Self-assembled nanoparticles, methods of making and using the same

Abstract

The application discloses self-assembled nanoparticles and a preparation method and application thereof, relates to the technical field of biological medicine manufacturing, and the self-assembled nanoparticles comprise nanoparticles self-assembled by polyphenol compounds and collagen in a weak alkaline environment. The self-assembled nanoparticles based on the polyphenol compound-collagen can induce the covalent crosslinking reaction between the polyphenol compound and the collagen in the weak alkaline environment, so that the reaction yield of the self-assembled nanoparticles is increased, and the stability of the self-assembled nanoparticles is enhanced.

Description

Technical Field

[0001] This invention relates to the technical field of biopharmaceutical manufacturing, specifically to a self-assembled nanoparticle, its preparation method, and its application. Background Technology

[0002] In recent years, ultraviolet (UV)-induced photoaging of the skin has become a significant concern for many. Prolonged exposure to UV radiation can induce the excessive production of reactive oxygen species and inflammatory factors, accelerating skin aging by inducing the expression of matrix metalloproteinases and promoting collagen degradation. This process leads to various manifestations of skin aging, including skin barrier dysfunction, wrinkles, roughness, erythema, and capillary thinning. Furthermore, UV radiation directly damages the DNA within host cells of the skin, leading to mutations and cellular dysfunction, and increasing the risk of skin cancer.

[0003] Current chemical sunscreens have been found to be environmentally harmful. For example, ingredients such as oxybenzone in cosmetics have been shown to disrupt the photosynthetic systems of coral symbiotic algae, leading to coral bleaching. Furthermore, some chemical sunscreens have been found to cause skin irritation and have potential carcinogenic risks. Therefore, in recent years, reducing natural compounds with good environmental friendliness and skin affinity, such as epigallocatechin gallate (EGCG), curcumin, and flavonoids, have been applied in preclinical and clinical studies of photoaging. These compounds play an important role in protecting against photoaging damage by disrupting the chain reaction of free radicals and providing hydrogen atoms to scavenge reactive oxygen species. For example, EGCG, a polyphenolic compound extracted from green tea, is known for its ability to scavenge ROS and reduce oxidative damage. In protecting against photoaging, EGCG can scavenge excess free radicals and reduce ROS levels in skin cells. Despite the advantages of these natural compounds in scavenging reactive oxygen species, they face challenges related to cellular uptake, which limits their effectiveness in scavenging intracellular ROS and restricts their application in the field of photoaging protection and treatment.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] The purpose of this invention is to provide a self-assembled nanoparticle, its preparation method, and its application. By self-assembling polyphenolic compounds and collagen in a weakly alkaline environment, this invention solves the problems of low yield of polyphenolic compounds and collagen particles, instability of nanoparticles after formation, and difficulty in purification in the prior art.

[0006] The present invention is achieved through the following technical solution: First, the embodiments of the present invention provide a self-assembled nanoparticle, including nanoparticles formed by the self-assembly of polyphenolic compounds and collagen in a weakly alkaline environment.

[0007] As an optional implementation, the weakly alkaline environment has a pH of 8 to 10.

[0008] As an alternative implementation, the polyphenolic compound includes epigallocatechin gallate (EGCG).

[0009] Secondly, this invention also provides a method for preparing self-assembled nanoparticles, comprising the following steps: S1: Collagen and epigallocatechin gallate (EGCG) were added to a weakly alkaline solution and allowed to stand for reaction to obtain an EGCG-collagen nanoparticle suspension. S2: The EGCG-collagen nanoparticle suspension was purified by a combination of centrifugation and resuspension, and the precipitate was collected to obtain EGCG-collagen nanoparticles.

[0010] As an alternative implementation, the weakly alkaline solution is a Tris buffer solution with a pH of 8 to 10 or double-distilled water.

[0011] As an optional implementation, in the reaction system of S1, the concentration ratio of epigallocatechin gallate (EGCG) to collagen is 4-8:1.

[0012] As an optional implementation, S2 includes placing the EGCG-collagen nanoparticle suspension in a centrifuge and centrifuging at a speed of 3000-5000 rcf for 2-4 minutes, removing the supernatant to obtain a precipitate; Resuspension and centrifugation: Add double-distilled water to the precipitate to fully resuspend the precipitate and obtain a new suspension. Place the new suspension in a centrifuge and centrifuge at 3000-5000 rcf for 2-4 minutes to collect the precipitate. Repeat the resuspension and centrifugation process at least twice, collect the precipitate, and obtain purified EGCG-collagen nanoparticles.

