CN110237254B - Preparation method and application of a kind of polymetallic oxygen cluster-food-derived antioxidant peptide photothermal material - Google Patents
Preparation method and application of a kind of polymetallic oxygen cluster-food-derived antioxidant peptide photothermal material Download PDFInfo
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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Abstract
本发明公开了一种多金属氧簇‑食源性抗氧化肽光热材料的制备方法，包括步骤：将大豆五肽水溶液和多金属氧簇水溶液混合，置于紫外光下照射5～10min，得光热材料；其中，所述多肽水溶液中的大豆五肽和所述多氧金属簇水溶液中的多氧金属簇的摩尔比为3:1～1:1。本发明的操作简单，所制备的超分子材料结构明确、尺寸均匀、生理稳定性高、细胞毒性低，具有优异的光热转换性质和抗菌活性，解决了目前技术上普遍存在的多金属氧簇光热材料光热性质不稳定的问题，同时该材料具有优异的抗菌活性，可以避免肿瘤光热治疗过程中炎症反应的发生。在多金属氧簇光热材料的开发及肿瘤光热治疗方面具有良好的应用前景。
The invention discloses a preparation method of a polymetallic oxygen cluster-food-derived antioxidant peptide photothermal material, comprising the steps of: mixing a soybean pentapeptide aqueous solution and a polymetallic oxygen cluster aqueous solution, placing it under ultraviolet light for 5-10 minutes, and then irradiating it for 5-10 minutes. A photothermal material is obtained; wherein, the molar ratio of the soybean pentapeptide in the polypeptide aqueous solution and the polyoxymetal cluster in the polyoxymetal cluster aqueous solution is 3:1 to 1:1. The method is simple in operation, the prepared supramolecular material has clear structure, uniform size, high physiological stability, low cytotoxicity, excellent photothermal conversion properties and antibacterial activity, and solves the problem of polymetallic oxygen clusters commonly existing in the current technology. The photothermal properties of photothermal materials are unstable, and the material has excellent antibacterial activity, which can avoid the occurrence of inflammatory reactions during tumor photothermal therapy. It has good application prospects in the development of polymetallic oxygen cluster photothermal materials and tumor photothermal therapy.
Description
Technical Field
The invention belongs to the technical field of materials and biomedicine, and particularly relates to a preparation method of a novel photothermal conversion material based on a polymetallic oxygen cluster and a food-borne antioxidant active peptide and application of the material in photothermal therapy and antibiosis.
Background
Cancer (malignant tumor) seriously harms human health. Despite the significant findings in Cancer treatment to date, overall Cancer-related mortality remains relatively stable (r.l. siegel, k.d. miller, a. Jemal, Cancer statistics,2015.Ca a Cancer Journal for Clinicians 2015,65, 5; d.l. hoyer, 75 years of mortalities in the United States 2012,88, 1). At present, the treatment means of cancer mainly comprises radiotherapy, chemotherapy, surgical resection and the like, however, the treatment means still has great limitations, such as great side effects, damage to normal cells of human body, drug resistance, high late stage metastasis rate and the like. In order to overcome this problem, people are working on developing novel cancer treatment means, and photothermal therapy is an emerging technology with significant effect on cancer treatment. The principle of photothermal therapy is to enrich the material with high light-heat conversion efficiency inNear the tumor tissue and converting light energy into thermal energy under external near infrared laser (650nm-950nm) irradiation to kill cancer cells (z.zhang, j.wang, c.chen, adv.mater. 2013,25, 3869; l.cheng, c.wang, l.feng, k.yang, z.liu, chem.rev.2014,114, 10869; d.quee, l.martinez Maestro, b.del Rosal, p.haro-Gonzalez, a.benayas, j.l.platza, e.martin Rodriguez, j.garcia Sole, Nanoscale 2015,6, 9494; h.j.kim, s.m.lee, k.h.park, c.h.mu.n, y.b.yk.yool, k.h.95, bio materials). Currently, photothermal therapeutics mainly include precious metal nanoparticles (l.kong, l.yang, c.q.xin, s.j.zhu, h.h.zhang, m.z.zhang, j.x.yang, l.li, h.p.zhou, y.p.tianan, Biosensors)& Bioelectronics 2018,108,14；Y.Wang,J.Xin,P.Peng,Y.Shi,D.Jian,J.Xu,J.Wu, B.Kirk,X.Wei,Journal of Materials Chemistry B 2018,6, 10.1039.C1038TB00233A；S.Kang,W.Shin,K.Kang,M.H.Choi,Y.J.Kim,Y.K. Kim,D.H.Min,H.Jang,Acs Applied Materials&Interfaces 2018,10,13819), graphene (w.gao, h.k.lee, j.hobley, t.liu, i.y.phang, x.y.link, angelbend Chemie 2015,127,4065), carbon nanotubes (j.song, f.wang, x.yang, b.ning, m.g.harp, s.h. Culp, s.hu, p.huang, l.nie, j.chen, Journal of the American Chemical Society 2016,138,7005), transition metal sulfides (mos.k.lee, j.g.blue, r.g.blue, r.h.blue, r.h. blue, r.g.blue, r.h.blue, r.g. blue, r.c.2CuS …) (L.Kong, L.Xing, B.Zhou, L.Du, X.Shi, Acs apple Mater Interfaces 2017,9, 15995-; x.deng, k.li, x.cai, b.liu, y.wei, k.deng, z.xie, z.wu, p.ma, z.hou, Advanced Materials 2017,29), porphyrin liposome (s.su, y.ding, y.li, y.wu, g.nie, Biomaterials 2016,80,169), near infrared absorbing organic dyes (m.lu, n.kang, c.chen, l.yang, y.li, m.hong, x.luo, l.ren, x.wang, Nanotechnology 2017,28,445710), and the like. However, many photothermal materials contain some uncertain problems such as low photothermal conversion efficiency, poor stability, undefined chemical structure and performance, difficult preparation, difficult storage, etc., and during photothermal treatment, the treatment temperature may reach 43 ℃ or even higher, and high temperature may cause tissue cell necrosis and pro-inflammatory reaction, easily causing bacterial infection (x.j.zhu, w.feng, j.chang, y.w.tan, j.c.li, m.chen, y.sun, f.y.li, nat.commun.2015,7, 10437; j.r.melamed, r.s.delestein, e.s.day, ACS nano.2015,9, 6). Therefore, a novel photo-thermal material with excellent photo-thermal performance and good performance is developedTherapeutic agents with good antimicrobial properties are of great value.
