Chitosan-PDRN-catechin compound nano-composite as well as preparation and application thereof

Abstract

The invention relates to a chitosan-PDRN-catechin compound nano-composite as well as preparation and application thereof. The preparation method comprises the following steps: dropwise adding a PDRN solution into a chitosan solution, and stirring to form chitosan-PDRN nano-particles; dropwise adding a catechin compound solution, and stirring to form a ternary composite reaction solution; and finally, centrifugally purifying to obtain the nano-composite. Synergistic interaction of all the components is achieved through a ternary composite system, the particle size of the prepared nano-composite ranges from 100 nm to 200 nm, and the nano-composite is low in polydispersity index, good in stability, excellent in anti-oxidation and anti-inflammatory activity, high in biological safety, capable of being widely applied to the fields of cosmetics, medicine and the like and used for improving skin oxidative stress and inflammation.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a nanocomposite and its preparation method, and particularly to a chitosan-PDRN-catechin nanocomposite and its preparation and application. Background Technology

[0002] With the fast pace of modern life and increasing environmental pressure, skin problems caused by oxidative stress and chronic inflammation are becoming increasingly prominent, such as photoaging, allergic dermatitis, acne, and slow wound healing, seriously affecting people's skin health and quality of life. Therefore, developing highly effective, safe functional skin care materials with both antioxidant and anti-inflammatory effects has become a research hotspot in the cosmetics and biomedical fields.

[0003] In existing technologies, commonly used antioxidants such as vitamin C and vitamin E suffer from poor stability, easy oxidation and inactivation, and weak skin permeability; anti-inflammatory agents such as glucocorticoids may cause adverse reactions such as skin atrophy and pigmentation with long-term use, and their safety is insufficient. To address these issues, researchers are attempting to combine natural active ingredients with carrier materials to construct novel delivery systems that improve the stability and bioavailability of active ingredients.

[0004] Chitosan (CS), a natural cationic polysaccharide, possesses excellent biocompatibility, biodegradability, and film-forming properties, making it an ideal drug delivery carrier material. Polydeoxyribonucleotides (PDRN), DNA fragments extracted from salmon sperm, have been proven to have significant anti-inflammatory and tissue repair and regeneration-promoting bioactivity. Catechins, especially epigallocatechin gallate (EGCG), are the core active ingredients in tea, exhibiting excellent antioxidant and anti-inflammatory properties and showing broad application prospects in the field of skin care.

[0005] However, both PDRN and catechin compounds suffer from easy degradation and low transdermal absorption, making direct application difficult to achieve optimal efficacy. Chinese invention patent application CN120732717A discloses an EGCG-DNA sodium composition with antioxidant and anti-aging effects, which can be compounded with ergothioneine, vitamin C derivatives, etc., and prepared by dissolution and volume adjustment. It can scavenge free radicals and promote collagen synthesis, and can be used in anti-aging serums, freeze-dried powders, and other cosmetics. The process is simple and the dosage form is adaptable. However, it is only a binary compound without synergistic design, failing to solve the problems of poor EGCG stability and low transdermal absorption; it relies on exogenous collagen supplementation and does not stimulate the skin's own repair ability, resulting in weak efficacy duration.

[0006] Currently, there are no known technical solutions for combining chitosan, PDRN, and catechin compounds to construct a stable ternary nanocomposite system through specific processes, in order to achieve synergistic efficacy enhancement, improve the stability of active ingredients, and increase bioavailability. Therefore, developing such novel nanocomposites has significant practical implications and application value. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing chitosan-PDRN-catechin nanocomposites with clear process steps, mild conditions, and easy scale-up.

[0008] Another object of the present invention is to provide a nanocomposite having specific physicochemical properties and stable performance, prepared by the above method.

