CN110960552B - A kind of single-atom nano-enzyme patch for skin wound and preparation method - Google Patents

A kind of single-atom nano-enzyme patch for skin wound and preparation method Download PDF

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CN110960552B
CN110960552B CN201911385416.9A CN201911385416A CN110960552B CN 110960552 B CN110960552 B CN 110960552B CN 201911385416 A CN201911385416 A CN 201911385416A CN 110960552 B CN110960552 B CN 110960552B
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张晓东
闫瑞娟
孙思
穆晓宇
刘爽杰
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Abstract

本发明公开了一种用于皮肤创伤的单原子纳米酶贴剂及制备方法，制备：配制Ce(NO3)3·6H2O水为溶液一；配制6‑氨基己酸和浓盐酸水为溶液二；将浓氨水加入溶液一，搅拌，加入溶液二，反应，得到CeO2团簇并配水悬浮液，将聚乙烯吡咯烷酮和K2PtCl4加入到水中，得到溶液三；将CeO2团簇水悬浮液加到溶液三中，反应，再滴入NaBH4水溶液；反应；得到单原子纳米酶，滴在活化亲水碳布上；干燥，得到含有单原子纳米酶的活化亲水碳布；在医用胶带上表面依次设置含有单原子纳米酶的活化亲水碳布和隔离纸，得到用于皮肤创伤的单原子纳米酶贴剂。本发明贴剂能显著促进伤口愈合与疤痕修复以及后期毛发再生，且无毒副作用。The invention discloses a single-atom nano-enzyme patch for skin wounds and a preparation method. The preparations include: preparing Ce(NO 3 ) 3 6H 2 O water as solution one; preparing 6-aminocaproic acid and concentrated hydrochloric acid water as Solution 2; add concentrated ammonia into solution 1, stir, add solution 2, react to obtain CeO 2 clusters and mix with water suspension, add polyvinylpyrrolidone and K 2 PtCl 4 to water to obtain solution 3; add CeO 2 clusters The aqueous suspension is added to the solution three, reacted, and then dripped into the NaBH 4 aqueous solution; react; obtain the single-atom nanozyme, drop it on the activated hydrophilic carbon cloth; dry to obtain the activated hydrophilic carbon cloth containing the single-atom nanozyme; Activated hydrophilic carbon cloth containing single-atom nano-enzyme and release paper are sequentially arranged on the upper surface of the medical tape to obtain a single-atom nano-enzyme patch for skin wounds. The patch of the invention can significantly promote wound healing, scar repair and hair regeneration in the later period, and has no toxic and side effects.

Description

Monoatomic nano enzyme patch for skin wounds and preparation method thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to an external patch.
Background
Skin trauma is a worldwide health problem, and presents an increasing challenge to clinical treatment[1,2]. Effectively restoring tissue integrity and function while minimizing the aesthetic impact after injury remains a central concern for clinical care. Regenerative healing of damaged tissue remains elusive, probably because most current wound healing therapies focus on structural restoration, and little is explored for microenvironment regulation of the wound site by the host's inherent regenerative capacity. Skin injury initiates a series of events in which free Radicals (RONS) are abundantly generated in the wound site microenvironment[3,4]Is part of its defense mechanism against pathogen invasion. Although this process is advantageous, if the overproduction of RONS is not controlled, wound repair and regeneration may be hindered, resulting in damage to biological macromolecules such as proteins, DNA, lipids and carbohydrates[5]. In addition, increased ron production at the site of injury may trigger a range of deleterious effects, such as cellular aging, fibrotic scarring and inflammation[6-8]. Since RONS have the role of double-edged sword, successful healing and regeneration of damaged tissue depends to a large extent on the optimal balance between the building and destructive abilities of RONS. Therefore, preparing a biomaterial surface with the ability to manage ron levels, control oxidative damage and relieve inflammatory responses in the microenvironment of the injury site is a potential therapeutic strategy to promote wound healing regeneration.
