Preparation method and application of polypeptide-cerium dioxide active nanoparticle complex

Abstract

The application relates to the technical field of biological medical nanomaterial manufacturing, and discloses a polypeptide-cerium dioxide active nanodot compound, which is prepared through electrostatic action of polypeptide (KTTKS) with the function of activating collagen and CeO2 nanodots with the functions of absorbing ultraviolet rays and removing free radicals. The nanodot compound has the advantages of easy cell uptake, ultraviolet resistance, high free radical removal activity, can significantly protect skin cells from ultraviolet damage, and can promote the synthesis of collagen in skin cells. The application has significant practical value in the fields of anti-skin photoaging skin care products and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medical nanomaterial manufacturing, in particular to a preparation method and application of a polypeptide-cerium dioxide active nanopoint compound. BACKGROUND

[0002] Skin is the largest organ of the human body and plays an important role in maintaining the normal operation of the body. With age, various organs of the human body will degenerate, and the skin also faces the problem of aging. Physiologically, skin aging is a systematic and complex process, mainly including intrinsic aging caused by time changes and extrinsic aging caused by environmental factors. Among them, skin photoaging caused by ultraviolet radiation in sunlight is the main cause of extrinsic skin aging, which directly or indirectly causes damage to biological molecules such as DNA, produces reactive oxygen species (ROS), causes adverse reactions such as reduction of collagen synthesis, and causes aging on the skin surface. Therefore, in the context of the increasing trend of global population aging, developing effective anti-skin photoaging strategies to delay skin aging is the common expectation of mankind.

[0003] Preparation of sunscreen with a substance having ultraviolet light absorption function as a functional additive is currently the most important anti-skin photoaging strategy. Currently commercialized sunscreens have many shortcomings. For example, traditional inorganic sunscreens such as TiO2 have been shown to produce photo-generated radicals under ultraviolet light irradiation and cause toxicity to cells, while organic chemical sunscreens can accumulate in the body and produce toxicity during the body's own metabolism. The current new type of nano sunscreen can reduce these toxicities and side effects through encapsulation, adsorption and other means, but the process of photoaging is often complex, and photo-generated radicals produced in the body due to light are still one of the important factors leading to cell necrosis and apoptosis, while existing sunscreens often only have the single function of absorbing ultraviolet light. Therefore, it is often of great significance to develop new multifunctional anti-skin photoaging materials. For this purpose, we aim to develop a new type of nano anti-skin photoaging agent with multiple functions, and prepare a new type of nano composite with the functions of absorbing ultraviolet light, removing free radicals, promoting collagen synthesis and the like. SUMMARY

[0004] In order to solve the problems in the prior art, the present application prepares a polypeptide-cerium dioxide active nanodot complex, which is prepared by electrostatic interaction of a polypeptide (KTTKS) having a collagen synthesis activating function and CeO2 nanodots having ultraviolet absorption and free radical scavenging activity. The nanodot complex has easy cell uptake, ultraviolet resistance, high free radical scavenging activity, can significantly protect skin cells from ultraviolet damage, and can promote collagen synthesis in skin cells. The present application has significant practical value in the field of anti-skin photoaging skin care products and the like.

[0005] The object of the present application will be achieved by the following technical solutions:

[0006] A preparation method of a polypeptide-cerium dioxide active nanodot complex, comprising the following steps:

[0007] S1. First, 8-12 mL of 0.24-0.26 M Ce(NO3)3·6H2O and 4-6 mL of 8-12% polyacrylic acid aqueous solution are mixed, then 14-16 mL of NH3·H2O is added, and stirring is carried out for 23-25 h;

[0008] S2. The obtained product is centrifuged and filtered at 3800-4200 rpm for 28-32 min to remove large particles, then concentrated by rotary evaporation at 38-42 ℃, and finally dialyzed using a 1 kDa dialysis bag for 46-50 h to obtain Ce NDs;

[0009] S3. The prepared Ce NDs (2-3 mg / mL) are combined with KTTKS peptide (1-2 mg / mL) in 8-12 mM phosphate buffer solution at pH 7.3-7.5 at room temperature, and stirring is carried out for 4-5 h to combine the Ce NDs with the KTTKS peptide;

[0010] S4. The PCHDs are obtained by centrifugal washing 3-4 times using a 1 kDa ultrafiltration tube.