[0009] Another object of the present invention is to provide the application of the nanocomposite in skin anti-oxidation and anti-inflammatory effects.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a chitosan-PDRN-catechin nanocomposite, characterized by comprising the following steps: S1: Dissolve chitosan in an aqueous acetic acid solution to obtain a chitosan solution; S2: Dissolve PDRN in deionized water to obtain a PDRN solution; S3: Dissolve catechin compounds in deionized water to obtain a catechin compound solution; S4: Add the PDRN solution dropwise to the chitosan solution and stir to obtain chitosan-PDRN nanoparticles; S5: Add the catechin compound solution dropwise to the chitosan-PDRN nanoparticles and stir to obtain a chitosan-PDRN-catechin compound reaction solution; S6: Centrifuge the reaction solution of the chitosan-PDRN-catechin compound, collect the supernatant, and obtain the chitosan-PDRN-catechin compound nanocomposite.

[0011] Preferably, the concentration of the acetic acid aqueous solution is 1% (v / v).

[0012] Preferably, in step S1, the preparation of the chitosan solution further includes ultrasonic treatment and centrifugation of the solution. The ultrasonic treatment time is 20-40 min, and the centrifugation speed is 1200-1600 rpm for 8-12 min.

[0013] Preferably, the mass ratio of chitosan to PDRN is (0.8-1.2):1.

[0014] Through extensive experiments, the applicant discovered that the mass ratio of chitosan to PDRN is limited to (0.8-1.2):1. This ratio enables precise matching of their charges and functions. The cationic groups of chitosan can fully interact with the anionic groups of PDRN to form structurally stable and uniformly dispersed binary nanoparticles. This ensures the structural integrity of the nanoparticles and maximizes the carrier advantages of chitosan and the anti-inflammatory and repairing activities of PDRN, laying a solid foundation for the subsequent construction of a ternary composite system.

[0015] Preferably, the mass ratio of the chitosan-PDRN nanoparticles to the catechin compound is (1.2-1.8):1.

[0016] Furthermore, the applicant discovered that setting the mass ratio of chitosan-PDRN nanoparticles to catechin compounds to (1.2-1.8):1 is key to achieving a synergistic effect of antioxidant and anti-inflammatory properties, allowing catechin compounds to fully bind with the binary nanoparticles through hydrophobic interactions. At this ratio, the ternary composite system perfectly integrates the bioactivities of the three components, achieving a synergistic effect of "1+1+1>3," while ensuring the composite particle size remains stable within the range of 100-200 nm, PDI < 0.2, and Zeta potential > +20 mV. This effectively prevents aggregation, improves skin permeability and bioavailability of active ingredients, and provides a reasonable loading of active ingredients, balancing efficacy and biosafety, thus meeting the application requirements of low-dose, high-efficiency cosmetics.

[0017] Preferably, the catechin compounds include one or more of epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate.

[0018] More preferably, the catechin compound is epigallocatechin gallate.

[0019] Preferably, in steps S4 and S5, the dropping rate is 0.8-1.5 mL / min.

[0020] Preferably, in step S4, the stirring is magnetic stirring, the stirring speed is 400-600 rpm, and the stirring time is 1.5-2.5 h.

[0021] Preferably, in step S5, the stirring is magnetic stirring, the stirring speed is 400-600 rpm, and the stirring time is 2.5-3.5 h.

[0022] Preferably, in step S6, the centrifugation is performed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10K, the centrifugation speed is 3500-4500 rpm, the centrifugation time is 25-35 min, and the centrifugation temperature is 20-30℃.

[0023] In a second aspect, the present invention provides a chitosan-PDRN-catechin nanocomposite prepared according to any of the foregoing methods, characterized in that its particle size is 100-200 nm, polydispersity index (PDI) < 0.2, and zeta potential > +20 mV.

[0024] This complex is formed by chitosan, PDRN and catechin compounds through molecular self-assembly and ionic cross-linking. The components work synergistically, which not only retains the bioactivity of individual components, but also produces a significant synergistic effect.

[0025] In a third aspect, the present invention provides an application of the above-mentioned nanocomposite in the preparation of skin antioxidant and anti-inflammatory agents.

[0026] Beneficial effects 1. This invention constructs a ternary nanocomposite system of chitosan-PDRN-catechin compounds. Through intermolecular interactions, the antioxidant and anti-inflammatory effects of the three compounds produce a synergistic effect of "1+1+1>3", which is significantly better than that of single components or binary complexes.

