection, the copolymer has the following structural general formula: # imgabs0 #, R1 and R4 are as shown in the specification, the value range of m + n is 400-3500, and m: n = 2: 1.

Description

Technical Field

[0001] The present invention relates to the field of polymer chemistry, and particularly to a hydrophilic organic copolymer and its application in the field of skin protection. Background Art

[0002] The components of liquid skin protectants vary depending on their uses, but common components include: film formers (such as polyurethanes, acrylic polymers) for forming a strong and flexible protective film; solvents (such as alcohol, water or butyl acetate) for dissolving the film former to facilitate application and drying; plasticizers (such as citrate esters) for enhancing the flexibility of the film layer and preventing cracking; antibacterial or disinfectant components (such as povidone iodine, chlorhexidine) for enhancing the anti-infection ability; soothing or moisturizing components (such as aloe vera extract, glycerin) for reducing skin irritation and keeping the skin comfortable. However, for existing liquid skin protectants, since their film formers are mainly composed of natural or synthetic polymers, the formed protective film is often too dense, lacks elasticity, has poor air permeability, weak skin adhesion, is prone to clog pores, and bacteria are likely to grow on the skin surface under the coverage of the film. In addition, due to the limitations of the physical and chemical properties of existing film formers, they often need to be used in combination with other types of film formers, and even plasticizers need to be added to improve the film-forming property. In addition, some film formers also need to introduce auxiliary components such as hydrophobic solvents and antibacterial disinfectants. The addition of these components not only results in complex added components, cumbersome manufacturing processes, poor product stability, but also some components are irritating or even harmful to the skin in existing liquid skin protectant products. Summary of the Invention

[0003] The present invention discloses a brand-new artificial synthetic hydrophilic skin surface rapid film-forming material. This film-forming material is an artificial synthetic super hydrophilic copolymer. It is formed by cross-linking polysaccharides or polysaccharide derivatives with polyamines or polypeptides to form a diblock copolymer. This polysaccharide-polyamine supermolecule diblock copolymer has a porous network structure. Its aqueous solution is a transparent solution or a homogeneous suspension at different pH values. This copolymer film-forming agent has good physical and chemical stability and is resistant to high-temperature and high-pressure sterilization. This copolymer has similar physical, chemical and biological characteristics to glycated proteins in the human body, so it has high biocompatibility and extremely low immunogenicity. Because of its huge molecular weight, it will not penetrate through the stratum corneum and cell membrane of the skin, has extremely high biological safety, and has no irritation to the skin or even skin wounds. The aqueous solution of this super hydrophilic copolymer is sprayed on the skin and skin wounds, and a transparent protective film rich in pores can be formed after drying. The super hydrophilic copolymer swells after absorbing water, resulting in an increase in its pore size, enhanced air permeability, and increased water evaporation. When drying, the water molecules bound to the super hydrophilic copolymer decrease, the swelling shrinks, the copolymer shrinks, and the pore size decreases, reducing water evaporation. Therefore, the formed transparent film can keep the skin breathable and regulate the water evaporation of the skin, thereby preventing excessive water evaporation from causing skin dryness. The thickness of the film formed by the copolymer can be adjusted by the concentration and spraying amount of the aqueous solution of the super hydrophilic copolymer. The surface of the super hydrophilic copolymer can carry positive charges, negative charges or both positive and negative charges at different pH values. Therefore, it can directly bind tightly to the negatively charged skin surface through charge attraction, so the adhesion is extremely good. At the same time, the strong positive charge on its surface has a broad-spectrum antibacterial effect, which can inhibit the growth of Gram-positive, negative, fungi and even viruses. At the same time, the skin protective film reduces or even blocks the adverse stimuli and damages caused by toxic and harmful substances (dust, pollen, spores, metal particles, etc.) in the environment to the skin. Therefore, this copolymer is a brand-new organic synthetic copolymer, with a positive charge on its molecular surface, which can tightly adsorb to the negatively charged skin through charge attraction to form a breathable film with pores, and this high-density positive surface charge endows it with a broad-spectrum antibacterial function. Therefore, this film-forming agent can be used alone without the need to additionally add organic solvents, plasticizers, disinfection and antibacterial components.

[0004] The chemical structure of the copolymer is as follows:

[0005]

[0006] Its synthesis process is as follows: BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 This is a finished product diagram of a 1L carboxymethyl cellulose-polylysine copolymer aqueous solution prepared in Example 1 of the present invention. Among them, A. Concentration 10 mg / ml, B. Concentration 100 mg / ml.

[0008] Figure 2 These are the photos of different concentrations of carboxymethyl cellulose-polylysine copolymer aqueous solution (eosin staining) before and after coating on a glass slide and drying in Example 2 of the present invention.

[0009] Figure 3 These are the optical microscope observations and high-resolution pictures of particles at different fields of view after drying the coating of different concentrations of carboxymethyl cellulose-polylysine copolymer aqueous solution (eosin staining) on a glass slide in Example 2 of the present invention.