[0013] Finally, this embodiment of the invention also provides an application of self-assembled nanoparticles, which are used to prevent and repair photoaging of the skin.

[0014] As an optional implementation, the method for preventing and repairing skin photoaging includes the ability of the self-assembled nanoparticles to load molecules of different sizes and / or to be biocompatible and / or to promote the uptake of the self-assembled nanoparticles by L929 cells and / or to achieve effective scavenging of reactive oxygen species.

[0015] As an alternative implementation, EGCG-collagen nanoparticles at concentrations of 500 ug / ml and below exhibit biocompatibility and the ability to scavenge reactive oxygen species.

[0016] Compared with the prior art, the embodiments of the present invention have the following advantages and beneficial effects: 1. This invention relates to self-assembled nanoparticles based on polyphenolic compounds and collagen. The synthesis of these nanoparticles involves the interaction of charged molecules and the covalent cross-linking reaction between polyphenolic compounds and collagen under weakly alkaline conditions. Under weakly acidic and neutral environments, this self-assembly system can initially form a small number of unstable self-assembled particles through positive and negative charge interactions. However, under weakly alkaline conditions, relying on the covalent cross-linking of polyphenolic compounds and collagen, self-assembled particles with significantly increased reaction yield and enhanced stability can be formed. Cell experiments have demonstrated that these particles exhibit good biocompatibility, cellular uptake capacity, and reactive oxygen species scavenging ability.

[0017] 2. This invention addresses the problems of low cellular uptake, poor stability, and insufficient bioavailability of polyphenolic compounds and collagen by designing EGCG-collagen self-assembled nanoparticles. The designed nanoparticles possess excellent cellular uptake and reactive oxygen species scavenging capabilities, and exhibit good loading capacity for drugs of different sizes, making them suitable for the prevention and repair of skin photoaging.

[0018] 3. Synthesis design of EGCG-collagen self-assembled nanoparticles in the embodiments of the present invention: EGCG and collagen can complete the initial self-assembly of particles through the interaction of charged molecules. At this time, by adjusting the proportion of particles, nanoparticles with relatively uniform distribution and relatively uniform size can be obtained.