Polymetallic oxygen clusters (POMs), as a type of inorganic polymetallic oxide nanoclusters, exhibit abundant physical and Chemical properties through precisely controlled synthesis (M.T. Pope, A.Muller, Angewandte Chemical International Edition 2010,30, 34; B.Li, W.Li, H.Li, L.Wu, Accounts of Chemical Research 2017,50,1391), has shown great potential in the biomedical field and has been used as antibacterial and antiviral drugs (J.Li, Z.Chen, M.Zhou, J.Jing, W.Li, Y.Wang, L.Wu, L.Wang, Y.Wang, M.Lee, Angewandte Chemie 2016,128,2638; J.Wang, Y.Liu, K.xu, Y.Qi, J.Zhong, K.Zhang, J.Li, E.Wang, Z.Wu, Acs Applied Materials & Interfaces 2014,6, 9785), magnetic imaging contrast agents (S.Zhang, Y.Zheng, S.Yin, J.Sun, B.Li, L.Wu, Chemistry-A European Journal 2017,23, 2) and protein inhibitors (S.Y.Lene, A.Leye, J.Sun, B.J.J.J.H, Biotech, H.J.J.J.J.J.J.J.J.J.J.J.J.application 2017,23, 2) as well as protein inhibitors (S.Y.Leye, H.Leye, H.J.Level, H.J.J.J.J.J.H.J.J.J.J.H.J.J.J.H.J.J.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.A. Ser. No. 1, B.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.A. 1, H.H.H.H.H.A. H.H.H.H.H.H.H.H.H.H.H.H.H.H.H.A. H.H.H.H.H.H.A.H.A. H.H.H.A. H.H.H.H.H.H.A. H.H.A. H.H.H.A. H.H.A. H.A. H.H.A. H.A. A. H.A. A. H.A. A. H.A. A. Polymetallic oxygen clusters can undergo efficient photothermal conversion upon reduction due to inter-valence charge transfer (IVCT) transitions into heteropolyblue, and thus POMs have begun to be used as photothermal therapeutic agents in the past two years (C.Zhang, W.Bu, D.Ni, C.Zuo, C.Cheng, Q.Li, L.Zhang, Z.Wang, J.Shi, Journal of the American Chemical Society 2016,138,8156, D.Ni, D.Jiang, H.F.Valdoinos, E.B.Ehlerding, B.Yu, T.E.Barnhart, P.Huang, W.Cai, Nano Letters 2017,17, 3282; S.Zhang, H.Chen, G.Zhang, X.Ko, S.Yin, B.Li, L.Walum, Chemistry 2017, Journal 6, Journal of Mat.8). As a new photo-thermal material, most POMs are unstable in heteropoly blue state after reduction and easy to oxidize when exposed to air, so that the photo-thermal conversion efficiency of the material is greatly reduced, and the single POMs are sensitive to acid, alkali and electrolyte and poor in biocompatibility, so that the development and application of the POMs are severely limited by the factors. Therefore, how to keep the structure and the photo-thermal performance of the material, and simultaneously inhibit the oxidation process and improve the physiological stability is a key technical problem to be solved by the POMs photo-thermal treatment material.
The Food-derived antioxidant peptide is extracted from various foods such as soybean, corn, peanut, milk, egg, fish and meat, has small molecular weight, easy absorption and high safety, can clear redundant free radicals, and is a natural antioxidant (X.J.Wang, X.Q.Zheng, N.K.Kopparau, W.S.Cong, Y.P.Deng, X.J.Sun, X.L.Liu, Process Biochemistry 2014,49, 1562; R.Yang, X.Li, S.Lin, Z.Zhang, F.Chen, Food Chemistry 2017,219,311). The antioxidant process can be mainly attributed to free radical quenching, can inhibit the formation of free radicals in food by delaying lipid oxidation, can be used as a nutritional health product to be added into functional food to improve health, and is widely applied to aspects such as food processing and storage, I.W.Hamley, Organic & biological Chemistry 2017,15,5867 and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of a novel polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material (rSP) with excellent photothermal performance and antibacterial performance, and the food-borne antioxidant peptide and the reduced polymetallic oxygen cluster supermolecule are assembled to hopefully prepare the novel photothermal treatment material with high photothermal conversion efficiency, high photothermal stability, good biocompatibility and antibacterial performance. .
In order to achieve the purpose, the invention provides a preparation method of a polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material, which comprises the following steps:
s1, dissolving soybean pentapeptide in water to prepare a polypeptide water solution with the concentration of the soybean pentapeptide of 1-6 mmol/L, wherein the amino acid sequence of the soybean pentapeptide is shown as SEQ ID:1, and the molecular weight is 590.68 g/mol;
s2, dissolving a Polyoxometalate (POM) in water to prepare a polyoxometalate aqueous solution with the concentration of 1-3 mmol/L;
s3, mixing the polypeptide aqueous solution obtained in the step S1 with the polyoxometalate aqueous solution obtained in the step S2, wherein the molar ratio of the soybean pentapeptide in the polypeptide aqueous solution to the polyoxometalate aqueous solution is 3: 1-1: 1, and uniformly mixing to obtain an assembly solution SP;
s4, placing the assembly solution SP obtained in the step S3 under ultraviolet light for irradiating for about 5-10 min, and obtaining a polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP; the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material is dark blue.