[0010] Figure 4 These are the SEM analysis result pictures after spraying 10 mg / mL carboxymethyl cellulose-polylysine copolymer aqueous solution on the sample stage, drying, and then performing ion sputtering coating in Example 2 of the present invention. A. 500x image, B. 10464x image.

[0011] Figure 5 These are the wound repair result pictures in Example 2 of the present invention. Experimental details include: Four 5-mm-diameter skin areas were removed from the back of C57BL / 6 mice using a skin punch. At position A, the wound was only rinsed with PBS. At position B, 25 mg / mL collagen aqueous solution was applied once a day. At position C, 25 mg / mL carboxymethyl cellulose-polylysine copolymer aqueous solution was applied once a day. At position D, 25 mg / mL carboxymethyl cellulose-polyethyleneimine copolymer aqueous solution was applied once a day. The experiment was carried out for 13 days, and photos were taken daily for recording.

[0012] Figure 6 These are the human skin injury repair result pictures in Example 2 of the present invention. For the scab-cracked wound, 30 mg / mL carboxymethyl cellulose-polylysine copolymer aqueous solution was applied in two coats (applied once first, and then applied again after drying). Photos were taken at the 0th hour, 3.5 hours, and 12.5 hours respectively.

[0013] Figure 7 These are the before-and-after comparative analysis pictures of the transdermal water loss value in Example 2 of the present invention.

[0014] Figure 8 These are the before-and-after comparison pictures of the oil content in Example 2 of the present invention.

[0015] Figure 9 These are the before-and-after comparison pictures of the acne area in Example 2 of the present invention.

[0016] Figure 10 These are the before-and-after comparative analysis pictures of the a* value of acne in Example 2 of the present invention.

[0017] Figure 11 These are the effect pictures of applying carboxymethyl cellulose-polylysine copolymer aqueous solution after cleaning the human forehead in Example 2 of the present invention.

[0018] Figure 12 It is the result diagram of the cytotoxicity experiment of carboxymethyl cellulose - polylysine copolymer.

[0019] Figure 13 It is the infrared spectra of carboxymethyl cellulose and dialdehyde carboxymethyl cellulose measured in Example 1 of the present invention.

[0020] Figure 14 It is the infrared spectra of carboxymethyl cellulose - L - polylysine copolymer and polylysine measured in Example 1 of the present invention. Specific Embodiments

[0021] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further elaborates on the present invention in detail with reference to specific embodiments and the accompanying drawings. However, those skilled in the art are aware that the present invention is not limited to the drawings and the following embodiments.

[0022] Example 1 Preparation

[0023] 1. Carboxymethyl cellulose (CMC) and polylysine CMC oxidation reaction:

[0024] 1) Add 180 L of deionized water to a 250 L reaction kettle, add 2.5 kg of sodium periodate, and continuously stir until completely dissolved.

[0025] 2) Add 9 kg of CMC to the dissolved sodium periodate solution, and add deionized water to the reaction system to 200 L.

[0026] 3) Continue to stir for 24 hours to obtain 200 L of oxidized carboxymethyl cellulose (OCMC) solution.

[0027] Cellulose, OCMC) solution.

[0028] Nucleophilic addition and reduction reaction:

[0029] 1) At room temperature, add 250 L of deionized water to a 500 L reaction kettle. Gradually add 10 kg of polylysine while stirring, and continuously stir until completely dissolved.

[0030] 2) Add the OCMC solution to the polylysine solution, and continuously stir to mix well.

[0031] 3) Continuously stir and react at 45 °C for 24 hours.

[0032] 4) Add 380 g of sodium borohydride in batches and react at 45 °C for 48 hours.

[0033] Washing and Purification:

[0034] 1) Adjust the pH value of the prepared intermediate product to 6.0, stir for 2 hours and then let it stand overnight.

[0035] 2) Remove the supernatant and wash it with deionized water until the conductivity is below 200 μS / cm.

[0036] 3) Collect the precipitate.

[0037] 2. Carboxymethyl Cellulose and Polyethyleneimine

[0038] CMC Oxidation Reaction:

[0039] 1) Add 180 L of deionized water to a 250 L reaction kettle, add 2.5 kg of sodium periodate, and continuously stir until completely dissolved.

[0040] 2) Add 9 kg of CMC to the dissolved sodium periodate solution, and add deionized water to the reaction system to make it 200 L.

[0041] 3) Continue to stir for 24 hours to obtain 200 L of OCMC solution.

[0042] Nucleophilic Addition and Reduction Reaction:

[0043] 1) At room temperature, add 250 L of deionized water to a 500 L reaction kettle. Gradually add 10 kg of polyethyleneimine while stirring, and continuously stir until completely dissolved.

[0044] 2) Add the OCMC solution to the polyethyleneimine solution, and continuously stir to mix well.

[0045] 3) Continuously stir and react at 45 °C for 24 hours.

[0046] 4) Add 380 g of sodium borohydride and react at 45 °C for 48 hours.

[0047] Washing and Purification:

[0048] 1) Adjust the pH value of the prepared intermediate product to 7.0, stir for 2 hours and then let it stand overnight.

[0049] 2) Remove the supernatant and wash it with deionized water until the conductivity is below 200 μS / cm.