[0019] 4. In the embodiments of this invention, the self-assembled nanoparticles exhibit good biocompatibility with skin cells and rapid cellular uptake capacity, being taken up by most L929 cells after 4 hours. They also demonstrate excellent reactive oxygen species (ROS) scavenging ability, effectively eliminating ROS in cells treated with 500 μM hydrogen peroxide for 2 hours after 4 hours of drug application. These biological properties indicate that it can be applied to the prevention and treatment of skin photoaging. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 Macroscopic images of EGCG-collagen nanoparticle suspensions with different configuration ratios in Example 1; Figure 2Bright-field microscopy images of EGCG-collagen nanoparticles with different configuration ratios in Example 1; in Figure 2 The ratio of a to b is 1:1. Figure 2 b corresponds to a ratio of 2:1. Figure 2 c ratio 4:1, Figure 2 d corresponds to a ratio of 6:1. Figure 2 e corresponds to a ratio of 8:1. Figure 2 f corresponds to a ratio of 10:1; Figure 3 The particle size distribution of EGCG-collagen nanoparticles with different configuration ratios in Example 1 is shown. Figure 4 Scanning electron microscope images of EGCG-collagen nanoparticles with different configuration ratios in Example 1; in Figure 4 a and Figure 4 d corresponds to a ratio of 4:1. Figure 4 b and Figure 4 e corresponds to a ratio of 6:1. Figure 4 c and Figure 4 f corresponds to a ratio of 8:1; Figure 4 d is Figure 4 A magnified view of part of a. Figure 4 e is Figure 4 A magnified view of b. Figure 4 f is Figure 4 A magnified view of c; Figure 5 This is a macroscopic view of the EGCG-collagen nanoparticle suspension under different pH conditions in Example 2; Figure 6 This is a comparison chart of the turbidity of EGCG-collagen nanoparticles under different pH conditions in Example 2; Figure 7 The particle size distribution of EGCG-collagen nanoparticles under different pH conditions in Example 2; Figure 8 This is a comparison diagram of the Zeta potentials of EGCG-collagen nanoparticles under different pH conditions in Example 2; Figure 9 These are scanning electron microscope images of EGCG-collagen nanoparticles under different pH conditions in Example 2. in Figure 9 a corresponds to a pH of 2. Figure 9 b corresponds to pH 5. Figure 9 c corresponds to a pH of 6. Figure 9 d and Figure 9 j corresponds to a pH of 7. Figure 9 e and Figure 9 k corresponds to a pH of 8. Figure 9 f and Figure 9 l corresponds to a pH of 9. Figure 9 g and Figure 9 m corresponds to a pH of 10. Figure 9 h and Figure 9 n corresponds to a pH of 11. Figure 9 i and Figure 9 o corresponds to a pH of 12; Figure 9 j is Figure 9 A magnified view of part of d. Figure 9 k is Figure 9 A magnified view of part of e. Figure 9 l is Figure 9 A magnified view of f. Figure 9 m is Figure 9 A magnified view of g. Figure 9 n is Figure 9 A magnified view of h. Figure 9 o for Figure 9 A magnified view of part of i; Figure 10 The graph shows the yield calculation and physical display of EGCG-collagen nanoparticles under different pH conditions in Example 2. Figure 11 This is a comparison of the loading efficiency of EGCG-collagen nanoparticles on fluorescent small molecule RhB and large molecule proteins BSA and IgG in Example 3. Figure 12 This is a loading diagram of the EGCG-collagen nanoparticles in Example 3; in Figure 12 a is the fluorescence image of RhB loaded onto EGCG-collagen nanoparticles. Figure 12 b is the bright-field plot of EGCG-collagen nanoparticles against RhB. Figure 12 c is Figure 12 a and Figure 12 b is a merged graph; in Figure 12 d shows the fluorescence pattern of IgG loading on EGCG-collagen nanoparticles. Figure 12 e is the bright-field plot of EGCG-collagen nanoparticles against BSA. Figure 12 f is Figure 12 d and Figure 12 The merged diagram of e; in Figure 12 g is the fluorescence image of IgG loaded onto EGCG-collagen nanoparticles. Figure 12 h is the bright-field plot of EGCG-collagen nanoparticles against BSA. Figure 12 i is Figure 12 g and Figure 12 The merged graph of h Figure 13 This is a comparison of cell viability of L929 cells treated with different concentrations of EGCG-collagen nanoparticles for 24 hours in Example 4. Figure 14 This is a comparison of cell viability and cell death staining after L929 cells were treated with different concentrations of EGCG-collagen nanoparticles for 24 hours in Example 4. Figure 15 This is a comparison of cell viability of HACAT cells treated with different concentrations of EGCG-collagen nanoparticles for 24 hours in Example 4. Figure 16 This is a comparison of cell viability and cell death staining after HACAT cells were treated with different concentrations of EGCG-collagen nanoparticles for 24 hours in Example 4. Figure 17 The image shows the fluorescence of L929 cells uptake of EGCG-collagen nanoparticles at different uptake times in Example 5. in Figure 17 a corresponds to 0 h, Figure 17 b corresponds to 0.5 h. Figure 17 c corresponds to 1 h. Figure 17 d corresponds to 2h, Figure 17 e corresponds to 4h. Figure 17 f corresponds to 8h; Figure 18 This is a graph showing the uptake efficiency of EGCG-collagen nanoparticles by L929 cells in Example 5. Figure 19 Fluorescence images of EGCG-collagen nanoparticles scavenging intracellular reactive oxygen species in L929 cells at different doses; Figure 20 A graph showing the ability of EGCG-collagen nanoparticles to scavenge reactive oxygen species in L929 cells at different doses. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0022] Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0023] Nanoparticle technology holds immense promise for transforming poorly absorbed and unstable physiologically active compounds into viable therapeutic agents. The small size of nanoparticles allows them to cross cell membranes through gaps or via endocytosis. Self-assembly technology promotes the spontaneous organization of molecules into nanoparticles through non-covalent interactions such as hydrogen bonding, electrostatic interactions, and hydrophobicity. Self-assembly offers advantages such as simplicity, cost-effectiveness, and high controllability, leading to its widespread application in nanoparticle preparation. Utilizing self-assembly technology, nanostructures with specific functions can be constructed that respond to changes in the cellular microenvironment, thereby enhancing cellular drug absorption. Therefore, converting free EGCG into nanoparticles via self-assembly technology may be an effective strategy to enhance its endocytosis, improve bioavailability, and improve therapeutic efficacy.