Preferably, the soybean pentapeptide of step S1 is synthesized by standard Fmoc solid phase synthesis strategy, and the purity of the soybean pentapeptide is determined by RP-C18 chromatography column and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Preferably, the polyoxometalate of step S2 is H3PMo12O40。
In a preferred mode, the preparation method of the polyoxometalate-food-borne antioxidant peptide photothermal material comprises the following steps:
s1, dissolving 11.8mg of soybean pentapeptide in 10mL of water, and stirring for 10min to obtain a polypeptide aqueous solution with the soybean pentapeptide concentration of 2 mmol/L; the amino acid sequence of the soybean pentapeptide is shown as SEQ ID: 1;
s2, weighing 36.5mg of polyoxometalate H3PMo12O40Dissolving in 10mL of water, and stirring for 10min to obtain a polyoxometalate aqueous solution with the concentration of 2 mmol/L;
s3, dropwise adding the polyoxometalate aqueous solution obtained in the step S2 into the polypeptide aqueous solution obtained in the step S1 at normal temperature, and stirring for 3 hours to obtain a co-assembled SP aqueous solution; the normal temperature is 24-26 ℃; the molar ratio of the soybean pentapeptide in the polypeptide aqueous solution to the polyoxometalate in the polyoxometalate aqueous solution is 1: 1;
s4, placing the SP aqueous solution obtained in the step S3 in ultraviolet light for 10min to obtain the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP.
The invention also provides the application of the polyoxometallate-food-borne antioxidant peptide photothermal material, and the polyoxometallate-food-borne antioxidant peptide photothermal material is used as an active ingredient for preparing a medicament for resisting escherichia coli; a medicine for resisting Escherichia coli is prepared by adding polyoxometallate-food-borne antioxidant peptide photothermal material; the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal treatment material is used as an active ingredient for preparing a medicament for photothermal treatment; a medicine for photothermal therapy is prepared by adding polyoxometallate-food-derived antioxidant peptide photothermal material; the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material is used for preparing a photothermal conversion material; a photothermal conversion material is prepared by adding polyoxometallate-food-derived antioxidant peptide photothermal material.
The invention has the beneficial effects that:
(1) the novel polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP prepared by the invention has good physiological stability and biocompatibility;
(2) the novel polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP prepared by the invention has good photothermal performance and high safety;
(3) compared with the traditional polyacid photothermal treatment agent, the novel polyoxometalate-food-borne antioxidant peptide photothermal material rSP prepared by the invention has better photothermal stability;
(4) the novel polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP prepared by the invention has good antibacterial activity, is expected to avoid inflammatory effect caused by high temperature in the photothermal treatment process, and has wide application prospect in the field of photothermal treatment;
(5) the novel polyoxometallate-food-borne antioxidant peptide photothermal material rSP prepared by the invention provides a wide application prospect for the application of polyoxometallate and polyoxometallate-based materials in the field of biomedicine.
Drawings
FIG. 1 shows the mass spectrometric identification of soybean pentapeptide described in step S1 of example 1 of the present invention.
Fig. 2 is an infrared spectrum of POM powder prepared by lyophilizing the POM solution prepared in step S2 of example 1, soybean pentapeptide powder prepared by lyophilizing the soybean pentapeptide solution prepared in step S1 of example 1, and rSP powder prepared by lyophilizing the rSP solution prepared in example 1 according to the present invention.
FIG. 3 is Zeta potential of rSP prepared in example 1 of the present invention in aqueous solution.
FIG. 4 is Zeta potential in aqueous solution of POM prepared by comparative example 2 of the present invention.
FIG. 5 is a graph showing the dynamic light scattering of 1mM rSP prepared in example 1 of the present invention in an aqueous solution.
FIG. 6 is a high-resolution TEM image of 1mM rSP prepared in example 1 of the present invention in an aqueous solution.
FIG. 7 is a cytotoxicity test of the POM solution prepared in comparative example 2, the rPOM solution prepared in comparative example 3, and the rSP solution prepared in example 1 against hepatoma cell HepG 2.
FIG. 8 is a graph showing UV absorption spectra of the POM solution prepared in comparative example 2, the rPOM solution prepared in comparative example 3, the rSP solution prepared in example 1, the rPOM solution prepared in comparative example 3 left in the air for 24 hours, and the rSP aqueous solution prepared in example 1 left in the air for 24 hours.
FIG. 9 shows the POM solution prepared in comparative example 2, the rPOM solution prepared in comparative example 3, the rSP solution prepared in example 1, the rPOM solution prepared in comparative example 3, and the rSP solution prepared in example 1 after being left in the air for 24 hours, respectively, at 808nm, 1W/cm and 24 hours after being left in the air2Photothermal curve under laser irradiation.
FIG. 10 is a graph showing the results of air standing for 24 hours for the POM solution prepared in comparative example 2, the rPOM solution prepared in comparative example 3, the rSP solution prepared in example 1, the rPOM solution prepared in comparative example 3, and the rSP solution prepared in example 1 at 808nm, 1W/cm and 24 hours after air standing2And (4) thermally imaging the picture under laser irradiation.
FIG. 11 is a graph showing the cytotoxicity test of rSP solution prepared in example 1 of the present invention against tumor cells HepG2 after 24-hour exposure to air.
FIG. 12 is a graph showing the cytotoxicity test of rPOM solution prepared in comparative example 3 of the present invention on tumor cells HepG2 after 24-hour placement in air.
FIG. 13 is a graph showing the effect of the change in optical density of E.coli with the culture time when the rSP solution prepared in example 1 of the present invention, the rPOM solution prepared in comparative example 3, the aqueous polypeptide solution prepared in comparative example 1, and a control were co-cultured with E.coli.
FIG. 14 is a graph showing the antibacterial effect of blank groups on E.coli.
FIG. 15 is a graph showing the antibacterial effect of rPOM solution prepared in comparative example 3 of the present invention on Escherichia coli.
FIG. 16 is a graph showing the antibacterial effect of the polypeptide solution prepared in comparative example 1 of the present invention on Escherichia coli.