At present, single-atom catalysis has become a hot point of research due to the nearly 100% atomic dispersity and the maximum metal utilization rate, and an important potential way is provided for remarkably improving the catalytic activity and selectivity[9]. In all monoatomic systems, the typical structure is a noble metal supported metal oxide nanomaterial. Ultra-small CeO2Cluster passing through Ce3+And Ce4+The rapid switching of ions has higher catalytic activity and shows remarkable advantages in the aspect of treating diseases related to oxidative stress[10]. And CeO2The (111) crystal face of the crystal has the lowest surface energy and the best stability[11]Especially of monoatomic Pt/CeO2(111) The catalyst also achieves low temperature by activation of surface lattice oxygen<100 ℃ C.) of catalytic reaction[12]And shows potential performance for application in biological systems. Through the research of domestic and foreign documents and patents, the relevant single atom Pt/CeO is not found2Research report of treating skin injury by catalyst, especially Pt/CeO2The research applied to the components of the biomedical devices is blank, and the research reports on the in-vivo curative effect and the toxic and side effect of the biomedical devices are lacked.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a monatomic nano-enzyme patch for skin wounds.
The second purpose of the invention is to provide a preparation method of the monatomic nanoenzyme patch for skin wounds.
The technical scheme of the invention is summarized as follows:
the preparation method of the monatomic nanoenzyme patch for skin wounds comprises the following steps:
(1) in proportion, 100-120mg of Ce (NO)3)3·6H2Dissolving O in 10mL of deionized water to obtain a solution I; dissolving 120-140mg of 6-aminocaproic acid and 5 mu L of concentrated hydrochloric acid in 1mL of deionized water under stirring to obtain a solution II; at room temperature, adding 2ml of concentrated ammonia water into the first solution, stirring uniformly, heating to 90-95 ℃, adding the second solution, reacting for 3-4 hours, cooling to room temperature, washing with water, and centrifuging to obtain CeO2Clustering;
(2) preparing 80-100mg/mL CeO according to the proportion2Aqueous suspension of clusters, 10-20mg of polyvinylpyrrolidone with MW 58000 and 10-40mg of K2PtCl4Adding the mixture into 5-10mL of water to obtain a solution III; 1mL of the CeO2Adding cluster water suspension into the third solution, reacting at 90-95 deg.C for 15-20 min, and adding 0.8mL of 50-100mM NaBH4An aqueous solution; reacting for 20-30 minutes at 90-95 ℃; cooling to room temperature, washing with water, and centrifuging to obtain monatomic nanoenzyme;
(3) dripping the water solution of the monatomic nanoenzyme on the activated hydrophilic carbon cloth, or coating the activated hydrophilic carbon cloth, or soaking the activated hydrophilic carbon cloth in the water solution of the monatomic nanoenzyme and taking out; drying to obtain activated hydrophilic carbon cloth containing the monatomic nanoenzyme;
(4) and sequentially arranging the activated hydrophilic carbon cloth containing the monatomic nanoenzyme and the release paper on the upper surface of the medical adhesive tape to obtain the monatomic nanoenzyme patch for the skin wound.
The monatomic nanoenzyme patch for skin wounds, prepared by the method above.
The invention has the advantages that:
the monatomic nano enzyme patch for skin wounds can obviously promote wound healing, scar repair and later-stage hair regeneration, and has no toxic or side effect.
Drawings
FIG. 1 is a schematic diagram of a monatomic nanoenzyme patch for skin wounds, FIG. 1a being a schematic diagram and FIG. 1b being a schematic front view, wherein 1 is a medical tape; 2. activated hydrophilic carbon cloth containing monatomic nanoenzyme; 3. and (4) release paper.
Fig. 2 is a representative picture of the treatment effect of the monatomic nanoenzyme patch for skin wounds-time dependent tissue healing for each group of mice.
Fig. 3 is a hematology analysis of the monatin nanoenzyme patch for skin trauma treatment of brain trauma mice for 12 days, 26 days. The results show the mean and standard deviation of white blood cells, red blood cells, hemoglobin, hematocrit, mean red blood cell volume, mean red blood cell hemoglobin amount, red blood cell mean hemoglobin concentration, platelets.