[0024] Collagen is the most abundant protein in mammals and plays a crucial role in various bodily functions. Due to its biodegradability, good bioactivity, and low immunogenicity, collagen has been widely used in surgical repair, wound healing, and skincare. Notably, recent studies have found that collagen can delay skin aging through antioxidant effects. Considering the benefits of both materials, a self-assembled nanoparticle of collagen and EGCG can be designed to produce a therapeutic formulation with beneficial properties in preventing and repairing photoaging of the skin.

[0025] Therefore, embodiments of the present invention provide a method for preparing self-assembled nanoparticles, comprising the following: EGCG-collagen nanoparticles were synthesized under aseptic conditions via an aqueous one-pot synthesis. The nanoparticles were constructed using epigallocatechin gallate (EGCG) and bovine collagen. The specific experimental steps were as follows: 10 μL of Collagen (1 mg / ml) and 10 μL of EGCG (4–8 mg / ml) were added sequentially to Tris (5 mM, pH 8–10). The mixture was allowed to stand for at least 2 hours to allow the reactants to fully react and form a turbid aqueous suspension. The suspension was centrifuged at 3000–5000 rcf for 2–4 minutes. The supernatant was carefully discarded, and the lower precipitate was resuspended in double-distilled water and centrifuged at 3000–5000 rcf for 2–4 minutes. This process was repeated at least twice. The precipitate was collected to obtain purified EGCG-collagen nanoparticles.

[0026] This invention addresses the problems of low yield, instability, and difficulty in purification of EGCG-collagen self-assembled nanoparticles in existing technologies by inducing covalent cross-linking between EGCG and collagen under weakly alkaline conditions and utilizing the positive and negative charge interactions between them. The synthesis design of EGCG-collagen self-assembled nanoparticles involves the initial self-assembly of particles through the interaction of charged molecules between EGCG and collagen. By adjusting the particle ratio, relatively uniformly distributed and sized nanoparticles can be obtained.

[0027] To better verify the effectiveness of the embodiments of the present invention, specific experiments will be designed below for verification: Example 1: Preliminary preparation of EGCG-collagen nanoparticles Using a one-pot aqueous solution method, different proportions of Collagen and EGCG were added sequentially to double-distilled water and allowed to stand for 1 hour to obtain an EGCG-collagen nanoparticle suspension (refer to...). Figure 1 After 60 minutes, the system changed from an initially clear solution to a turbid suspension, indicating that the initial self-assembly of the nanoparticles was complete. (Optical microscope, reference...) Figure 2 The results showed that in each reaction system, the reaction groups with EGCG:collagen concentration ratios of 4:1 to 8:1 produced a larger number of particles with relatively uniform size and distribution. The 2:1 and 10:1 groups had relatively lower particle yields, and the 10:1 group exhibited relatively uneven particle size. No particle formation was observed in the 1:1 group. Since EGCG:collagen concentration ratios of 4:1 to 8:1 consistently yielded a relatively large number of relatively uniformly sized particles, this concentration ratio will be considered for subsequent experiments. Dynamic light scattering (refer to...) Figure 3 The results showed that EGCG-collagen nanoparticles with a concentration ratio of 4:1 exhibited good dispersibility, with a particle size distribution of approximately 200–500 nm. (Scanning electron microscopy, see reference...) Figure 4 The results showed that the EGCG-collagen nanoparticles with a concentration ratio of 4:1 maintained a spherical structure and were well distributed after drying. The particle size obtained by SEM imaging was slightly larger than that obtained by dynamic light scattering. This may be because the particles shrink during SEM dehydration and are prone to collapse due to surface tension, resulting in an increased apparent particle size. The above results indicate that the aqueous "one-pot method" can be used to initially prepare nanoparticles with uniform size and distribution.