FIG. 17 is a graph showing the antibacterial effect of rSP solution prepared in example 1 of the present invention on Escherichia coli.
FIG. 18 is a schematic diagram of the preparation process and application of photothermal tumor treatment and inflammation inhibition by the novel polyoxometalate-food-borne antioxidant peptide photothermal material rSP.
Detailed Description
The invention is realized by the following technical scheme: soy pentapeptide was first synthesized using standard Fmoc solid phase synthesis strategy. Purity was determined using RP-C18 chromatography columns and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The soybean pentapeptide and the polyoxometalate aqueous solution are simply mixed and stirred, fully assembled together in the solution and then freeze-dried by a freeze dryer, so as to prepare the polyoxometalate-soybean pentapeptide hybrid material SP. And (3) carrying out photoreduction on the SP aqueous solution under ultraviolet irradiation for 10 minutes, wherein the color of the solution is changed from yellow to dark blue in the irradiation process, and finally obtaining the reduced heteropolyblue material (rSP). The rSP and the single heteropoly blue material rPOM are characterized by adopting an infrared spectrum (FT-IR), an Elemental Analysis (EA), a Dynamic Light Scattering (DLS) and a Transmission Electron Microscope (TEM), and the cytotoxicity of the rSP material on human hepatoma cells HepG2 is evaluated by an MTT (3- (4, 5-dimethylthiazole-2-acyl) -2, 5-diphenyltetrazole ammonium bromide) method, so that the rSP is proved to have high biocompatibility on a cellular level and can be applied to further biological application. The photothermal performance and the photothermal stability of the prepared material are evaluated by comparing the rSP with a single heteropoly blue material rPOM solution and the temperature change of the solutions after being exposed in the air for 24 hours under the 808nm laser irradiation, the photothermal treatment effect of the prepared material on tumor cells is further researched, the cytotoxicity is determined by an MTT colorimetric method, and the research result proves that the rSP can be used as a high-efficiency and stable tumor photothermal treatment agent. We subsequently evaluated the antibacterial activity of rSP materials against e.coli (e.coli), whose excellent antibacterial effect is expected to inhibit bacterial infection induced by photothermal therapy of hyperthermia processes. The novel photothermal therapeutic agent with excellent photothermal performance and antibacterial performance can be obtained through simple supramolecular assembly, and has potential application value in photothermal therapy and antibacterial aspect.
The method comprises the following steps of,
1) the soybean pentapeptide is synthesized by adopting a standard Fmoc solid phase synthesis strategy, and the purity of the soybean pentapeptide is measured by using an RP-C18 chromatographic column and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Dissolving soybean pentapeptide in water to prepare a polypeptide water solution with the concentration of the soybean pentapeptide of 1-6 mmol/L, wherein the amino acid sequence of the soybean pentapeptide is shown in figure 1;
2) dissolving a Polyoxometalate (POM) in water to prepare a polyoxometalate aqueous solution with the concentration of 1-3 mmol/L;
3) the equimolar ratio of the soybean pentapeptide aqueous solution obtained in the step 1) and the step 2) to the polyoxometalate POM aqueous solution is 3: 1-1: 1, mixing, shaking and stirring to obtain an assembly solution SP;
4) irradiating the SP solution obtained in the step 3) for 5-10 minutes under ultraviolet light until a dark blue photothermal material rSP is obtained.
In this process, the polyoxometallate used is H3PMo12O40This anionic cluster compound is very common and can be used directly as a raw material.
The main advantages of the present invention include the following aspects: (1) the prepared novel photo-thermal material rSP has good physiological stability and biocompatibility; (2) the prepared novel photothermal material rSP has good photothermal performance and high safety; (3) compared with the traditional polyacid photothermal treatment agent, the prepared novel photothermal material rSP has better photothermal stability; (4) the prepared novel photo-thermal material rSP has good antibacterial activity, is expected to avoid inflammatory effect caused by high temperature in the photo-thermal treatment process, and has wide application prospect in the photo-thermal treatment field; (5) the prepared novel photo-thermal material rSP provides a wide application prospect for the application of polyoxometallate and polyoxometallate-based materials in the biomedical field. The photothermal therapeutic agent rSP prepared according to the present invention can also be used for: use of a novel photothermal therapeutic agent (rSP) in photothermal conversion materials; the application of the novel photothermal therapeutic agent rSP in the aspect of photothermal treatment of tumors; the application of a novel photothermal therapeutic agent rSP in the aspects of antibiosis and antiphlogosis.
FIG. 1 is a mass spectrum of soybean pentapeptide. The short peptides were synthesized using standard Fmoc solid phase synthesis methods. The purity was determined by using an RP-C18 column and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
FIG. 2 is an infrared spectrum of POM, soybean pentapeptide and rSP. Can be seen in 3300, 2933, 2823, 1630, 1425, 1350, 1325, 1297cm-1Characteristic vibrational peaks ascribed to methyl/methylene; at 1672cm-1Characteristic peaks ascribed to amine groups; at 1203cm-1、1128cm-1Characteristic peaks ascribed to imine and carbon-sulfur bonds; at 1064cm-1(P-Od)，967cm-1(Mo＝Od)，867cm-1(Mo-Ob-Mo), and 791cm-1(Mo-Oc-Mo) a characteristic peak attributed to the inorganic cluster, indicating that the soybean pentapeptide and POM framework structures remain good in the hybrid composite.
FIG. 3 shows the Zeta potential of rSP in aqueous solution, which is the Zeta potential value of rSP in aqueous solution, and is +22.4 mV.
FIG. 4 shows Zeta potential of POM in an aqueous solution, and Zeta-potential value of POM in an aqueous solution. 32.5 mV. The results in fig. 3 and 4 show that the polypeptide with positive charge covers the surface of the polyoxometalate, so that the surface of the prepared composite material rSP is positively charged, and the supermolecule co-assembly enhances the stability of the single POM material, so that the rSP is suitable for in vivo application.