[0050] 3) Collect the precipitate.

[0051] 3. Hydroxypropyl Methylcellulose and Polylysine

[0052] CMC Oxidation Reaction:

[0053] 1) Add 180 L of deionized water to a 250 L reactor, and add 2.5 kg of sodium periodate. Stir continuously until completely dissolved.

[0054] 2) Add 9 kg of CMC to the dissolved sodium periodate solution, and add deionized water to the reaction system to make it up to 200 L.

[0055] 3) Continue stirring for 24 hours to obtain 200 L of OCMC solution.

[0056] Nucleophilic addition and reduction reaction:

[0057] 1) At room temperature, add 250 L of deionized water to a 500 L reactor. Gradually add 10 kg of polylysine while stirring, and continue stirring until completely dissolved.

[0058] 2) Add the OCMC solution to the polylysine solution, and continue stirring to mix well.

[0059] 3) Stir and react at 45 °C for 24 hours.

[0060] 4) After adding 380 g of sodium borohydride, react at 45 °C for 48 hours.

[0061] Washing and purification:

[0062] 1) Adjust the pH value of the prepared intermediate product to 10.0 - 11.0, stir for 2 hours, and then let it stand overnight.

[0063] 2) Remove the supernatant, and wash with deionized water until the conductivity is below 200 μS / cm.

[0064] 3) Collect the precipitate.

[0065] 4. Hydroxypropyl methylcellulose and polyethyleneimine

[0066] CMC oxidation reaction:

[0067] 1) Add 180 L of deionized water to a 250 L reactor, and add 2.5 kg of sodium periodate. Stir continuously until completely dissolved.

[0068] 2) Add 9 kg of CMC to the dissolved sodium periodate solution, and add deionized water to the reaction system to make it up to 200 L.

[0069] 3) Continue stirring for 24 hours to obtain 200 L of OCMC solution.

[0070] Nucleophilic addition and reduction reaction:

[0071] 1) At room temperature, add 250 L of deionized water to a 500 L reactor. Gradually add 10 kg of polyethyleneimine while stirring, and continue stirring until completely dissolved.

[0072] 2) Add the OCMC solution to the polylysine solution and continuously stir to mix well.

[0073] 3) Continuously stir and react at a temperature of 45 °C for 24 hours.

[0074] 4) After adding 380 g of sodium borohydride, react at 45 °C for 48 hours.

[0075] Washing and purification:

[0076] 1) Adjust the pH value of the prepared intermediate product to 10.0 - 11.0, stir for 2 hours and then let it stand overnight.

[0077] 2) Remove the supernatant and wash with deionized water until the conductivity is below 200 μS / cm.

[0078] 3) Collect the precipitate.

[0079] The finished product of the carboxymethyl cellulose - polylysine copolymer solution is as Figure 1 shown. As Figure 1 can be seen, the solution is yellow at a concentration of 10 mg / mL and dark brown at a concentration of 100 mg / mL. Figure 13 and Figure 14 are the infrared spectra of carboxymethyl cellulose, dialdehyde carboxymethyl cellulose, carboxymethyl cellulose - L - polylysine copolymer, and polylysine. After carboxymethyl cellulose (Carboxymethyl Cellulose, CMC) is oxidized by sodium periodate (NaIO4), it reacts with polylysine (Poly - L - lysine, PLL), and finally is reduced by sodium borohydride (NaCNBH3) to form a stable cross - linked product of reduced amine: carboxymethyl cellulose - L - polylysine copolymer. Fourier Transform Infrared Spectroscopy (FTIR) can characterize the changes in functional groups during this reaction. The following is the analysis of key absorption peaks:

[0080] 1. Characteristic peaks of CMC before reaction:

[0081] Symmetric stretching vibration of carboxylate (–COO -): ~1600 cm -1 (arrow 1) and ~1420 cm -1 (arrow O - H (hydroxyl) stretching vibration: ~3200 - 3600 cm -1 (arrow 3)

[0082] 2. Dialdehyde CMC (OCMC) formed after oxidizing CMC with sodium periodate

[0083] Aldehyde group (–CHO) C=O stretching vibration: ~1720 - 1740 cm -1 (Arrow 4)

[0084] 3. React with polylysine (Schiff base formation), and after reduction by sodium borohydride, a carboxymethylcellulose-L-polylysine copolymer is formed

[0085] C-N stretching vibration (arrow 5) appears at ~1250 cm -1 , indicating that the Schiff base has been completely reduced by sodium borohydride to a stable C-N bond. The amide I (1640 - 1680 cm -1 ) of polylysine and the symmetric stretching vibration of the carboxylate (–COO-) of carboxymethylcellulose (~1600 cm -1 ) (arrow 1) overlap, resulting in an obvious change in the peak shape of the carboxymethylcellulose-L-polylysine copolymer. (Arrow 7) The characteristic peak (1720 - 1740 cm -1 ) of the aldehyde group disappears after reaction with PLL, indicating that the aldehyde group has participated in the reaction. The vibration of the linked N-H (lysine side chain) (arrow 8): ~3200 - 3400 cm -1 and the O-H (hydroxyl group) stretching vibration of carboxymethylcellulose ~3200 - 3600 cm -1 (Arrow 1) overlap, resulting in an obvious change in the peak shape of the carboxymethylcellulose-L-polylysine copolymer (arrow 6).