[0028] Example 2: Improved Yield and Stability of EGCG-Collagen Nanoparticles In neutral or weakly acidic environments, uniformly sized and evenly distributed ECGC-collagen nanoparticles can be initially obtained using a one-pot aqueous solution method. However, the turbidity of the nanoparticle suspension obtained by this method is significantly reduced after centrifugation (see reference). Figure 5 The results indicated that the nanoparticles obtained by this preparation method had poor stability, and it was impossible to obtain purified nanoparticles with stable yields. Therefore, further improvements are needed in the synthesis method of EGCG-collagen nanoparticles. Before the self-assembly of EGCG and collagen, 5mM Tris buffer at different pH values ​​(2, 5, 6, 7, 8, 9, 10, 11, 12) were added, followed by the sequential addition of Collagen and EGCG. After standing for 24 hours, an EGCG-collagen nanoparticle suspension was obtained (refer to...). Figure 5 The suspension results showed that the color of the EGCG-collagen self-assembly system changed under weakly alkaline conditions, suggesting that EGCG and collagen undergo further covalent reactions under weakly alkaline conditions, thereby stabilizing the particles. Particles synthesized under different pH conditions were centrifuged multiple times, and after centrifugation, they were resuspended in double-distilled water to obtain a resuspended EGCG-collagen nanoparticle suspension (refer to...). Figure 5 The turbidity of the resuspended solution was measured using an ELISA reader at a wavelength of 600 nm. A comparison of turbidity under different pH conditions is shown in the figure (see [reference]). Figure 6 The results showed that in neutral and weakly acidic environments, although EGCG and collagen could form particles through the binding of positive and negative charges, the solution became clear after multiple centrifugations, and the number of particles decreased significantly, indicating that particles formed solely through the binding of positive and negative charges are unstable. In contrast, the solution remained turbid after multiple centrifugations in a weakly alkaline environment, indicating that the particles are stabilized through covalent cross-linking in this environment. Furthermore, the highest turbidity was observed in the solution after centrifugation within the pH range of 8-10, suggesting that the largest number of particles were formed within this pH range. In strongly acidic and strongly alkaline environments, the number of particles decreased significantly due to collagen denaturation and the protonation of negative charges and deprotonation of positive charges. Dynamic light scattering (see reference) Figure 7 The results showed that EGCG-collagen nanoparticles under weakly alkaline conditions had similar particle sizes to those under neutral conditions, with the particle size mainly distributed between 200 and 500 nm. Zeta potential results (see reference) Figure 8 After 24 hours of reaction, the Zeta potential of the particles under weakly alkaline conditions (pH=9) was approximately -33 mV, while that under neutral conditions (pH=7) was approximately -15 mV. It is generally believed that particles with an absolute Zeta potential <20 mV have a higher risk of instability, while those with an absolute Zeta potential >30 mV exhibit good stability. This suggests that particles synthesized under weakly alkaline conditions are significantly more stable than those synthesized under neutral conditions. (Scanning electron microscopy, reference...) Figure 9 The results showed that the highest yield of nanoparticles, with uniform size and distribution, was observed under pH conditions of 8-10. The number of nanoparticles decreased significantly under neutral and weakly acidic conditions, and further decreased as the pH approached strong acid and strong alkaline conditions, consistent with previous experimental results. Comparing the yield of nanoparticles under different conditions, the yield under weakly alkaline conditions increased from 13.21% to 25.9%. In conclusion, relying on the covalent cross-linking of EGCG and collagen under weakly alkaline conditions, EGCG-collagen nanoparticles with enhanced stability and increased yield can be obtained from the initial synthesis of nanoparticles.

[0029] Example 3: Loading capacity of EGCG-collagen nanoparticles Before adding 10 µL of EGCG (10 mg / mL), add 2 µL of Rhodamine B (RhB) (50, 75, 100, 200, or 500 µg / mL). -1 10 μL of collagen (2.5 mg / mL) was mixed with 10 μL of Tris (5 mM pH=9) buffer and loaded into EGCG-collagen nanoparticles. The loading efficiency of RhB in EGCG-collagen nanoparticles was determined by subtracting the loading amount from the total amount of cargo added. Fluorescence intensity was measured at 600 nm on an equal volume of supernatant obtained from the initial centrifugation step with an excitation wavelength of 560 nm to determine the unloading amount. Using the same protocol, fluorescence intensity was measured at 550 nm excitation and 590 nm emission for different concentrations of rhodamine isothiocyanate-labeled bovine serum albumin (BSA-RBITC) (50, 75, 100, 200, or 500 µg / mL) labeled with fluorescein isothiocyanate. -1 The loading rate of IgG-FITC and the fluorescence intensity at 495 nm excitation and 525 nm emission were measured for different concentrations of IgG-FITC (50, 75, 100, 200, or 500 µg mL). -1 The load factor, such as (refer to) Figure 10 As shown in the figure. The results showed that the nanoparticles had high loading rates (over 90%) for both RhB and IgG-FTIC, while the loading rate for BSA-RBITC was relatively low (around 60%). After centrifuging the RhB- and BSA-RBITC-loaded nanoparticles, the supernatant was removed, and the solution was resuspended in double-distilled water. The particle fluorescence pattern was observed under confocal microscopy. It was observed that the particle size of RhB-loaded particles did not change significantly, remaining at around 400 nm (refer to the reference). Figure 11 The particle size of particles loaded with IgG-FITC and BSA-RBITC is slightly larger, around 700 nm (refer to...). Figure 11This may be due to the increased volume of the nanoparticles caused by loading larger IgG and BSA. In summary, EGCG-collagen nanoparticles can load molecules of different sizes, suggesting that they could be used as a delivery system for drug delivery.