FIG. 4 shows the biochemical analysis of blood of mice with skin trauma treated with monatomic nanoenzyme patch for 12 days and 26 days. The results show the mean and standard deviation of the ratios of glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total protein, blood urea nitrogen, serum albumin, creatinine, globulin, albumin and globulin.
Detailed Description
Hydrophilic carbon cloth (commercial product, Shanghai river Sen electric Co., Ltd.)
Activating the hydrophilic carbon cloth:
cutting the hydrophilic carbon cloth into a rectangle with the length of 15mm and the width of 8mm, putting the rectangle into a mixed solution (the volume ratio is 1: 3) of concentrated nitric acid and concentrated sulfuric acid, and refluxing for 3h at 80 ℃ to obtain the activated hydrophilic carbon cloth.
The present invention will be further described with reference to the following examples.
Example 1
The preparation method of the monatomic nanoenzyme patch for skin wounds comprises the following steps:
(1) 110mg of Ce (NO)3)3·6H2Dissolving O in 10mL of deionized water to obtain a solution I; dissolving 130mg of 6-aminocaproic acid and 5 μ L of concentrated hydrochloric acid in 1mL of deionized water under stirring to obtain a solution two; at room temperature, adding 2ml of concentrated ammonia water into the first solution, stirring uniformly, heating to 93 ℃, adding the second solution, reacting for 3.5 hours, cooling to room temperature, washing with water, and centrifuging to obtain CeO2Clustering;
(2) formulation 90mg/mL of CeO2Aqueous suspension of clusters, 15mg of polyvinylpyrrolidone MW 58000 and 25mgK2PtCl4Adding the mixture into 8mL of water to obtain a solution III; 1mL of the CeO2Adding the cluster water suspension into the third solution, reacting at 93 ℃ for 18 minutes, and then adding 0.8mL of newly prepared 80mM NaBH4An aqueous solution; reaction at 93 deg.C for 25 min; cooling to room temperature, washing with water, and centrifuging to obtain monatomic nanoenzyme;
(3) dripping 0.5mL of water solution of the monatomic nanoenzyme of 50mg/mL on the activated hydrophilic carbon cloth, and drying to obtain the activated hydrophilic carbon cloth containing the monatomic nanoenzyme;
(4) sequentially arranging the activated hydrophilic carbon cloth 2 containing the monatomic nanoenzyme and release paper 3 (glassine release paper) (see fig. 1a and fig. 1b) on the upper surface of the medical adhesive tape 1 to obtain the monatomic nanoenzyme patch (Pt/CeO for short) for skin wounds2Groups).
Example 2
The preparation method of the monatomic nanoenzyme patch for skin wounds comprises the following steps:
(1) 100mg of Ce (NO)3)3·6H2Dissolving O in 10mL of deionized water to obtain a solution I; dissolving 6-aminocaproic acid 120mg and concentrated hydrochloric acid 5 μ L in deionized water 1mL under stirring to obtain solution two; at room temperature, adding 2ml of concentrated ammonia water into the first solution, stirring uniformly, heating to 90 ℃, adding the second solution, reacting for 4 hours, cooling to room temperature, washing with water, and centrifuging to obtain CeO2Clustering;
(2) 80mg/mL CeO is prepared2Aqueous suspension of clusters, 10mg of polyvinylpyrrolidone MW 58000 and 10mgK2PtCl4Adding the mixture into 5mL of water to obtain a solution III; 1mL of the CeO2Adding the cluster water suspension into the third solution, reacting at 90 deg.C for 20 min, and adding 0.8mL of 50mM NaBH4An aqueous solution; reacting at 90 ℃ for 30 minutes; cooling to room temperature, washing with water, and centrifuging to obtain monatomic nanoenzyme;
(3) coating 0.5mL of water solution of 50mg/mL of monatomic nanoenzyme on activated hydrophilic carbon cloth, and drying to obtain the activated hydrophilic carbon cloth containing the monatomic nanoenzyme;
(4) and sequentially arranging the activated hydrophilic carbon cloth containing the monatomic nano-enzyme and release paper (glassine release paper) on the upper surface of the medical adhesive tape to obtain the monatomic nano-enzyme patch for the skin wound.
