Preparation method and application of polypeptide-cerium dioxide active nanodot compound

Abstract

The invention relates to the technical field of manufacturing of biomedical nano materials, and discloses a polypeptide-cerium dioxide active nano dot compound. The polypeptide-cerium dioxide active nanodot compound is prepared by electrostatic interaction of polypeptide (KTTKS) with a collagen synthesis activating function and CeO2 nanodots with ultraviolet ray absorbing and free radical scavenging activity. The nano-dot compound has the advantages of easiness in cellular uptake, ultraviolet resistance and efficient free radical scavenging activity, can obviously protect skin cells from being damaged by ultraviolet rays, and can promote collagen synthesis in the skin cells. The technology disclosed by the invention has remarkable practical value in the fields of application of skin photoaging-resistant skin care products and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical nanomaterial manufacturing, in particular to a preparation method and application of a polypeptide-cerium dioxide active nanodot complex. Background Art

[0002] The skin is the largest organ in the human body and plays a vital role in maintaining the normal functioning of the body. As we age, various organs in the human body degenerate, and the skin also faces the problem of aging. From a physiological perspective, skin aging is a systematic and complex process, mainly consisting of intrinsic aging caused by time and extrinsic aging caused by environmental factors. Among them, photoaging of the skin caused by ultraviolet radiation in sunlight is the most important cause of extrinsic skin aging. It directly or indirectly causes damage to biomolecules such as DNA, generates reactive oxygen species (ROS), and causes adverse reactions such as reduced collagen synthesis, resulting in the apparent aging of the skin. Therefore, against the backdrop of the increasingly aging trend of the global population, the development of effective anti-photoaging strategies to delay skin aging is a common expectation of mankind.

[0003] Formulating sunscreens with UV-absorbing substances as functional additives is currently the leading strategy for combating photoaging. However, currently available commercial sunscreens suffer from numerous shortcomings. For example, traditional inorganic sunscreens, such as TiO2, have been shown to generate photogenerated free radicals under UV exposure, leading to cellular toxicity. Organic chemical sunscreens also accumulate and become toxic during their metabolism. New nano-sunscreens can mitigate these toxicities and side effects through methods such as encapsulation and adsorption. However, the photoaging process is complex, and photogenerated free radicals generated by light in the body remain a key driver of cell necrosis and apoptosis. However, many existing sunscreens often possess only a single UV-absorbing function. Therefore, developing novel multifunctional anti-photoaging materials is crucial. To this end, we aimed to develop novel multifunctional nano-complexes that not only absorb UV light but also scaveng free radicals and promote collagen synthesis. Summary of the Invention

[0004] To address the challenges of the existing technology, the present invention has developed a peptide-cerium dioxide active nanodot complex. This complex is formed through the electrostatic interaction of a peptide (KTTKS) that activates collagen synthesis with CeO2 nanodots that absorb UV light and scavenge free radicals. This nanodot complex exhibits easy cellular uptake, UV resistance, and highly effective free radical scavenging activity, significantly protecting skin cells from UV damage and promoting collagen synthesis within skin cells. This technology has significant practical value in applications such as anti-photoaging skincare products.

[0005] The purpose of the present invention is achieved through the following technical solutions: A method for preparing a polypeptide-cerium dioxide active nanodot complex comprises the following steps: S1. First, mix 8-12 mL of 0.24-0.26 M Ce(NO3)3·6H2O and 4-6 mL of 8-12% polyacrylic acid aqueous solution, then add 14-16 mL of NH3·H2O and stir to react for 23-25 ​​h. S2. The obtained product was centrifuged at 3800-4200 rpm for 28-32 min to remove large particles, and then concentrated by rotary evaporation at 38-42 °C. Finally, it was dialyzed using a 1 kDa dialysis bag for 46-50 h to obtain Ce NDs. S3. The prepared Ce NDs (2-3 mg / mL) and KTTKS peptide (1-2 mg / mL) were reacted in 8-12 mM phosphate buffer (pH 7.3-7.5) at room temperature with stirring for 4-5 h to allow Ce NDs to bind to the KTTKS peptide. S4. Use 1 kDa ultrafiltration tube to centrifuge and wash 3-4 times to obtain PCHDs.