FIG. 5 is a graph of dynamic light scattering of 1mM rSP in aqueous solution. The results showed that the mean hydrodynamic diameter of 1mM rSP in aqueous solution was 60 ± 15.4nm (PDI ═ 0.066).
FIG. 6 is a high resolution TEM image of 1mM rSP in an aqueous solution. The size of the rSP is proved to be about 60nm, the transmission electron microscope picture is consistent with the dynamic light scattering result, and the rSP of the assembly of the soybean pentapeptide and the POM is proved to be a spherical aggregate of about 60 nanometers.
FIG. 7 shows cytotoxicity tests of POM, rPOM and rSP against hepatoma cell HepG 2. The cytotoxicity of POM, rPOM and rSP against HepG2 cells at different concentrations was evaluated by MTT colorimetry. The experimental results show that even when the concentration is increased to 104At nM, cell viability remained at 90%On the other hand, this indicates that the POM, rPOM and rSP of the present invention have high biocompatibility at the cellular level and may be suitable for further biological applications.
FIG. 8 is a graph showing ultraviolet absorption spectra of an aqueous solution after POM, rPOM, rSP and rPOM were left in air for 24 hours and in rSP air for 24 hours. It can be seen that rPOM and rSP have strong absorption at the 600-900 nm absorption band, the near infrared absorption disappears after the rPOM is placed in the air for 24 hours, and the rSP still remains unchanged after the rPOM is placed in the air for 24 hours.
FIG. 9 shows photothermal curves of an aqueous solution after POM, rPOM, rSP, and rPOM were left in air for 24 hours and in rSP air for 24 hours under 808nm laser irradiation.
FIG. 10 is a thermal image of POM, rPOM, rSP, rPOM after being left in air for 24 hours, rSP after being left in air for 24 hours, under 808nm laser irradiation. Irradiating with near infrared laser (1.0W/cm at 808 nm)2) The temperature of the rPOM and rSP solutions rises rapidly to 57.1 ℃ within 10 minutes, while the temperature rise of pure POM changes to less than 5 ℃ under the same condition, which proves that the photo-thermal conversion efficiency of the reduced polyoxometalate and rSP materials is high. The solution of rPOM after being placed in the air for 24 hours has insignificant temperature change under the same laser irradiation conditions, which indicates that the rPOM has poor photo-thermal stability, while the solution of rSP after being exposed in the air for 24 hours still has significant temperature rise under the same laser irradiation conditions, which confirms that the assembly rSP of the peptide and the POM has excellent photo-thermal stability.
FIG. 11 is a graph showing cytotoxicity test of rSP, an aqueous solution of rSP left in the air for 24 hours, against tumor cells HepG 2. The result of the cytotoxicity test of the rSP and the rSP aqueous solution which is placed in the air for 24 hours on HepG2 liver cancer tumor cells tested by the MTT method is obtained. The results prove that the HepG2 liver cancer tumor cells are obviously apoptotic under the condition that rSP is placed in the air for 24 hours, and the assembly rSP of the peptide and the POM has excellent tumor cell photothermal treatment effect.
FIG. 12 is a graph showing cytotoxicity test of rPOM in air for 24 hours in an aqueous solution of rPOM against tumor cells HepG 2. The result of the cytotoxicity test of the rPOM aqueous solution of the invention on HepG2 liver cancer tumor cells is tested by using an MTT method and placing the rPOM aqueous solution for 24 hours in the air. The results prove that HepG2 liver cancer tumor cells under the rPOM condition are obviously apoptotic, and the group does not have obvious cell death after being placed in the rPOM air for 24 hours.
FIG. 13 is a test chart of the antibacterial activity of rPOM, soybean pentapeptide and rSP against E.coli.
FIG. 14 is a graph showing the antibacterial effect of blank groups on E.coli.
FIG. 15 is a graph showing the antibacterial effect of rPOM prepared in comparative example 3 of the present invention on Escherichia coli.
FIG. 16 is a graph showing the antibacterial effect of soybean pentapeptide prepared in comparative example 1 of the present invention on Escherichia coli.
Fig. 17 is a graph showing the antibacterial effect of the photothermal material rSP prepared in the example of the present invention on escherichia coli. Compared with the control group, no obvious antibacterial effect is found by using the soybean pentapeptide or the rPOM alone, and the growth of the Escherichia coli in the rSP group is obviously inhibited. The figure of the number of escherichia coli in the LB solid culture medium is consistent with the result of the antibacterial activity test, which shows that the photothermal therapeutic agent rSP has excellent antibacterial performance.
FIG. 18 depicts the preparation of novel polyoxometalate-food-derived antioxidant peptide photothermal material rSP and the use of the prepared material in photothermal treatment of tumors and inhibition of inflammation.
The invention is further illustrated by the following specific examples.
Example 1
A preparation method of polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP comprises the following steps:
s1, synthesizing soybean pentapeptide by adopting a standard Fmoc solid phase synthesis strategy, and determining the purity of the soybean pentapeptide by using an RP-C18 chromatographic column and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, wherein the mass spectrometry result is shown in figure 1; weighing 11.8mg of the soybean pentapeptide, dissolving in 10mL of water, and stirring for 10min to obtain a polypeptide aqueous solution with the soybean pentapeptide concentration of 2 mmol/L; the amino acid sequence of the soybean pentapeptide is shown as SEQ ID: 1;
s2, weighing 36.5mg of polyoxometalate H3PMo12O40Dissolving in 10mL of water, and stirring for 10min, obtaining a polymetallic oxygen cluster aqueous solution with the concentration of 2 mmol/L;
s3, dropwise adding the polyoxometalate aqueous solution obtained in the step S2 into the polypeptide aqueous solution obtained in the step S1 at normal temperature, and mixing soybean pentapeptide with H3PMo12Is 1:1, and is stirred for 3 hours under the condition to obtain a co-assembled SP water solution; the normal temperature is 24-26 ℃;
s4, placing the SP aqueous solution obtained in the step S3 in ultraviolet light for reduction for 10min to obtain a polyoxometalate-food-borne antioxidant peptide photothermal material rSP; the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP is a dark blue solution.