Example 3
The preparation method of the monatomic nanoenzyme patch for skin wounds comprises the following steps:
(1) 120mg of Ce (NO)3)3·6H2Dissolving O in 10mL of deionized water to obtain a solution I; dissolving 140mg of 6-aminocaproic acid and 5 μ L of concentrated hydrochloric acid in 1mL of deionized water under stirring to obtain a solution two; at room temperature, adding 2ml of concentrated ammonia water into the first solution, stirring uniformly, heating to 95 ℃, adding the second solution, reacting for 3 hours, cooling to room temperature, washing with water, and centrifuging to obtain CeO2Clustering;
(2) 100mg/mL CeO is prepared2Aqueous suspension of clusters, 20mg of polyvinylpyrrolidone MW 58000 and 40mgK2PtCl4Adding the mixture into 10mL of water to obtain a solution III; 1mL of the CeO2Adding the cluster water suspension into the third solution, reacting at 95 ℃ for 15 minutes, and then adding 0.8mL of newly prepared 100mM NaBH4An aqueous solution; reacting at 95 ℃ for 20 minutes; cooling to room temperature, washing with water, and centrifuging to obtain monatomic nanoenzyme;
(3) soaking the activated hydrophilic carbon cloth in 0.5mL of water solution of 50mg/mL of monatomic nanoenzyme, and taking out; drying to obtain activated hydrophilic carbon cloth containing the monatomic nanoenzyme;
(4) and sequentially arranging the activated hydrophilic carbon cloth containing the monatomic nano-enzyme and release paper (glassine release paper) on the upper surface of the medical adhesive tape to obtain the monatomic nano-enzyme patch for the skin wound.
Example 4
Experiment of monatomic nanoenzyme patch for treating skin injury
Male mice (C57BL/6,19-21g) were randomly divided into 4 groups (14 per group):
in the group of the lesions, the number of the lesions,
carbon cloth control group (activated hydrophilic carbon cloth);
CeO2control (CeO prepared in step (1) of example 1)2The clusters were then formed into patches prepared in steps (3) and (4) of example 1.
Pt/CeO2Treatment group (prepared in example 1).
All mice were anesthetized with chloral hydrate (10%, 4 mL/kg). After shaving the head of the mouse, a sagittal incision of about 12mm was made in the scalp. Each experimental material was attached to the wound of a mouse and replaced every two days. A picture of the mouse wound was taken during isoflurane gas anesthesia as shown in figure 2.
Pt/CeO2The wounds of the treated mice were completely repaired on day 8, while small wounds remained on the scalp of the mice of the injured group and the carbon cloth control group. Furthermore, with CeO2Comparison of control mice with Pt/CeO2The treatment group significantly promoted scar healing and epidermal protective hair regeneration. Animal experiments show that the monatomic nano-enzyme patch (Pt/CeO) for skin trauma2Treatment group) can significantly improve the wound healing efficiency and enhance the skin tissue regeneration effect.
Experiments prove that the monatomic nano-enzyme patch for skin wounds, prepared in examples 2 and 3, can remarkably promote scar healing and regeneration of epidermal protective hair, can remarkably improve wound healing efficiency, and can enhance skin tissue regeneration effect.
Example 5
Research on toxic and side effects of monatomic nanoenzyme patch for skin wounds
Male mice (C57BL/6,19-21g) were randomly divided into 4 groups (10 per group):
in the normal control group, the control group was,
injury group
CeO2Control (CeO prepared in step (1) of example 1)2The clusters were then formed into patches prepared in steps (3) and (4) of example 1.
Pt/CeO2Treatment group (prepared in example 1).
Blood samples were collected by eye bleeds on days 12 and 26 after wound treatment, requiring one aliquot to be stored separately and one aliquot to be pretreated with anticoagulant. After obtaining the sample, the blood sample pretreated by the anticoagulant is used for hematology examination by a blood cell counter, and the blood sample without the anticoagulant is centrifuged to prepare serum for biochemical index detection and analysis.