[0006] As a further embodiment of the present invention, the polypeptide-cerium dioxide active nanodot complex is used in sun protection.

[0007] The photosensitizer achieves tumor immunotherapy by stimulating the body's immune system.

[0008] The beneficial effects of the present invention are: This peptide-cerium dioxide active nanodot complex is produced through the electrostatic interaction of a peptide (KTTKS) that activates collagen synthesis with CeO2 nanodots that absorb UV light and scavenge free radicals. This nanodot complex exhibits easy cellular uptake, UV resistance, and highly effective free radical scavenging activity, significantly protecting skin cells from UV damage and promoting collagen synthesis within skin cells. This invention has significant practical value in applications such as anti-photoaging skincare products. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0010] Figure 1 a is the attenuated total reflection infrared spectra of Ce NDs and PCHDs; Figure 1 b is the Zeta potential diagram of PCHDs loaded with different amounts of KTTKS peptide; Figure 1 c is the transmission electron microscopy image of PCHDs; Figure 1 d is the UV-visible spectra of Ce NDs, PCHDs, and KTTKS; Figure 2 a is the molecular structure formula of KTTKS peptide and FITC-KTTKS; Figure 2 b is the fluorescence emission spectrum of FITC-PCHDs and PCHDs; Figure 2 c is the regression curve of the fluorescence intensity of FITC-KTTKS; Figure 2 d is the fluorescence emission spectrum of unreacted FITC-KTTKS after dilution; Figure 3 Fluorescence imaging of DHF cells taking up different concentrations of FITC-KTTKS; Figure 4 Fluorescence imaging of DHF cells taking up different concentrations of FITC-PCHDs; Figure 5 The effect of different concentrations of PCHDs on HDF cell activity; Figure 6 is the SOD enzyme activity at different concentrations of PCHDs; Figure 7 The activity of HDF cells after co-incubation with different concentrations of PCHDs and irradiation with UV light; Figure 8 Changes in collagen expression in HDF cells incubated with different concentrations of PCHDs before (a) and after (b) UV irradiation. DETAILED DESCRIPTION

[0011] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0012] If specific experimental steps or conditions are not specified in the examples, the procedures or conditions of conventional experimental steps described in the literature in the field can be followed. All raw materials and instruments used are commercially available, including but not limited to those used in the examples of this application.

[0013] Synthesis of Cerium Dioxide Nanodots (Ce NDs) First, 10 mL of 0.25 M Ce(NO₃)₃·6H₂O and 5 mL of 10% polyacrylic acid aqueous solution were mixed, followed by the addition of 15 mL of NH₃·H₂O and stirring for 24 hours. The resulting product was centrifuged at 4000 rpm for 30 minutes to remove large particles, then concentrated by rotary evaporation at 40°C and dialyzed using a 1 kDa dialysis bag for 48 hours to obtain Ce NDs.

[0014] Synthesis of Peptide-Cerium Dioxide Nanodot Complexes (PCHDs) The prepared Ce NDs (2 mg / mL) and KTTKS peptide (1 mg / mL) were reacted in 10 mM phosphate buffered saline (PBS) (pH 7.4) at room temperature with stirring for 4 h to allow binding of the Ce NDs to the KTTKS peptide. PCHDs were then obtained by centrifugation and washing three times using a 1 kDa ultrafiltration tube.

[0015] FITC-PCHDs were prepared by replacing KTTKS peptide with FITC-fluorescently modified FITC-KTTKS peptide under the same reaction conditions and conjugated to Ce NDs. This was used to evaluate the KTTKS peptide binding capacity of Ce NDs. A standard curve was generated by preparing FITC-KTTKS peptide solutions of known concentrations. The reaction solution, free of FITC-PCHDs, was ultrafiltration-filtered. Fluorescence intensity was measured using a fluorescence spectrometer and compared with the standard curve to calculate the peptide binding capacity of Ce NDs.