[0011] As a further scheme of the present application, the polypeptide-cerium dioxide active nanodot complex is applied in sun protection.

[0012] The photosensitizer realizes immunotherapy of tumors by stimulating the immune system of the body.

[0013] The present application has the following beneficial effects:

[0014] The polypeptide-cerium dioxide active nanodot complex is prepared by electrostatic interaction of polypeptide (KTTKS) with the function of activating collagen synthesis and CeO2 nanodot with the functions of absorbing ultraviolet and free radical scavenging. The nanodot complex has the advantages of easy cell uptake, anti-ultraviolet, high efficient free radical scavenging activity, can significantly protect skin cells from ultraviolet damage, and can promote the synthesis of collagen in skin cells. The application has significant practical value in the field of anti-skin photoaging skin care products and the like. BRIEF DESCRIPTION OF DRAWINGS

[0015] The drawings constituting a part of the specification illustrate the present application and, together with the description, serve to explain the principles of the application. They are intended solely for purposes of illustration and should not be considered restrictive in any way.

[0016] Figure 1 a is an attenuated total reflection infrared spectrum of Ce NDs and PCHDs;

[0017] Figure 1 b is a Zeta potential diagram of PCHDs loaded with different amounts of KTTKS peptide;

[0018] Figure 1 c is a transmission electron microscope diagram of PCHDs;

[0019] Figure 1 d is an ultraviolet-visible spectrum diagram of Ce NDs, PCHDs and KTTKS;

[0020] Figure 2 a is the molecular structure formula of KTTKS peptide and FITC-KTTKS;

[0021] Figure 2 b is the fluorescence emission spectrum of FITC-PCHDs and PCHDs;

[0022] Figure 2 c is the fluorescence intensity regression curve of FITC-KTTKS;

[0023] Figure 2 d is the fluorescence emission spectrum of unreacted FITC-KTTKS after dilution;

[0024] Figure 3 is the fluorescence imaging diagram of the uptake of different concentrations of FITC-KTTKS by HDF cells;

[0025] Figure 4 is the fluorescence imaging diagram of the uptake of different concentrations of FITC-PCHDs by HDF cells;

[0026] Figure 5 is the effect of different concentrations of PCHDs on the activity of HDF cells;

[0027] Figure 6 SOD enzyme activity of PCHDs at different concentrations;

[0028] Figure 7 HDF cell viability after co-incubation of PCHDs at different concentrations and UV irradiation;

[0029] Figure 8 Collagen expression changes of HDF cells incubated with PCHDs at different concentrations before and after UV irradiation. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed description is illustrative only, and is intended to provide further description in order to provide a fuller enabling and complete disclosure of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0031] Unless otherwise indicated, the procedures and conditions in the examples described herein are carried out according to conventional procedures described in the literature. The starting materials or instruments used are conventional products available on the market, including but not limited to the starting materials or instruments used in the examples described herein.

[0032] Synthesis of ceria nanodots (Ce NDs)

[0033] First, 10 mL of 0.25 M Ce(NO3)3·6H2O and 5 mL of 10% polyacrylic acid aqueous solution were mixed, then 15 mL of NH3·H2O was added, and the reaction was stirred for 24 h. The obtained product was centrifuged at 4000 rpm for 30 min to remove large particles, then concentrated by rotary evaporation at 40 °C, and finally dialyzed using a 1 kDa dialysis bag for 48 h to obtain Ce NDs.

[0034] Synthesis of polypeptide-ceria nanodot complexes (PCHDs)

[0035] The prepared Ce NDs (2 mg / mL) were mixed with KTTKS peptide (1 mg / mL) in 10 mM pH 7.4 phosphate buffer (PBS) at room temperature for 4 h to allow the binding of Ce NDs and KTTKS peptide. PCHDs were obtained by centrifugation and washing 3 times using a 1 kDa ultrafiltration tube.