[0086] The molecular weight of glucoside is 162.14 g / mol, and the molecular weight of carboxymethyl is 58.03 g / mol. Therefore, the formula for calculating the molecular weight of the carboxymethylcellulose unit is: Molecular weight of carboxymethylcellulose unit = 162.14 + (substitution rate of carboxymethyl × 58.03). When the substitution rate is 0.9, the molecular weight of the carboxymethylcellulose unit is 214.37 g / mol. When the molecular weight of the selected carboxymethylcellulose is between 80000 and 750000, the total number (m + n) of the units containing carboxymethylcellulose is approximately between 400 and 3500. Only when the hydroxyl groups on C2 and C3 are not substituted by carboxymethyl can the hydroxyl groups on C2 and C3 be selectively oxidized to aldehyde groups, and at the same time, the connecting bond between C2 and C3 is broken by ring-opening oxidation to form 2,3-dialdehyde carboxymethylcellulose. Therefore, when the substitution rate is 0.9, only 1 / 3 of the carboxymethylcellulose units have hydroxyl groups on both C2 and C3 at the same time. So m:n = 2:1. Since the molecular weight of the lysine unit in poly-L-lysine is 128.17 g / mol, the molecular weight of the poly-L-lysine used in the synthesis can be between 1200 and 350000. Therefore, the number of lysine units (E) contained in the poly-L-lysine in the copolymer is approximately between 10 and 2500.

[0087] Example 2 Film Formation

[0088] Preparation of Film Formation

[0089] 1) Prepare carboxymethyl cellulose - polylysine copolymer solutions with concentrations of 50 mg / mL, 100 mg / mL, and 200 mg / mL, as well as pure water (0 mg / mL) as a control. Add 4% eosin working solution (Solarbio, Cat#G1120) to each sample and vortex - mix. Then, use a triangular glass spreading rod to take 100 μL of the sample and evenly spread it on the surface of a clean glass slide. Observe and use a camera to record photos. After completely drying in a dust - free environment at 25°C, use the camera to take photos of the samples again, and observe and take photos for recording using a stereoscopic phase - contrast microscope. The carboxymethyl cellulose - polylysine copolymer solutions all showed good film - forming ability at different concentrations, and could form a complete and stable film layer on the surface of the glass slide, while the control group of water could not form a film. As Figure 3 shown, the edge of the film layer of the dried carboxymethyl cellulose - polylysine copolymer solution is well - defined, evenly distributed on the surface of the glass slide, and there is no peeling or cracking phenomenon, indicating its good stability. The small amount of particulate matter present in the film layer mainly comes from the impurities that were not completely removed from the surface of the glass slide, rather than the solution itself, which indicates that the carboxymethyl cellulose - polylysine copolymer solution does not introduce additional impurities or particulate matter during the film - forming process.

[0090] 2) Evenly apply 20 μL of the carboxymethyl cellulose - polylysine copolymer solution with a concentration of 10 mg / mL on the surface of a scanning electron microscope sample disk. After completely drying in a dust - free environment at 25°C, perform scanning electron microscope analysis after ion sputtering coating. The results are as Figure 4 shown: The 10 mg / mL carboxymethyl cellulose - polylysine copolymer solution can form a complete covering film on the surface of the sample disk after being applied. And the film formed by the carboxymethyl cellulose - polylysine copolymer presents a porous network structure under the scanning electron microscope.

[0091] The situations of different concentrations of carboxymethyl cellulose - polylysine copolymer aqueous solutions (eosin - stained) coated on the glass slide before and after drying are as Figure 2 shown. The optical microscope observations of different fields of view and the high - resolution map of particulate matter after drying of different concentrations of carboxymethyl cellulose - polylysine copolymer aqueous solutions (eosin - stained) coated on the glass slide are as Figure 3 shown. The results of scanning electron microscope analysis after spraying 10 mg / mL carboxymethyl cellulose - polylysine copolymer aqueous solution on the sample stage, drying, and then ion sputtering coating are as Figure 4 shown.

[0092] Antibacterial Experiment

[0093] 1) Take the 24-hour fresh slant culture of the test bacteria, wash it off with PBS, and dilute it with PBS to about 5.0×10 5 CFU / mL to 4.5×10 6 CFU / mL bacterial suspension for standby. Take a sterile test tube, first add 5.0 mL of 5 mg / mL carboxymethyl cellulose-polylysine copolymer aqueous solution, place it in a water bath at 20°C ± 1°C for 5 minutes, then add 0.1 mL of the test bacterial suspension, mix quickly and start timing immediately. After the test bacteria and the sample interact for 24 hours, respectively pipette 1.0 mL of the test bacteria and sample mixture and inoculate 2 petri dishes, and pour the culture medium. When the number of bacteria cannot be counted, make 10-fold serial dilutions with PBS, select appropriate dilution degrees, respectively pipette 1.0 mL and inoculate 2 petri dishes for viable bacteria culture and counting. At the same time, use PBS instead of the sample for parallel tests as the positive control. The number of recovered colonies in the positive control is between 1.0×10 4 CFU / mL and 9.0×10 4 CFU / mL. Take the same batch of PBS and culture medium as the negative control. All test samples and control samples are cultured at 36°C ± 1°C. For bacterial vegetative forms, observe the final results after 48 hours of culture; for Candida albicans, observe the final results after 72 hours of culture. Repeat the test 3 times and calculate the antibacterial rate.