The infrared spectrum structure representation of the rSP powder prepared by freeze-drying the polyoxometalate-food-borne antioxidant peptide photothermal material rSP solution prepared in this example is shown in fig. 2; can be seen in 3300, 2933, 2823, 1630, 1425, 1350, 1325, 1297cm-1Characteristic vibrational peaks ascribed to methyl/methylene; at 1672cm-1Characteristic peaks ascribed to amine groups; at 1203cm-1、1128cm-1Characteristic peaks ascribed to imine and carbon-sulfur bonds; at 1064cm-1(P-Od)，967cm-1(Mo＝Od)，867cm-1(Mo-Ob-Mo), and 791cm-1(Mo-Oc-Mo) characteristic peaks ascribed to inorganic clusters, indicating soybean pentapeptide and POM (i.e., H)3PMo12) The framework structure remains good in the hybrid composite. 1mL of the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP solution prepared in example 1 was taken, and the particle size and Zeta potential of the rSP in the aqueous solution were measured by a Malvern dynamic light scattering instrument. The results of the Zeta potential test are shown in fig. 3; the zeta potential value of SP in aqueous solution is +22.4 mV.
The polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP solution prepared in the example 1 is dripped on an ultrathin copper mesh, and the copper mesh is dried in the air. And observing and analyzing the treated dry rSP on the copper mesh by using a high-resolution transmission electron microscope. The transmission electron microscope pictures and the dynamic light scattering test results are shown in fig. 5 and 6; FIG. 5 is a graph showing dynamic light scattering of 1mM rSP in aqueous solution, and the results show that 1mM rSP in aqueous solutionThe mean hydrodynamic diameter was 60 ± 15.4nm (PDI ═ 0.066); FIG. 6 is a high resolution TEM image of 1mM rSP in water, demonstrating that the size of rSP is about 60nm, the TEM image is consistent with the dynamic light scattering results, and demonstrating soybean pentapeptide and POM (i.e., H)3PMo12O40) The assembly of (3) rSP is a spherical aggregate of about 60 nm.
1mL of the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP aqueous solution prepared in the example was put in a sample cell (cross-sectional area 1 cm)2) In the middle, the power consumption is 1W/cm2The solution was irradiated with 808nm laser for 10 minutes and the temperature of the solution was recorded every 1 minute by a thermal imager. The cooling curve of the solution at room temperature after laser irradiation was recorded, and similarly every 1 minute. As shown in fig. 9, the photothermal temperature increase curve of the rSP aqueous solution shows that the rSP aqueous solution can be rapidly increased in temperature by laser irradiation, and is expected to be used for photothermal therapy.
1mL of the photothermal material rSP aqueous solution prepared in this example was left to stand for 24 hours, and then placed in a sample cell (cross-sectional area 1 cm)2) In the middle, the power consumption is 1W/cm2The solution was irradiated with 808nm laser for 10 minutes and the temperature of the solution was recorded every 1 minute by a thermal imager. The cooling curve of the solution at room temperature after laser irradiation was recorded, and similarly every 1 minute. As shown in fig. 9, which is a photo-thermal temperature-rising curve of the rSP aqueous solution after being left for 24 hours, it can be seen that the rSP aqueous solution after being left for 24 hours can still be rapidly heated under laser irradiation, and maintain good photo-thermal performance.
Comparative example 1
Preparing an aqueous soy pentapeptide Solution (SHCMN) comprising the steps of:
s1, dissolving soybean pentapeptide in water to prepare a polypeptide water solution with the concentration of the soybean pentapeptide of 2mmol/L, wherein the amino acid sequence of the soybean pentapeptide is shown as SEQ ID: 1.
Comparative example 2
Preparing an aqueous Polyoxometalate (POM) solution comprising the steps of:
s1, taking 36.5mg of polyoxometalate H3PMo12O40Dissolving in 10mL water, stirring for 10min to obtain the solution with concentration of2mmol/L of polyoxometalate aqueous solution.
As shown in FIG. 4, POM (i.e., H) was added to the aqueous solution of polyoxometalate clusters prepared in this comparative example3PMo12O40) The zeta potential value in aqueous solution is-32.5 mV; the zeta potential value of the aqueous rSP solution prepared in connection with example 1 was +22.4 mV; the polypeptide with positive charge on the surface is covered on the surface of the polyoxometalate to ensure that the surface of the prepared composite material rSP is positively charged, and the supermolecule co-assembly enhances the single POM (namely H)3PMo12O40) The stability of the material makes rSP suitable for in vivo applications.
Comparative example 3
Preparing a reduced polyoxometalate aqueous solution comprising the steps of:
s1, irradiating the polyoxometalate POM aqueous solution in the comparative example 2 for 5-10 minutes under ultraviolet light to obtain reduced polyoxometalate POM aqueous solution rPOM.
Comparative example 4
Preparing soybean pentapeptide-polyoxometalate assembly aqueous solution, comprising the steps of:
s1, mixing the soybean pentapeptide aqueous solution in the comparative example 1 and the polyoxometalate aqueous solution in the comparative example 2 in a molar ratio of 1:1, and uniformly mixing to obtain a soybean pentapeptide-polyoxometalate assembly solution SP.
The infrared spectrum structural characteristics of the soybean pentapeptide solution, the POM solution and the rSP solution prepared in the comparative example 1, the comparative example 2 and the example 1 are shown in figure 2. Can be seen in 3300, 2933, 2823, 1630, 1425, 1350, 1325, 1297cm-1Characteristic vibrational peaks ascribed to methyl/methylene; at 1672cm-1Characteristic peaks ascribed to amine groups; at 1203cm-1、1128cm-1Characteristic peaks ascribed to imine and carbon-sulfur bonds; at 1064cm-1(P-Od)，967cm-1(Mo＝Od)，867cm-1(Mo-Ob-Mo), and 791cm-1(Mo-Oc-Mo) a characteristic peak attributed to the inorganic cluster, indicating that the soybean pentapeptide and POM framework structures remain good in the hybrid composite.