[0036] FITC-KTTKS peptide modified with FITC fluorescence was used to replace KTTKS peptide to prepare FITC-PCHDs under the same reaction conditions to bind with Ce NDs, for evaluating the loading of Ce NDs on KTTKS peptide. A standard curve was prepared by preparing a solution of FITC-KTTKS peptide with a known concentration, and the reaction solution was obtained by removing FITC-PCHDs using ultrafiltration, and the fluorescence intensity was measured using a fluorescence spectrometer, and compared with the standard curve to finally calculate the loading of Ce NDs on the polypeptide.

[0037] Cell culture and cell viability assay

[0038] All cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 1% penicillin-streptomycin. All cells were cultured in a cell incubator at 37 °C, 5% CO2, and the medium was replaced every 2 days. Human dermal fibroblasts (HDF) were used as a cell model to determine the cytotoxicity of PCHDs using the MTT method. HDF cells were seeded at 10 4 000 cells per well in a 96-well plate and grown overnight, then 0, 25, 50, 75, 100 μg / mL of PCHDs were added respectively, with five replicates for each concentration. After 12 h of culture, the medium was replaced with serum-free DMEM medium and MTT was added, and incubated in the cell incubator for 4 h. Then the medium was removed, 150 μL of DMSO was added to each well, and the formazan was fully dissolved by shaking at room temperature for 15 min. Finally, the absorbance at 490 nm was read on a microplate reader.

[0039] PCHDs cellular uptake

[0040] HDF cells were seeded in a 24-well plate at a density of 30,000 cells per well, and after 12 h of culture, they reached 70-80% confluence. FITC-KTTKS or PCHDs modified with FITC-KTTKS were added to the culture medium, and incubated at 37 °C in a 5% CO2 incubator for 8 h. Then the culture medium was removed and the cells were fixed with 5% formaldehyde for 20 min, and washed with PBS for 3 times. Then they were stained with Hoescht dye for 20 min, washed with PBS for 3 times, and finally observed under a fluorescence microscope.

[0041] Superoxide dismutase (SOD) activity test

[0042] Superoxide dismutase (SOD) activity was tested using a commercial detection kit (Shanghai Yuan Ye Biological Technology Co., Ltd.). NBT colorimetric method was used for measurement. In the experiment, different concentrations of PCHDs were incubated with the kit detection solution, and finally the absorbance of the solution was measured, and the SOD-like enzyme activity value of the polypeptide nanodot complex was calculated.

[0043] PCHDs UV protection assay

[0044] The protective effect of PCHDs on cell photodamage was evaluated by measuring cell viability after UV irradiation. Briefly, HDF cells were seeded at 10 4 cells per well on 96-well microplates and incubated with PCHDs at 25, 50, 75, 100 pg / mL for 8 h. After that, cells were irradiated with a UV lamp for 60 min using unirradiated cells as negative control. Then the plates were kept in a 5% C02 incubator at 37 °C for 24 h. Finally, cell viability was determined by MTT assay.

[0045] Collagen expression assay

[0046] First, HDF cells were cultured overnight on 24-well plates with 10 5 cells per well. Then PCHDs were added to the cells at a final concentration of 0, 25, 50, 75, 100 pg / mL, respectively. After 3 days of culture, the medium was removed and the amount of collagen deposited by the cells was measured. In the collagen amount test, cells were first fixed in situ with 70% ice ethanol for 10 min, then washed with distilled water. Next, the cells were immersed in a Sirius red picric acid staining solution and incubated gently at 4 °C overnight. Then the staining solution was washed off with water, and the cells were treated with 1 M NaOH at room temperature for 10 min to dissolve the collagen dye complex. Finally, 100 pL aliquots of each sample were transferred to a 96-well plate in duplicate, and the absorbance at 490 nm was measured on a microplate reader.