[0094] 2) Antibacterial rate calculation formula:

[0095]

[0096] In the formula:

[0097] X - antibacterial rate, %;

[0098] A0 - the amount of recovered bacteria in the positive control group, in CFU / mL;

[0099] A1 - the amount of recovered bacteria in the test group, in CFU / mL.

[0100] 3) Result determination

[0101] When the antibacterial rate is ≥ 50% - 90%, it is judged to have antibacterial effect; when the antibacterial rate is ≥ 90%, it is judged to have strong antibacterial effect.

[0102] 4) Results and conclusions

[0103] Table 1 The effects of 5 mg / mL carboxymethyl cellulose-polylysine copolymer aqueous solution and the control group on Escherichia coli, Staphylococcus aureus and Candida albicans for 24 hours

[0104]

[0105] As shown in Table 1, the 5 mg / mL aqueous solution of carboxymethyl cellulose-polylysine copolymer has a strong antibacterial effect on Escherichia coli 8099, Staphylococcus aureus ATCC 6538, and Candida albicans ATCC 10231.

[0106] 1. Cell experiment

[0107] According to the requirements of in vitro experiments for promotional efficacy, a suitable cell line was selected as the detection model and cultured in the corresponding complete medium until the logarithmic growth phase. Subsequently, after adjusting the cell density to an appropriate range, the cells were inoculated into culture dishes, and sample groups, negative control groups, and positive control groups were set up, with at least 3 replicates in each group to ensure data reliability. The complete medium with different concentrations of carboxymethyl cellulose-polylysine copolymer was used as the sample group, the pure complete medium was used as the negative control, and the complete medium supplemented with special efficacy components was used as the positive control. The cell culture temperature was 37 °C, the CO2 concentration was 5%, and the humidity was maintained above 95%. On the 7th day, cell samples were collected, and immunofluorescence (IF) was used to test the generation of barrier-related protein (FLG), the generation of tight junction protein (ZO-1), and the expression of hydration-related protein (AQP3) in keratinocytes. Enzyme-linked immunosorbent assay (ELISA) was used to test the expression levels of specific indicators of macrophage-inhibiting inflammatory factors (TNF-α, IL-6), and statistical analysis was performed.

[0108] (1) Moisturizing

[0109] Experiments were carried out according to the parameters shown in Table 2, and the experimental results are shown in Table 3. There was a significant difference in the expression of AQP3 between the sample addition amount of 0.013% and above and the non-added group. It can be concluded that a sample addition amount of 0.013% and above has a good moisturizing effect.

[0110] Table 2 Experimental detection scheme for the specific detection index AQP3 of moisturizing efficacy

[0111]

[0112] Table 3 Experimental results of the specific detection index AQP3 of moisturizing efficacy

[0113]

[0114]

[0115] (2) Repair

[0116] Experiments were conducted according to the parameters shown in Table 4, and the experimental results are shown in Table 5. There was a highly significant difference in the expression of ZO-1 between the samples with an addition amount of 0.0089% and above and those without addition; as shown in Table 6, there was a significant difference in the expression of FLG between the samples with an addition amount of 0.013% and above and those without addition. It can be concluded that a good repair effect exists when the addition amount of the sample is 0.013% and above.

[0117] Table 4 Experimental detection scheme for specific detection indicators ZO1 and FLG of repair efficacy

[0118]

[0119] Table 5 Experimental results of specific detection indicator ZO1 for repair efficacy

[0120]

[0121] Table 6 Experimental results of specific detection indicator FLG for repair efficacy

[0122]

[0123] (3) Soothing

[0124] Experiments were conducted according to the parameters shown in Table 7, and the experimental results are shown in Table 8. There was a significant difference in the expression of TNF-α between the samples with an addition amount of 0.027% and above and those without addition; as shown in Table 9, there was a significant difference in the expression of IL-6 between the samples with an addition amount of 0.027% and above and those without addition. It can be concluded that a good soothing effect exists when the addition amount of the sample is 0.027% and above.