[0030] Example 4: Biocompatibility of EGCG-collagen nanoparticles Mouse fibroblasts (L929) and human immortalized keratinocytes (HACAT) were used as models in this experiment. The biocompatibility of EGCG-collagen nanoparticles was assessed using CCK-8 assay and cell viability / deadness staining. L929 cells were seeded at a density of 1 × 10⁴ cells / well in 96-well plates and incubated for 24 hours. Subsequently, the original culture medium was replaced with medium containing different concentrations of EGCG-collagen nanoparticles, with serum-containing DMEM medium serving as a negative control. After 24 hours of culture, the cells were washed three times with PBS, and then 10 µL of CCK-8 reagent and 100 µL of serum-containing DMEM medium were added to each well. The cells were incubated at 37 ℃ in the dark for 2 hours, and the absorbance was read at 450 nm using a microplate reader. This experiment was repeated three times. Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100% (reference) Figure 12 Cell viability assays for HACAT cells were performed using the same method (see [reference]). Figure 14 L929 cells were fed at a rate of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 24-well plates and incubated for 24 hours. Subsequently, the original culture medium was replaced with medium containing different concentrations of EGCG-collagen nanoparticles, with serum-containing DMEM medium used as a negative control. After 24 hours of culture, cells were washed twice with PBS, and then 0.5 mL of cell viability / death staining solution was added to each well. The cells were incubated at 37 °C in the dark for 20 minutes. The cell viability / death staining solution was then removed, and the cells were washed twice with PBS. 0.5 mL of PBS was added, and the cells were immediately observed under a fluorescence microscope. Live cells showed green fluorescence, and dead cells showed red fluorescence (see reference). Figure 13 Cell viability staining of HACAT cells was performed using the same method (see [reference]). Figure 15 The results in summary show that EGCG-collagen nanoparticles at concentrations of 500 ug / ml and below exhibit good biocompatibility.

[0031] Example 5: Cellular uptake of EGCG-collagen nanoparticles L929 cells were used as a model. Cells were cultured at a density of 2.5 × 10⁻⁶. 4L929 cells were seeded at a density of 1 cell / well in 24-well plates and incubated for 24 hours. The culture medium was then replaced with medium containing 250 μg / ml of nanoparticles, and incubation was continued for 0, 0.5, 1, 2, 4, and 8 hours, respectively. The cytoskeleton and nucleus of the cells were stained with FITC-labeled phalloidin and DAPI staining solutions, respectively. The uptake of RhB-loaded EGCG-collagen nanoparticles by L929 cells was observed using a fluorescence microscope. Excitation wavelengths: FITC-labeled phalloidin (488 nm, green fluorescence), RhB-loaded EGCG-collagen nanoparticles (560 nm, red fluorescence), DAPI (358 nm, blue fluorescence) (refer to...). Figure 16 For quantitative analysis, flow cytometry can be used to detect the fluorescence intensity of intracellular nanoparticles (see [reference]). Figure 17 The results in summary show that L929 cells basically completed the cellular uptake of nanoparticles 4 hours after drug administration.