[0027] 2. The nano-encapsulation structure can effectively protect PDRN and catechin compounds from degradation by light, heat and oxygen, extend the storage life and action time of active ingredients, and ensure that the complex does not easily agglomerate in solution and has good dispersion stability.

[0028] 3. The particle size of the complex is controlled at 100-200nm, with a large specific surface area. Chitosan has good skin affinity, which can significantly improve the skin penetration and cell absorption efficiency of active ingredients, and can exert the best efficacy at a lower dose.

[0029] 4. The entire preparation process is carried out in the aqueous phase, without the need for toxic organic solvents. The reaction conditions are mild, the operation steps are simple, the required equipment is conventional, and it is easy to scale up production, which is in line with the principles of green chemistry.

[0030] 5. All raw materials are of natural origin or approved bioactive substances, with good biocompatibility and biodegradability. The nanocomposite has been verified by cytotoxicity tests to have extremely low cytotoxicity and is highly safe when applied to skin care products. Attached Figure Description

[0031] Figure 1 Appearance images of chitosan-PDRN-epigallocatechin gallate nanocomposites in Example 1 and Comparative Example 1; Figure 2 Particle size distribution of the chitosan-PDRN-epigallocatechin gallate nanocomposite prepared in Example 1; Figure 3A bar chart comparing the DPPH radical scavenging rates of the nanocomposite prepared in Example 1 with those of single epigallocatechin gallate and chitosan-PDRN binary complex. Figure 4 The results of the cytotoxicity test of the nanocomposite prepared in Example 1 on human immortalized keratinocytes (HaCaT) are shown in the figure. Figure 5 The figure shows the results of the determination of the inhibition rate of inflammatory factors in the RAW264.7 macrophage inflammation model induced by lipopolysaccharide (LPS) in the nanocomposite prepared in Example 1 and the comparison between free epigallocatechin gallate and chitosan-PDRN binary complex. Figure 6 The nanocomposite prepared in Example 1 and the comparison of HDF cells after treatment with free epigallocatechin gallate and chitosan-PDRN binary complex in a cell scratch assay and after 24 hours of culture are shown in the figure. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1 A method for preparing a chitosan-PDRN-catechin nanocomposite includes the following steps: (1) Accurately weigh 6 mg of chitosan and dissolve it in 6 mL of 1% (v / v) acetic acid aqueous solution. Stir at 500 rpm for 2 h until completely dissolved, then sonicate for 30 min, and centrifuge at 1500 rpm for 10 min to obtain a chitosan solution of 1 mg / mL.

[0035] (2) Accurately weigh 6 mg of PDRN, dissolve it in 6 mL of deionized water, and vortex until completely dissolved to obtain a 1 mg / mL PDRN solution.

[0036] (3) Accurately weigh 8 mg of epigallocatechin gallate, dissolve it in 8 mL of deionized water, and vortex until completely dissolved to obtain a 1 mg / mL epigallocatechin gallate solution.

[0037] (4) Under magnetic stirring at 500 rpm, 6 mL of PDRN solution was slowly added dropwise to 6 mL of chitosan solution at a rate of 1 mL / min using a syringe pump. After the addition was complete, stirring was continued for 2 h to obtain chitosan-PDRN nanoparticles.

[0038] (5) Keep the magnetic stirring at 500 rpm and slowly add 8 mL of epigallocatechin gallate solution to the above nanoparticle solution at a rate of 1 mL / min using a syringe pump. After the addition is complete, continue stirring for 3 h to obtain chitosan-PDRN-epigallocatechin gallate reaction solution.

[0039] (6) Transfer the reaction solution to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10K, centrifuge at 25℃ and 4000rpm for 30min, and collect the supernatant, which is the chitosan-PDRN-epigallocatechin gallate nanocomposite.

[0040] Comparative Example 1: The difference from Example 1 is that in step (4), 6 mL of PDRN solution is slowly added dropwise to 3 mL of chitosan solution.