[0125] Table 7 Experimental detection scheme for specific detection indicators TNF-α and IL-6 of soothing efficacy

[0126]

[0127] Table 8 Experimental results of specific detection indicator TNF-α for soothing efficacy

[0128]

[0129]

[0130] Table 9 Experimental results of specific detection indicator IL-6 for soothing efficacy

[0131]

[0132] (4) Cytotoxicity experiment

[0133] Resuscitate NHEK keratinocytes, digest and collect the cells after at least one passage, and adjust the cell density to 1×10 5cells / mL. Inoculate 100 μL per well into a 96-well culture plate and incubate overnight for cell attachment. Add carboxymethyl cellulose-polylysine copolymer at 0.1, 0.25, 0.5, 1, and 2 mg / mL respectively, with 9 replicates for each concentration. The well without sample addition serves as the control group, and the wells with cell culture medium containing different concentrations of the sample but without cells added serve as the blank control. Continue culturing for 48 h, then add 10 μL of CCK-8 solution to each well, mix well, and continue incubating at 37 °C for 1 h. Measure the absorbance at 450 nm and use 650 nm as the reference wavelength for dual-wavelength measurement. Calculate the cell viability, where cell viability = (OD value of the sample group - OD value of the sample blank control) / (OD value of the control group - OD value of the control blank control) × 100%; the results are as Figure 12 shown that carboxymethyl cellulose-polylysine copolymer has no cytotoxicity to NHEK cells.

[0134] 2. Toxicology experiments

[0135] (1) Repeated skin irritation experiment

[0136] Detect according to Chapter 4, Section 6 of the "Technical Specifications for Cosmetics Safety" (2015 Edition). Before the experiment, clip the hair on both sides of the spine of the experimental animals on the back, with the hair removal area being 3 cm × 3 cm and the application area being 2.5 cm × 2.5 cm. Apply carboxymethyl cellulose-polylysine copolymer with a water content of 50% on the left side of the skin, and the right side serves as the blank control. Apply it once a day for 14 consecutive days. Starting from the second day, the hair should be clipped before each application, and the residual test substance should be removed with purified water. Observe the results 1 hour later, score according to Table 10, and treat the control area and the test area in the same way. Judge according to the results in Table 11. As shown in the test results in Table 12, according to the "Technical Specifications for Cosmetics Safety" (2015 Edition), the test substance, namely carboxymethyl cellulose-polylysine copolymer, is non-irritating to the skin of Japanese white rabbits.

[0137] Table 10 Skin irritation reaction score

[0138]

[0139]

[0140] Table 11 Classification of skin irritation intensity

[0141]

[0142] Table 12 Results of repeated skin irritation test of the test substance on Japanese white rabbits

[0143]

[0144] Note: The integral mean value is reserved to 2 decimal places. Average integral per animal per day:

[0145] (2) Acute Eye Irritation Test

[0146] 1) The test substance was a carboxymethyl cellulose - polylysine copolymer with a water content of 50%. The eyes of the animals were examined 24 h before the test. All the selected animals had no eye irritation symptoms, corneal defects, and conjunctival injuries. Gently pull down the lower eyelid of the left eye of the Japanese white rabbit, and apply 0.1 g of the test substance into the conjunctival sac of the animal's eye, making the upper and lower eyelids close passively for 1 s. Do not rinse for 24 h after applying the test substance, and do not treat the right eye as a self - control. Examine the animal's eyes at 1 h, 24 h, 48 h, and 72 h after applying the test substance, and further examine the eyes of all animals with sodium fluorescein after the 24 - h examination. If no irritation reaction occurs at 72 h, terminate the test.

[0147] 2) Scoring criteria for eye damage and classification of raw material eye irritation reactions: For the scoring criteria of eye damage and classification of raw material eye irritation reactions in the "Technical Specifications for Cosmetics Safety" (2015 Edition), Chapter 6 Toxicological Test Methods, 5 Acute Eye Irritation / Corrosion Test, the scoring criteria for eye damage are shown in Table 13, and the classification of raw material eye irritation reactions is shown in Table 14. Observe the acute eye irritation test of the test substance on Japanese white rabbits, calculate the integral according to Table 10, and classify the integral results according to Table 11.

[0148] 3) Test conclusion: The results of the acute eye irritation test of the test substance on Japanese white rabbits are shown in Table 15. According to the "Technical Specifications for Cosmetics Safety" (2015 Edition), under the non - rinsing condition, this sample has no irritation to the eyes of Japanese white rabbits.

[0149]

[0150]

[0151]

[0152] Table 15 Results of the Acute Eye Irritation Test of the Test Substance on Japanese White Rabbits

[0153]

[0154]

[0155] (3) Skin Phototoxicity Test

[0156] 1) The test substance was a carboxymethyl cellulose - polylysine copolymer with a water content of 50%. Use a radiometer to measure the light intensity (mW / cm 2 ) at 6 points in the irradiation area on the back of the experimental animals, and calculate the average value. The average light intensity of the positive substance irradiated by UVA (wavelength 320 nm - 400 nm) is 5.5340 mW / cm 2, the irradiation time was 30 minutes and 7 seconds; the average light intensity of the test substance irradiation was 6.5062 mW / cm 2 , and the irradiation time was 25 minutes and 37 seconds.

[0157] 2) 18 h - 24 h before the formal test, depilate the skin on both sides of the animal's spine. The skin at the test site should be intact, without damage or abnormality. The depilated area for each piece is about 2 cm × 2 cm. The experimental positions can refer to Figure 5 the four regions ABCD of

[0158] 3) Clinical observation: After the irradiation, observe the skin reactions at 1 h, 24 h, 48 h, and 72 h respectively. Determine the skin reaction score of each animal according to Table 16. The positive control group is carried out in the same way.