[0032] Example 6: Reactive Oxygen Scavenging by EGCG-Collagen Nanoparticles The ROS scavenging ability of EGCG-collagen nanoparticles was assessed using 2,7-dichlorofluorescein diacetate (DCFH-DA). L929 nanoparticles were loaded at 2.5 × 10⁻⁶ cells per well. 4 Cells were seeded into 24-well plates. The culture medium was replaced with medium containing nanoparticles at concentrations of 0 μg / ml, 125 μg / ml, 250 μg / ml, and 500 μg / ml, and incubated for 4 hours. The wells were then rinsed three times with PBS, and 500 μL of H2O2 was added for further incubation for 2 hours. Plates without added nanoparticles or hydrogen peroxide were used as the particle group. The supernatant was removed, and 500 μL of DCFH-DA was added to each well for incubation for 30 minutes. The staining solution was removed, and the cells were rinsed three times with PBS. Hoechst staining was then added for 15 minutes, the staining solution was removed, and the cells were rinsed three times with PBS. Cell fluorescence was imaged using an inverted fluorescence microscope. Under the microscope, green fluorescence represented reactive oxygen species intensity, and blue should represent cell nuclei (refer to...). Figure 18 Fluorescence intensity was assessed by flow cytometry (see reference). Figure 19 The results in summary show that 500 μg / ml of nanoparticles, administered for 4 hours, can effectively scavenge reactive oxygen species. Note: Figure 18 The blurred content at the bottom of each small image indicates a magnification of "100μm", which is unrelated to the technical solution.

[0033] In summary, the embodiments of the present invention successfully constructed self-assembled nanoparticles using EGCG and collagen, which have the following significant effects: (1) Good biocompatibility: The raw materials of nanoparticles are derived from natural compounds, which have good biocompatibility and have no effect on cell growth and proliferation; (2) Rapid cell delivery capability: Based on the endocytosis mechanism of nanoparticles, EGCG-collagen nanoparticles can load drugs and deliver them into cells within 4 hours; (3) Good reactive oxygen species scavenging ability: Relying on the ability of EGCG to scavenge excess free radicals, EGCG-collagen nanoparticles can reduce the ROS level in skin cells and play a role in protecting and repairing skin photoaging.

[0034] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A self-assembled nanoparticle, characterized in that, This includes nanoparticles that are self-assembled from polyphenolic compounds and collagen in a weakly alkaline environment.

2. The self-assembled nanoparticle according to claim 1, characterized in that, The pH value is 8-10 in the weakly alkaline environment.

3. The self-assembled nanoparticle according to claim 1, characterized in that, The polyphenolic compounds include epigallocatechin gallate (EGCG).

4. A method for preparing self-assembled nanoparticles as described in any one of claims 1-3, characterized in that, Includes the following steps: S1: Collagen and epigallocatechin gallate (EGCG) were added to a weakly alkaline solution and allowed to stand for reaction to obtain an EGCG-collagen nanoparticle suspension. S2: The EGCG-collagen nanoparticle suspension was purified by a combination of centrifugation and resuspension, and the precipitate was collected to obtain EGCG-collagen nanoparticles.

5. The method for preparing self-assembled nanoparticles according to claim 4, characterized in that, The weakly alkaline solution is a Tris buffer solution with a pH of 8 to 10 or double-distilled water.

6. The method for preparing self-assembled nanoparticles according to claim 4, characterized in that, In the S1 reaction system, the concentration ratio of epigallocatechin gallate (EGCG) to collagen is 4~8:

1.

7. The method for preparing self-assembled nanoparticles according to claim 4, characterized in that, S2 includes placing the EGCG-collagen nanoparticle suspension in a centrifuge and centrifuging at a speed of 3000-5000 rcf for 2-4 minutes, removing the supernatant to obtain a precipitate; Resuspension and centrifugation: Add double-distilled water to the precipitate to fully resuspend the precipitate and obtain a new suspension. Place the new suspension in a centrifuge and centrifuge at 3000-5000 rcf for 2-4 minutes to collect the precipitate. Repeat the resuspension and centrifugation process at least twice, collect the precipitate, and obtain purified EGCG-collagen nanoparticles.

8. An application of the self-assembled nanoparticles as described in any one of claims 1-3, characterized in that, The self-assembled nanoparticles are used to prevent and repair photoaging of the skin.

9. The application of the self-assembled nanoparticles according to claim 8, characterized in that, The method for preventing and repairing photoaging of the skin includes the ability of the self-assembled nanoparticles to load molecules of different sizes and / or to be biocompatible and / or to promote the uptake of the self-assembled nanoparticles by L929 cells and / or to achieve effective scavenging of reactive oxygen species.

10. The application of the self-assembled nanoparticles according to claim 9, characterized in that, EGCG-collagen nanoparticles at concentrations of 500 ug / ml and below exhibit biocompatibility and the ability to scavenge reactive oxygen species.

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