[0016] Cell culture and cell viability assay All cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were cultured in a cell culture incubator at 37°C and 5% CO2, and the medium was changed every 2 days. Human dermal fibroblasts (HDF) were used as a cell model to determine the cytotoxicity of PCHDs using the MTT assay. HDF cells were plated at 10 cells per well. 4Cells were seeded into 96-well plates and grown overnight. PCHDs were then added at concentrations of 0, 25, 50, 75, and 100 μg / mL, with each concentration replicated five times. After 12 hours of culture, the medium was replaced with serum-free DMEM supplemented with MTT and incubated in a cell culture incubator for 4 hours. The medium was then removed, and 150 μL of DMSO was added to each well. The plates were shaken at room temperature for 15 minutes to fully dissolve the formazan. The absorbance was then read at 490 nm on a microplate reader.

[0017] Cellular uptake of PCHDs HDF cells were seeded at a density of 30,000 per well in 24-well plates and cultured for 12 hours to reach 70-80% confluence. FITC-KTTKS or FITC-KTTKS-modified PCHDs were added to the culture medium and incubated at 37°C in a 5% CO2 incubator for 8 hours. The culture medium was then removed and the cells were fixed with 5% formaldehyde for 20 minutes and washed three times with PBS. They were then stained with Hoechst dye for 20 minutes, washed three times with PBS, and observed under a fluorescence microscope.

[0018] Superoxide dismutase (SOD) activity test Superoxide dismutase (SOD) activity was measured using a commercially available assay kit (Shanghai Yuanye Biotechnology Co., Ltd.) using the NBT colorimetric method. Different concentrations of PCHDs were incubated with the assay solution. The absorbance of the solution was measured, and the SOD-like enzyme activity of the peptide nanodot complex was calculated using the response matrix.

[0019] PCHDs UV protection test The protective effect of PCHDs against cell photodamage was evaluated by measuring cell viability after UV irradiation. 4 Cells were seeded into each well of a 96-well microplate and grown overnight. Cells were then incubated with 25, 50, 75, or 100 μg / mL of PCHDs for 8 hours. Unirradiated cells were then irradiated with UV light for 60 minutes, with unirradiated cells serving as negative controls. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours. Finally, cell viability was determined using the MTT assay.

[0020] Collagen expression determination First, HDF cells were cultured overnight in 24-well plates, with 10 cells seeded per well. 5The cells were then treated with PCHDs at final concentrations of 0, 25, 50, 75, and 100 μg / mL. After 3 days of culture, the culture medium was removed and the amount of collagen deposited by the cells was measured. For the collagen production assay, cells were first fixed in situ with 70% ice-cold ethanol for 10 minutes and then washed with distilled water. The cells were then stained with Sirius Red Picric Acid Solution and incubated overnight at 4°C with gentle shaking. The staining solution was then washed off with water, and the cells were then treated with 1 M NaOH for 10 minutes at room temperature to dissolve the collagen-dye complex. Finally, 100 μL aliquots of each sample were transferred in duplicate to a 96-well plate, and the absorbance at 490 nm was measured on a microplate reader.

[0021] Experimental results This study used the KTTKS peptide as an anti-photoaging active ingredient and prepared peptide-cerium dioxide hybrid nanodot complexes (PCHDs) conjugated to ceria nanodots (CeNDs). KTTKS peptide is a signaling peptide widely used in skincare products. It promotes extracellular matrix (ECM) production and the expression of type I and type III collagen in fibroblasts, thereby exerting anti-aging effects. CeO2 nanodots are highly biocompatible particles with excellent UV absorption and reactive oxygen species scavenging capabilities, showing promising biomedical applications. CeNDs were first synthesized by alkaline precipitation. PCHDs were then prepared by electrostatically binding the basic peptide KTTKS (predominantly a cationic form at pH values ​​below its isoelectric point) and the polyacrylic acid on the CeND surface (predominantly a carboxyl anion form under weakly alkaline conditions) to the CeNDs surface.