[0041] Performance testing 1. Appearance The ternary nanocomposite chitosan-PDRN-epigallocatechin gallate solutions prepared in Example 1 and Comparative Example 1 were placed at room temperature, and the appearance results were compared as follows: Figure 1 As shown: Example 1 was clear and free of precipitate at room temperature.

[0042] Comparative Example 1 showed obvious precipitation at room temperature.

[0043] 2. Particle size Measurements were taken using a dynamic light scattering (DLS) instrument; results are shown below. Figure 2 As shown: The average hydrated particle size of the chitosan-PDRN-catechin nanocomposite is 158.8 ± 5.6 nm.

[0044] 3. Antioxidant activity verification (DPPH method) (1) The nanocomposite obtained in Example 1 was diluted with deionized water to prepare a 10% (v / v) sample solution; at the same time, a 40 μg / mL EGCG solution (single component control group) and a 60 μg / mL chitosan-PDRN binary complex solution (binary control group) were prepared.

[0045] (2) Take 2 mL of sample solution, EGCG solution, and chitosan-PDRN binary complex solution respectively, mix them with 2 mL of 0.1 mM DPPH ethanol solution, vortex mix them, and react them at room temperature in the dark for 30 min.

[0046] (3) The absorbance value was measured at a wavelength of 517 nm (sample A). An equal volume of anhydrous ethanol was used instead of DPPH solution as the sample background control (sample A background), and an equal volume of deionized water was used instead of sample solution as the DPPH background control (control A).

[0047] (4) Antioxidant activity calculation: DPPH free radical scavenging rate (%) = [1 - (A sample - A sample background) / A control] × 100%.

[0048] The results are as follows Figure 3 The results show that the DPPH free radical scavenging rate of the nanocomposite prepared in Example 1 is as high as 80.3%, which is significantly higher than that of the single EGCG solution and the chitosan-PDRN binary complex solution, proving that the ternary nanocomposite has excellent antioxidant activity and significant synergistic effect.

[0049] 4. Cytotoxicity assay (CCK8) To assess the biocompatibility of the nanocomposite of the present invention, cytotoxicity tests were conducted using human immortalized keratinocytes (HaCaT).

[0050] (1) Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were placed at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells per well in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere completely.

[0051] (2) Remove the original culture medium and add fresh complete culture medium containing different concentrations (1%, 5%, 10%, 20%) of the nanocomposite of Example 1. Set up 5 replicates for each group, and set up blank zeroing wells without cells and normal control groups without samples.

[0052] (3) After culturing for 24 hours, aspirate the culture medium from the wells, add 110 μL of fresh complete culture medium and 10 μL of LCK-8 reagent to each well, and put the 96-well plate back into the incubator to incubate in the dark for 1-2 hours.

[0053] (4) Carefully aspirate the culture medium from the wells, add 150 μL DMSO to each well, and shake for 10 minutes to fully dissolve the formazan crystals.

[0054] (5) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0055] (6) Cell survival rate calculation: Cell survival rate (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%.

[0056] The results are as follows Figure 4 As shown, when the concentration of the nanocomposite was below 10%, the survival rate of HaCaT cells was above 100%, with no significant difference from the normal control group (p>0.05); even when the concentration reached 20%, the cell survival rate remained above 90%, indicating that the nanocomposite had extremely low cytotoxicity and good biosafety.

[0057] 5. Anti-inflammatory activity test (ELISA method) (1) Mouse macrophages (RAW264.7) were seeded in 24-well plates and cultured overnight until the cells adhered.

[0058] (2) Cells were pretreated for 2 hours by adding 10% of the nanocomposite from Example 1, 10 μM dexamethasone (positive control), 40 μg / mL free EGCG, and 60 μg / mL chitosan-PDRN binary complex solution respectively.

[0059] (3) Except for the normal control group, each of the other groups was given 100 ng / mL of lipopolysaccharide (LPS) to stimulate the cells for 24 h to construct an inflammation model.

[0060] (4) Collect the cell culture supernatant and measure the concentration of TNF-α in the supernatant according to the instructions of the mouse TNF-α ELISA kit.