[0047] Experimental results

[0048] In this study, KTTKS peptide was used as an anti-aging active ingredient and polypeptide-cerium dioxide hybrid nanodot complexes (PCHDs) combined with cerium dioxide nanodots (CeNDs) were prepared. KTTKS peptide is a signal peptide widely used in skincare products, which can promote the production of extracellular matrix (ECM) and the expression of type I and III collagen in fibroblasts, thereby playing an anti-aging role in the skin. CeO2 nanodots are a kind of high biocompatibility particles with superior UV absorption capacity and active oxygen scavenging capacity, showing good application potential in the biomedical field. First, CeNDs were synthesized by alkaline precipitation method, then using the characteristics of basic polypeptide KTTKS mainly existing in cationic form at a lower pH and the characteristics of polyacrylic acid on the surface of CeNDs mainly existing in carboxyl anion form under weak alkaline conditions, they were electrostatically combined on the surface of CeNDs to prepare PCHDs.

[0049] 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 1 b) 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.

[0050] 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.

[0051] Cellular uptake

[0052] 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 3As shown, FITC-KTTKS has weak cellular uptake ability, only showing weak green fluorescence at high concentration, while FITC-PCHDs show certain fluorescence intensity at 10 μg / mL (equivalent to 10% wt loading of polypeptide) Figure 4 ), and the fluorescence intensity increases with the increase of FITC-PCHDs concentration, indicating that PCHDs have good cellular uptake ability and Ce NDs have good carrying capacity for KTTKS peptide.

[0053] Cytotoxicity assay

[0054] Considering the requirement of material safety for biomedical applications, we then determined the cytotoxicity of PCHDs by MTT colorimetric method. Succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet Formazan and deposit in cells, and DMSO-solubilized Formazan has strong absorbance value at 490 nm wavelength. The change of related degree value produced by cells after MTT treatment under different conditions can evaluate the proliferation of cells, thus reflecting the cytotoxicity of materials. As shown in Figure 5 , HDF cells treated with different concentrations of PCHDs have similar activity as the control group, and the activity of cells still maintains at about 95% when the concentration of PCHDs is 100 μg / mL, indicating that PCHDs have very low cytotoxicity.

[0055] Superoxide dismutase (SOD) activity test

[0056] Then, we studied the superoxide dismutase-like (SOD-like) activity of PCHDs. SOD is a key enzyme in vivo to eliminate reactive oxygen species (ROS). SOD can catalyze superoxide anion radical (·O 2- ) to be dismutated into oxygen (O2) and hydrogen peroxide (H2O2). CSOD activity is determined by NBT colorimetric method, which uses riboflavin to generate ·O 2- under light. Briefly, riboflavin can catalyze O2 to generate ·O 2- under light, and ·O 2- reacts with NBT to generate monomethyl succinate (yellow). Monomethyl succinate is further reduced by a reducing agent to generate blue methylene violet. Nanoscale enzymes with SOD-like activity can eliminate ·O 2- , thus reducing the color of the solution. The stronger the SOD activity of nanoscale enzymes, the lighter the color of the solution. As shown in Figure 6As 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.

[0057] Cell photoprotection ability

[0058] 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 7 The 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.

[0059] Collagen synthesis

[0060] 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.

[0061] 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, 8-12 mL of 0.24-0.26 M Ce(NO3)3·6H2O and 4-6 mL of 8-12% polyacrylic acid aqueous solution were mixed, and then 14-16 mL of NH3·H2O was added and stirred for reaction. S2, the obtained product was centrifuged to remove large particles and then concentrated by rotary evaporation, and finally dialyzed using a 1 kDa dialysis bag to obtain CeNDs; S3. CeNDs prepared at a concentration of 2-3 mg / mL and KTTKS peptide prepared at a concentration of 1-2 mg / mL were reacted in a phosphate buffer at room temperature with stirring to allow CeNDs 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 stirring reaction time is 23-25 ​​hours.

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 the phosphate buffer is 8-12 mM, the pH is 7.3-7.5, and the stirring reaction time at room temperature is 4-5 h.

5. 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.

6. Use of the polypeptide-cerium dioxide active nanodot complex prepared by the method for preparing the polypeptide-cerium dioxide active nanodot complex according to any one of claims 1 to 5 in the preparation of anti-photoaging skin care products.

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