[0022] In order to prove the successful synthesis of PCHDs, attenuated total reflection infrared spectroscopy, Zeta potential and UV-visible spectrophotometry were used to characterize PCHDs. Figure 1 a), 1695 cm -1 stretching vibration of the C=O bond at 1550 cm -1 Symmetric stretching vibration of COO- at 1400 cm -1 The asymmetric stretching vibration of COO- at 1200 cm indicates that polyacrylic acid was successfully synthesized on the surface of Ce NDs. In the infrared spectrum of PCHDs, the -1 stretching vibration of the CN bond at 1650cm -1 The stretching vibration of the C=O bond in the amide bond and the 3000 cm -1 The stretching vibration of the saturated carbon-hydrogen bond at the position of KTTKS increased, indicating that the KTTKS peptide has been successfully modified on the surface of Ce NDs. Zeta potential ( Figure 1b) shows that as the loading capacity of Ce NDs for KTTKS peptide increases, its Zeta potential gradually increases and is close to 0 mV at a loading capacity of 10%, indicating that the loading capacity of Ce NDs for KTTKS peptide is around 10%. Figure 1 The c shows that the synthesized PCHDs have a particle size of less than 10 nm and good dispersion. Figure 1 d) shows that both Ce NDs and PCHDs have strong absorption capabilities for UVB and part of UVA. Comparing the spectra of Ce NDs and PCHDs, the absorption wavelength of PCHDs is red-shifted compared with that of Ce NDs, and the absorption has a significant increase in 200-260 nm, which happens to be the absorption band of KTTKS, once again indicating the successful synthesis of PCHDs.

[0023] We also used FITC-KTTKS instead of KTTKS to prepare PCHDs. Figure 2 The results showed that PCHD exhibited fluorescence compared to unloaded CeNDs. Finally, the fluorescence intensity regression curve of unabsorbed FITC-KTTKS versus FITC-KTTKS in solution revealed that the loading capacity of CeNDs for KTTKS was approximately 10% by weight. These results demonstrate the successful preparation of peptide-ceria nanodot complexes.

[0024] Cellular uptake We then used cell uptake experiments to verify the ability of PCHDs to enter cells. We used human dermal fibroblasts (HDF) as a cell model and replaced the KTTKS peptide with FITC-modified KTTKS peptide FITC-KTTKS for easier imaging. Figure 3 As shown in the figure, FITC-KTTKS has a weak cellular uptake ability and only exhibits weak green fluorescence at higher concentrations, while FITC-PCHDs modified with FITC exhibits a certain fluorescence intensity ( Figure 4 ), and the fluorescence intensity increased with the increase of FITC-PCHDs concentration, which indicated that PCHDs had good ability to be taken up by cells and Ce NDs had good carrying capacity for KTTKS peptide.

[0025] Cytotoxicity assay Considering the requirements for material safety in biomedical applications, we then used the tetrazolium salt (MTT) colorimetric method to determine the cytotoxicity of PCHDs. Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells. DMSO-dissolved formazan has a strong absorbance value at a wavelength of 490 nm. The changes in the correlation values ​​produced by cells after MTT treatment under different conditions can be used to evaluate cell proliferation, thereby reflecting the cytotoxicity of the material. Figure 5 As shown in the figure, HDF cells treated with different concentrations of PCHDs had similar activities to those of the control cells. When the PCHDs concentration was 100 μg / mL, the cell activity was still maintained at around 95%, indicating that PCHDs had extremely low cytotoxicity.

[0026] Superoxide dismutase (SOD) activity test Next, we studied the superoxide dismutase (SOD)-like activity of PCHDs. SOD is a key enzyme in the elimination of reactive oxygen species (ROS). SOD can catalyze the production of superoxide anion radicals (·O 2- ) is disproportionated into oxygen (O2) and hydrogen peroxide (H2O2). CSOD activity was determined by the NBT colorimetric method, which utilizes the photogenerated O 2- In short, under light conditions, riboflavin can catalyze the conversion of O2 to ·O 2- , ·O 2- It can react with NBT to generate monomethylsuccinic acid (yellow). Monomethylsuccinic acid is further reduced by reducing agents to generate blue gentian violet. Nanozymes with SOD-like activity will remove O 2- , thereby reducing the color of the solution, and the enzyme activity is evaluated and calculated by monitoring the solution color. The stronger the SOD activity of the nanozyme, the lighter the solution color. Figure 6 As shown in the results, PCHDs have significant SOD-like enzyme activity. At a concentration of 100 μg / mL, the SOD activity exceeded 40 U, ​​indicating that they have excellent free radical scavenging ability.