FIG. 8 is the UV absorption spectra of the POM solution prepared in comparative example 2, the rPOM solution prepared in comparative example 3, the rSP solution prepared in example 1, the rPOM solution prepared in comparative example 3, which was allowed to stand in the air for 24 hours, and the rSP aqueous solution prepared in example 1, which was allowed to stand in the air for 24 hours, showing that the rPOM solution and the rSP solution have strong absorption at the 600-900 nm absorption band, the near infrared absorption of the rPOM solution disappeared after the rPOM solution was allowed to stand in the air for 24 hours, and the rSP solution remained unchanged after the rSP solution was allowed to stand in the air for 24 hours.
1mL of the aqueous solution of Polyoxometalate (POM) prepared in comparative example 2 was placed in a sample cell (cross-sectional area 1 cm)2) In the middle, the power consumption is 1W/cm2The solution was irradiated with 808nm laser for 10 minutes and the temperature of the solution was recorded every 1 minute by a thermal imager. The cooling curve of the solution at room temperature after laser irradiation was recorded, and similarly every 1 minute. As shown in fig. 9, the photo-thermal temperature rise curve of the POM aqueous solution shows that the temperature change of the POM aqueous solution is not significant under laser irradiation.
1mL of the rPOM aqueous solution prepared in comparative example 3 was taken and placed in a sample cell (cross-sectional area 1 cm)2) In the middle, the power consumption is 1W/cm2The solution was irradiated with 808nm laser for 10 minutes and the temperature of the solution was recorded every 1 minute by a thermal imager. The cooling curve of the solution at room temperature after laser irradiation was recorded, and similarly every 1 minute. Fig. 9 shows the photothermal temperature increase curve of the rPOM aqueous solution, and it can be seen that the rPOM aqueous solution rapidly increases in temperature under laser irradiation.
1mL of the rPOM aqueous solution prepared in comparative example 3 was left to stand for 24 hours, and then placed in a sample cell (cross-sectional area 1 cm)2) In the middle, the power consumption is 1W/cm2The solution was irradiated with 808nm laser for 10 minutes and the temperature of the solution was recorded every 1 minute by a thermal imager. The cooling curve of the solution at room temperature after laser irradiation was recorded, and similarly every 1 minute. As shown in fig. 9, the photothermal temperature rise curve of the rPOM aqueous solution after 24 hours of standing was shown, and it was found that the temperature rise of the rPOM aqueous solution under laser irradiation did not change significantly after 24 hours of standing. It is seen that the photothermal properties significantly decline after 24 hours of storage.
FIG. 10 shows the POM solution prepared in comparative example 2, the rPOM solution prepared in comparative example 3, and the rPOM solution prepared in example 1rSP solution, rPOM solution prepared in comparative example 3, and aqueous solution prepared in example 1, after 24 hours of air standing, were placed at 808nm, 1W/cm2Thermal imaging pictures under laser irradiation, near infrared laser irradiation (1.0W/cm at 808 nm)2) The temperature of the rPOM solution and the rSP solution rises rapidly to 57.1 ℃ within 10 minutes, while the temperature of the pure POM solution rises by less than 5 ℃ under the same condition, which proves that the photo-thermal conversion efficiency of the reduced polymetallic oxygen cluster rPOM and rSP materials is high. The solution of rPOM after being placed in the air for 24 hours has insignificant temperature change under the same laser irradiation conditions, which indicates that the rPOM has poor photo-thermal stability, while the solution of rSP after being exposed in the air for 24 hours still has significant temperature rise under the same laser irradiation conditions, which confirms that the assembly rSP of the peptide and the POM has excellent photo-thermal stability.
Example 2
Polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP cytotoxicity test
HepG2 liver cancer tumor cells are selected for cytotoxicity test. HepG2 tumor cells were seeded in 96-well plates with 10 cells per well4Selecting DMEM as culture medium, injecting 100uLDMEM solution into each hole, and placing in 5% CO2And cultured in a cell culture box at 37 ℃ for 24 hours. Samples (1250nM,2500nM,5000nM,10000nM) of POM solution, rPOM solution and rSP solution (100 uL) at different concentrations were added to the cell wells, incubated with HepG2 cells, and further incubated for 24 hours. Adding 20uL of MTT solution, culturing for 4 hours under the same condition, sucking out the culture solution in each hole, adding 150uL of DMSO into each hole, measuring the absorption value of the absorption peak at 570nm by using an enzyme-labeling instrument, and calculating the cell survival rate according to the OD value of each experimental sample group/the OD value of a control group multiplied by 100 percent; wherein the rSP solution is the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP prepared in the embodiment of the invention; the POM solution is the metal oxygen cluster aqueous solution POM prepared in comparative example 2 of the invention; the rPOM solution is the polymetallic oxygen cluster aqueous solution rPOM prepared in comparative example 3 of the invention. The results are shown in FIG. 7, where the control group is the cell survival rate in the blank experiment, and the results show that even when the concentration is increasedTo 104The viability of the cells was still maintained around 90% at nM, indicating that the POM, rPOM and rSP of the present invention are highly biocompatible at the cellular level and may be suitable for further biological applications.