[0159] 4) Judgment criteria and scoring: Conduct the skin reaction scoring for the skin phototoxicity test according to the relevant regulations of Chapter 6, Section 7, Skin Phototoxicity Test in the Cosmetics Safety and Technology Specifications (2015 Edition). When the number of animals with a skin reaction score of 2 or more in the areas that were irradiated after applying the test substance while no skin reaction occurred in the areas that only applied the test substance without irradiation is 1 or more, the test substance is judged to have phototoxicity.

[0160] 5) Results: The results of the skin phototoxicity test of the positive control substance on guinea pigs are shown in Table 17; the results of the skin phototoxicity test of the test substance on guinea pigs are shown in Table 18. As shown in Table 18, no skin reaction occurred in all areas that only applied the test substance without irradiation, and the sum of the skin reaction scores in all areas that were irradiated after applying the test substance was less than 2. The test results were evaluated according to Chapter 6, Toxicological Test Methods, Section 7, Skin Phototoxicity Test in the Cosmetics Safety and Technology Specifications (2015 Edition). The test substance showed no phototoxicity to the skin of guinea pigs.

[0161] Table 16 Skin Irritation Reaction Scoring

[0162]

[0163]

[0164] Table 17 Results of the Skin Phototoxicity Test of Methoxypsoralen on Guinea Pigs

[0165]

[0166] Note: Headers 1, 2, 3, 4 areFigure 1 The test area shown

[0167] Table 18 Results of phototoxicity test of carboxymethyl cellulose - polylysine copolymer (test substance) on guinea pig skin

[0168]

[0169]

[0170] 3. Wound repair experiment

[0171] For C57BL / 6 mice, a skin punch was used to remove a 5 - millimeter - diameter skin on the back to obtain 4 circular skin flap excision wounds. Starting from day 0 of the experiment, after photographing the wounds daily, the test samples were applied to the skin wounds, and the time when the wounds smeared with the test samples completely scabbed was recorded. When the scab of any of the test samples applied to the wound completely fell off, it was the end point of the experiment for this experimental animal, and the time was recorded.

[0172] As Figure 5 shown, on the 12th day, the scab of the wound at site D completely fell off, on the 13th day, the scab of the wound at site C completely fell off, while at this time, the scabs of the wounds at sites A and B partially fell off. The experiment shows that the aqueous solutions of carboxymethyl cellulose - polylysine copolymer and carboxymethyl cellulose - polyethyleneimine copolymer have better wound repair effects compared with PBS and collagen solution, which is beneficial to the wound repair of mice.

[0173] (1) Acne treatment and oil control experiment

[0174] 1) Qualified subjects were selected according to the subject inclusion and exclusion criteria. The subjects voluntarily signed a written informed consent form, understood the test process and voluntarily participated in the test. 33 subjects were recruited, and finally 31 subjects completed the test and were included in the statistics. Among them, there were 26 females and 5 males, with an age range of 23 - 50 years old and an average age of 34.4 ± 8.7 years.

[0175] 2) The subjects sat quietly in a waiting room at a temperature of (21 ± 1)°C and (50 ± 10)% RH for 30 min. The tester used VISIA - 7 to take full - face photos of the subjects, and used TM Hex, SM 815, CM 825 probes to measure the baseline values.

[0176] 3) The subjects received the test articles and a test article usage diary, and applied them continuously for 28 days.

[0177] 4) Conduct the usage compliance check after 28 days; the subjects sit quietly for 30 minutes in the waiting room at a temperature of (21±1)°C and a relative humidity of (50±10)%. The tester takes full-face photos of the subjects using VISIA-7 and uses TM Hex, SM 815, CM 825 probe to test the baseline values.

[0178] 5) Data statistics: The statistical analysis software is SPSS. Descriptive statistics are performed on the measured values, and a normal distribution test is conducted. If the requirements of normal distribution are met, paired T-tests are used; otherwise, rank sum tests are used. All statistical methods use two-tailed tests, and the confidence level of the tests is 0.05.

[0179] Table 19 Test indicators

[0180]

[0181] Table 20 Summary of transdermal water loss value data

[0182]

[0183] Table 21 Comparative analysis results of transdermal water loss values

[0184]

[0185] Significance annotation method: "n.s" (p≥0.05); "*" (0.01≤p<0.05); "**" (0.001≤p<0.01); "***" (p<0.001)

[0186] It can be seen from Figure 7 that after 28 days of using the sample carboxymethyl cellulose-polylysine copolymer, the transdermal water loss value of the subjects decreased very significantly. It shows that after continuous use of this sample, the transdermal water loss value has been significantly improved compared with the initial value, and the improvement rate is 8.74%.

[0187] Table 22 Summary of oil content data

[0188]

[0189] Table 23 Comparative analysis results of oil content

[0190]

[0191] Significance annotation method: "n.s" (p≥0.05); "*" (0.01≤p<0.05); "**" (0.001≤p<0.01); "***" (p<0.001)

[0192] It can be seen fromFigure 8 It can be seen that after 28 days of using the sample carboxymethyl cellulose-polylysine copolymer, the oil content of the subjects decreased extremely significantly. This indicates that after continuous use of this sample, the oil content has been significantly improved compared to the initial value, with an improvement rate of 38.85%.