[0027] Cell photoprotection ability To investigate the UV protection ability of PCHDs, HDF cells were incubated with different concentrations of PCHDs and then irradiated with UV light for 60 min. Unirradiated HDF cells were used as negative control ( Figure 7The results showed that UV light exposure caused severe damage to the cells, reducing cell viability to less than 40% of untreated cells. The presence of PCHDs significantly enhanced the cells' resistance to UV damage. A dose of 25 μg / mL of PCHDs restored cell viability to over 70%, and 50 μg / mL restored cell viability to over 90%. These results demonstrate that the peptide ceria nanodot complex has a strong protective effect against UV-induced skin cell damage.

[0028] Collagen synthesis After verifying the anti-photodamage function of PCHDs in skin cells, we further studied the efficacy of PCHDs in promoting collagen expression in HDF cells. Figure 8 As shown in Figure a, PCHDs can significantly promote the expression of collagen in skin cells without UV exposure, and the trend of increasing collagen expression increases with the increase of PCHDs concentration. When cells are incubated with PCHDs at a concentration of 100 μg / ml, collagen expression is observed to increase by more than 35%. We then evaluated the effect of UV exposure on collagen expression. Figure 8 As shown in Figure b, under UV irradiation, collagen expression in HDF cells was strongly inhibited, decreasing to approximately 30% of the control group. In the presence of PCHDs, the cells' collagen synthesis capacity showed a significant recovery. When the PCHD concentration was increased to 100 μg / ml, intracellular collagen expression activity was fully restored, increasing more than twofold compared to HDF cells irradiated with UV light. These results demonstrate that PCHDs not only minimize UV-induced cell damage but also significantly promote collagen expression.

[0029] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a polypeptide-cerium dioxide active nanodot complex, characterized in that: The following steps are involved: S1. First, Ce(NO3)3·6H2O and polyacrylic acid aqueous solution were mixed, and then NH3·H2O was added and stirred for reaction; S2. The obtained product was centrifuged to remove large particles and then concentrated by rotary evaporation. Finally, Ce NDs were dialyzed using a 1 kDa dialysis bag. S3. The prepared Ce NDs and KTTKS peptide were reacted in a phosphate buffer at room temperature with stirring to allow the Ce NDs to bind to the KTTKS peptide; S4. Use 1 kDa ultrafiltration tube for centrifugation and washing to obtain PCHDs.

2. The method for preparing the polypeptide-cerium dioxide active nanodot complex according to claim 1, characterized in that: In step S1, the Ce(NO3)3·6H2O is 8-12 mL with a concentration of 0.24-0.26 M, the polyacrylic acid aqueous solution is 4-6 mL with a mass concentration of 8-12%, and the NH3·H2O is 14-16 mL. The stirring reaction time is 23-25 ​​h.

3. The method for preparing the polypeptide-cerium dioxide active nanodot complex according to claim 1, characterized in that: In step S2, the centrifugal filtration speed is 3800-4200 rpm, the time is 28-32 min, the temperature of the rotary evaporation concentration is 38-42° C., and the dialysis bag dialysis time is 46-50 h.

4. The method for preparing the polypeptide-cerium dioxide active nanodot complex according to claim 1, characterized in that: In step S3, the concentration of Ce NDs is 2-3 mg / mL, and the concentration of KTTKS peptide is 1-2 mg / mL.

5. The method for preparing the polypeptide-cerium dioxide active nanodot complex according to claim 1, characterized in that: In step S3, the concentration of the phosphate buffer is 8-12 mM, the pH is 7.3-7.5, and the stirring reaction time is 4-5 h at room temperature.

6. The method for preparing the polypeptide-cerium dioxide active nanodot complex according to claim 1, characterized in that: In step S4, the number of centrifugal washing in the ultrafiltration tube is 3-4 times.

7. Use of the polypeptide-cerium dioxide active nanodot complex prepared by the preparation method of the polypeptide-cerium dioxide active nanodot complex according to any one of claims 1 to 6 in preventing skin photoaging.

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