Example 3
The polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP is used for in vitro treatment of HepG2 liver cancer tumor cells, and HepG2 liver cancer tumor cells are used for photothermal treatment. HepG2 tumor cells were seeded in 96-well plates with 10 cells per well4Selecting DMEM as culture medium, and placing in 5% CO2And cultured in a cell culture box at 37 ℃ for 24 hours. And taking rPOM solution, rSP solution, rPOM solution empty placed in the air for 24 hours and rSP solution placed in the air for 24 hours, respectively adding the rPOM solution, the rSP solution empty and the rSP solution into the cell hole, and co-culturing with HepG2 cells. After 1 hour, the cells were irradiated with a 808nm laser for 10 minutes, further cultured for 24 hours, added with MTT solution, further cultured for 4 hours under the same conditions, added with DMSO, and measured for an absorption peak at 570nm using a microplate reader, and the cell survival rate was calculated from OD value ═ lg (1/trans). Here, rSP and rPOM were allowed to stand in the air for 24 hours to sufficiently oxidize them. The results are shown in FIGS. 11 and 12; in fig. 11, HepG2 liver cancer tumor cells in the rSP solution and rSP solution treatment group placed in the air for 24 hours were significantly apoptotic, indicating that the assembly of soybean pentapeptide and POM polyoxometalate-food-borne antioxidant peptide photothermal material rSP has excellent tumor cell photothermal treatment effect; in FIG. 12, the liver cancer cells of HepG2 in the rPOM solution-treated group were significantly apoptotic, while the liver cancer cells of HepG2 were treated after 24 hours of air-exposure in the rPOM solution without significant cell death.
Example 4
The rSP solution prepared in example 1, the aqueous polypeptide solution prepared in comparative example 1, the rPOM solution prepared in comparative example 3 and a blank pure water solution were taken for co-culture with E.coli (Escherichia coli), in which 10 co-cultures were co-cultured on each plate medium8CFU Escherichia Coli (CFU) with sample concentration of 100uM, wherein the culture medium is beef extract peptone culture medium, culturing at 37 deg.C for 24 hr, testing optical density variation curve of Escherichia coli with culture time, and dissolving three solutionsThe liquid is cultured on a solid LB culture medium, and after the liquid is cultured for 48 hours in an incubator at 37 ℃, the colony phenomenon is observed and the picture is taken for recording. The results are shown in FIGS. 13 to 17; the polypeptide aqueous solution prepared in the comparative example 1, the rPOM solution prepared in the comparative example 3 and the blank pure water solution all showed obvious growth of Escherichia coli colonies, while the rSP solution prepared in the example 1 showed a very small number of Escherichia coli colonies in the experimental group, which indicates that the rSP solution prepared in the example 1 has a very good inhibitory effect on the growth of Escherichia coli.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Sequence listing
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<120> preparation method and application of polyoxometalate-food-borne antioxidant peptide photothermal material
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1 5
Claims (9)
1. A preparation method of a polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material is characterized by comprising the following steps:
s1, dissolving soybean pentapeptide in water to prepare a polypeptide water solution with the concentration of the soybean pentapeptide of 1-6 mmol/L, wherein the amino acid sequence of the soybean pentapeptide is shown as SEQ ID: 1;
s2, dissolving the polyoxometalate in water to prepare a polyoxometalate aqueous solution with the concentration of 1-3 mmol/L;
s3, mixing the polypeptide aqueous solution obtained in the step S1 with the polyoxometalate aqueous solution obtained in the step S2 to obtain an assembly solution SP; wherein the molar ratio of the soybean pentapeptide in the polypeptide aqueous solution to the polyoxometalate in the polyoxometalate aqueous solution is 3: 1-1: 1;
s4, irradiating the assembly solution SP obtained in the step S3 in ultraviolet for 5-10 min to obtain a polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP;
in step S2, the polyoxometalate is H3PMo12O40。
2. The method for preparing the polyoxometalate-food-borne antioxidant peptide photothermal material of claim 1, wherein the soybean pentapeptide of step S1 is synthesized by standard Fmoc solid phase synthesis strategy, and the purity of the soybean pentapeptide is determined by RP-C18 chromatographic column and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
3. The method for preparing the polyoxometalate-food-borne antioxidant peptide photothermal material according to claim 1, comprising the steps of:
s1, dissolving 11.8mg of soybean pentapeptide in 10mL of water, and stirring for 10min to obtain a polypeptide aqueous solution with the soybean pentapeptide concentration of 2 mmol/L; the amino acid sequence of the soybean pentapeptide is shown as SEQ ID: 1;
s2, weighing 36.5mg of polyoxometalate H3PMo12O40Dissolving in 10mL of water, and stirring for 10min to obtain a polyoxometalate aqueous solution with the concentration of 2 mmol/L;
s3, dropwise adding the polyoxometalate aqueous solution obtained in the step S2 into the polypeptide aqueous solution obtained in the step S1 at normal temperature, and stirring for 3 hours to obtain a co-assembled SP aqueous solution; the normal temperature is 24-26 ℃; the molar ratio of the soybean pentapeptide in the polypeptide aqueous solution to the polyoxometalate in the polyoxometalate aqueous solution is 1: 1;
s4, placing the SP aqueous solution obtained in the step S3 in ultraviolet light for 10min to obtain the polymetallic oxygen cluster-food-borne antioxidant peptide photothermal material rSP.
4. The application of the polyoxometalate-food-borne antioxidative peptide photothermal material prepared by the method of claim 1, wherein the polyoxometalate-food-borne antioxidative peptide photothermal material is used as an active ingredient for preparing an anti-escherichia coli drug.
5. The use of the polyoxometalate-food derived antioxidant peptide photothermal material prepared by the method of claim 1, wherein the polyoxometalate-food derived antioxidant peptide photothermal material is used as an active ingredient for the preparation of a drug for photothermal therapy.
6. The use of the polyoxometalate-food derived antioxidant peptide photothermal material prepared by the method of claim 1, wherein the polyoxometalate-food derived antioxidant peptide photothermal material is used for preparing a photothermal conversion material.
7. An anti-escherichia coli drug, which is characterized in that the polyoxometalate-food-borne antioxidant peptide photothermal material prepared by the preparation method of the polyoxometalate-food-borne antioxidant peptide photothermal material according to claim 1 is added during drug preparation.
8. A drug for photothermal therapy, characterized in that the polyoxometalate-food-borne antioxidative peptide photothermal material prepared by the method for preparing a polyoxometalate-food-borne antioxidative peptide photothermal material according to claim 1 is added during the preparation of a drug.
9. A photothermal conversion material, wherein the polyoxometalate-food-borne antioxidative peptide photothermal material prepared by the method of claim 1 is added during the preparation process.
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