[0061] The results are as follows Figure 5 As shown, LPS stimulation significantly induced a large amount of TNF-α secretion. Compared with the LPS model group, pretreatment with the nanocomposite of this invention significantly inhibited the release of TNF-α, and its effect was superior to that of free EGCG and the CS-PDRN binary complex. This fully demonstrates that the nanocomposite of this invention has a strong anti-inflammatory ability and can effectively regulate the expression of inflammatory factors.

[0062] 6. Cell scratch test (1) HDF cells were seeded at high density in 6-well plates and incubated for 24 hours. Then, a vertical “scar” was made in the center of the cell layer in each well using a 200 μL sterile pipette tip.

[0063] (2) Gently wash away the exfoliated cells with PBS, and add culture medium containing 10% of the nanocomposite from Example 1, free EGCG (40 μg / mL), chitosan-PDRN binary complex solution (60 μg / mL) and blank control (complete culture medium).

[0064] (3) The healing of the scratches was observed and photographed under an inverted microscope at 0, 12 and 24 hours.

[0065] The results are as follows Figure 6 As shown, after 24 hours of culture, the scratch healing rate of the nanocomposite group was significantly higher than that of the blank control group, the free EGCG group, and the CS-PDRN binary complex group. This indicates that the ternary nanocomposite provided by this invention can significantly promote the migration and proliferation of skin cells, exhibiting excellent repair and regeneration capabilities in an in vitro model, suggesting its great application potential in anti-skin aging and wound healing.

Claims

1. A preparation method of a chitosan-PDRN- catechin compound nanocomposite, characterized in that, Comprising the following steps: S1: dissolving chitosan in aqueous acetic acid to obtain a chitosan solution; S2: dissolving PDRN in deionized water to obtain a PDRN solution; S3: dissolving a catechin compound in deionized water to obtain a catechin compound solution; S4: adding the PDRN solution dropwise to the chitosan solution and stirring to obtain chitosan-PDRN nanoparticles; S5: adding the catechin compound solution dropwise to the chitosan-PDRN nanoparticles and stirring to obtain a chitosan-PDRN-catechin compound reaction solution; S6: centrifuging the chitosan-PDRN-catechin compound reaction solution, collecting the supernatant, and obtaining the chitosan-PDRN-catechin compound nanocomposite.

2. The method for preparing the chitosan-PDRN-catechins nanocomposite according to claim 1, characterized in that, The mass ratio of chitosan to PDRN is (0.8-1.2):

1.

3. The method for preparing the chitosan-PDRN-catechin nanocomposite according to claim 1, characterized in that, The mass ratio of the chitosan-PDRN nanoparticles to the catechin compound is (1.2-1.8):

1.

4. The method for preparing the chitosan-PDRN-catechin nanocomposite according to claim 1, characterized in that, The catechin compound includes one or more of epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate.

5. The method for preparing the chitosan-PDRN-catechin nanocomposite according to claim 1, characterized in that, In steps S4 and S5, the dropwise addition speed is 0.8-1.5 mL / min.

6. The method for preparing the chitosan-PDRN-catechin nanocomposite according to claim 1, characterized in that, In step S4, the stirring is magnetic stirring at a stirring speed of 400-600 rpm for 1.5-2.5 h.

7. The method for preparing the chitosan-PDRN-catechin nanocomposite according to claim 1, characterized in that, In step S5, the stirring is magnetic stirring at a stirring speed of 400-600 rpm for 2.5-3.5 h.

8. The method for preparing the chitosan-PDRN-catechin nanocomposite according to claim 1, characterized in that, In step S6, the centrifugation uses an ultrafiltration core centrifuge tube with a molecular weight cutoff of 10K, the centrifugation speed is 3500-4500 rpm, the centrifugation time is 25-35 min, and the centrifugation temperature is 20-30℃.

9. The chitosan-PDRN-proanthocyanidins nanocomplex prepared by the method according to any one of claims 1-8, characterized in that, The chitosan-PDRN-catechin compound nanocomposite has a particle size of 100-200 nm, a polydispersity index <0.2, and a Zeta potential >+20 mV.

10. The chitosan-PDRN-catechin compound nanocomposite of claim 9 for use in skin antioxidant and anti-inflammatory applications.

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