[0193] Table 24 Summary of Acne Area Data

[0194]

[0195]

[0196] Table 25 Comparative Analysis Results of Acne Area

[0197]

[0198] Significance annotation method: "n.s" (p≥0.05); "*" (0.01≤p<0.05); "**" (0.001≤p<0.01); "***" (p<0.001)

[0199] It can be seen from Figure 9 It can be seen that after 28 days of using the sample carboxymethyl cellulose-polylysine copolymer, the acne area of the subjects decreased extremely significantly. This indicates that after continuous use of this sample, the acne area has been significantly improved compared to the initial value, with an improvement rate of 31.25%.

[0200] Table 26 Summary of Acne a* Value Data

[0201]

[0202] Table 27 Comparative Analysis Results of Acne a* Value

[0203]

[0204] Significance annotation method: "n.s" (p≥0.05); "*" (0.01≤p<0.05); "**" (0.001≤p<0.01); "***" (p<0.001)

[0205] It can be seen from Figure 10 It can be seen that after 28 days of using the sample carboxymethyl cellulose-polylysine copolymer, the acne a* value of the subjects decreased very significantly. This indicates that after continuous use of this sample, the acne a* value has been significantly improved compared to the initial value, with an improvement rate of 7.32%.

[0206] Figure 11 It is the effect picture of applying the aqueous solution of carboxymethyl cellulose-polylysine copolymer after cleaning the upper forehead of a person. It can be seen from Figure 11It can be seen that after washing the face every day, apply the aqueous solution of carboxymethyl cellulose-polylysine copolymer (20 mg / ml) of the sample on the forehead, once in the morning and once in the evening every day. After 4 days, the oiliness of the forehead skin is significantly reduced, and the number of acne is significantly reduced.

Claims

1. A copolymer synthesized from a selectively oxidized cellulose derivative and a polyamine polymer, characterized in that, The copolymer has the following general structural formula: wherein R1 and R4 are as shown above, the value range of m + n is 400 - 3500, and m:n = 2:

1.

2. The method for preparing the copolymer according to claim 1, characterized in that, The method comprises the following steps: providing an oxidized cellulose derivative having an aldehyde moiety; reacting the oxidized cellulose derivative with a polyamine polymer through a polymerization reaction to form a copolymer containing an imine derivative; reducing the imine structure on the copolymer to an amine to obtain a cellulose derivative-polyamine copolymer, the cellulose derivative-polyamine copolymer having both amino and carboxyl or hydroxyl functional groups; the cellulose derivative-polyamine copolymer can carry a positive charge, a negative charge, or both positive and negative charges in different pH environments.

3. The method according to claim 2, characterized in that, The copolymer synthesized from the selectively oxidized cellulose derivative and the polyamine polymer is a diblock copolymer.

4. The method according to claim 2, wherein The aldehyde moiety is produced by selectively oxidizing the hydroxyl groups on C2 and C3 of the glucose unit, and the oxidation does not produce more carboxyl groups than aldehyde groups or cause the breakage of the polysaccharide chain.

5. The method according to claim 2, characterized in that The selectively oxidized cellulose derivative is selected from at least one of oxidized carboxymethyl cellulose, carboxyethyl cellulose, carboxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxymethyl cellulose, and hydroxyethyl cellulose.

6. The method according to claim 2, wherein Wherein the selectively oxidized cellulose derivative is a selectively oxidized polysaccharide, selected from at least one of selectively oxidized carboxymethyl cellulose, selectively oxidized carboxyethyl cellulose, selectively oxidized carboxypropyl cellulose, selectively oxidized hydroxypropyl methyl cellulose, selectively oxidized hydroxymethyl cellulose, and selectively oxidized hydroxyethyl cellulose, and not more than 50% of the glucose units of the selectively oxidized cellulose derivative are selectively oxidized to form a 2,3-dialdehyde moiety capable of reacting with the amino polymer.

7. The method according to any one of claims 2-6, characterized in that, The amino polymer is selected from at least one of polylysine, branched polyethyleneimine, and polyallylamine; Preferably, the cellulose derivative-polyamine copolymer carries a positive charge, a negative charge, or both positive and negative charges in different pH environments; Preferably, the cellulose derivative-polyamine copolymer is soluble in water.

8. The method according to any one of claims 2-6, characterized in that, The nitrogen content of the cellulose derivative-polyamine copolymer is not more than 20%.

9. Use of the copolymer according to claim 1 or the copolymer prepared by the method according to any one of claims 2 - 8, characterized in that, Applying it to antibacterial, the bacteria include Gram-negative, Gram-positive, and fungi.

10. Use of the copolymer according to claim 1 or the copolymer prepared by the method according to any one of claims 2-8, characterized in that, Applying it to promote skin wound healing, skin moisturizing, skin acne removal, or skin oil removal.

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