As one of the possible approaches to treating skin lesions, we further examined the toxic reactions of the monatomic nanoenzyme patch for skin wounds, including blood routine examination and blood biochemical index detection. Typical hematological indices and blood biochemical data (fig. 3 and 4) show that each index in the treated mice was similar to that of normal mice and was within the healthy range. Thus, in the experimental studies of the present invention, the single atom Pt/CeO2The nano enzyme is prepared into a patch which is safe and effective for external application in skin injury treatment and basically has no side effect.
Reference documents:
[1]C.K.Sen,G.M.Gordillo,S.Roy,R.Kirsner,L.Lambert,T.K.Hunt,et al.,Human skin wounds:a major and snowballing threat to public health and the economy,Wound Repair Regen.17(2009)763-771.
[2]R.J.Hay,N.E.Johns,H.C.Williams,I.W.Bolliger,R.P.Dellavalle,D.J.Margolis et al.,The global burden of skin disease in 2010:an analysis of the prevalence and impact of skin conditions,J.Invest.Dermatol 134(2014)1527-1534.
[3]P.Niethammer,C.Grabher,A.T.Look,T.J.Mitchison,A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish,Nature 459(2009)996-999.
[4]M.Schaefer,S.Werner,Oxidative stress in normal and impaired wound repair,Pharmacol.Res.58(2008)165-171.
[5]C.K.Tsang,Y.Liu,J.Thomas,Y.Zhang,X.F.S.Zheng,Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance,Nat.Commun.5(2014)3446.
[6]T.Finkel,N.J.Holbrook,Oxidants,oxidative stress and the biology of ageing,Nature 408(2000)239-247.
[7]A.Pellicoro,P.Ramachandran,J.P.Iredale,J.A.Fallowfield,Liver fibrosis and repair:immune regulation of wound healing in a solid organ,Nat.Rev.Immunol.14(2014)181-194.
[8]M.Mittal,M.R.Siddiqui,K.Tran,S.P.Reddy,A.B.Malik,Reactive oxygen species in inflammation and tissue injury,Antioxid.Redox Sign.20(2014)1126-1167.
[9]Peterson E J,DeLaRiva A T,Lin S,et al.Low-temperature carbon monoxide oxidation catalysed by regenerable atomically dispersed palladium on alumina.Nature communications,2014,5:4885.
[10]Li Y,He X,Yin J J,et al.Acquired superoxide-scavenging ability of ceria nanoparticles.Angewandte Chemie International Edition,2015,54(6):1832-1835；
[11]Xie Q,Zhang H,Kang J,et al.Oxidative dehydrogenation of propane to propylene in the presence of HCl catalyzed by CeO2 and NiO-modified CeO2nanocrystals.ACS Catalysis,2018,8(6):4902-4916.
[12]Nie L,Mei D,Xiong H,et al.Activation of surface lattice oxygen in single-atom Pt/CeO2 for low-temperature CO oxidation.Science,2017,358(6369):1419-1423.

Claims (2)

1.用于皮肤创伤的单原子纳米酶贴剂的制备方法，其特征是包括如下步骤：1. the preparation method of the single-atom nanozyme patch for skin wound is characterized in that comprising the steps: (1)按比例，将100-120mg Ce(NO3)3·6H2O溶于10mL去离子水，得到溶液一；在搅拌下将120-140mg6-氨基己酸和5μL浓盐酸溶解在1mL去离子水中，得到溶液二；在室温下，将2ml浓氨水加入到溶液一中，搅拌均匀，加热至90-95℃，加入溶液二，反应3-4小时，冷却至室温，水洗，离心，得到CeO2团簇；(1) Dissolve 100-120 mg Ce(NO 3 ) 3 ·6H 2 O in 10 mL of deionized water in proportion to obtain solution 1; dissolve 120-140 mg of 6-aminocaproic acid and 5 μL of concentrated hydrochloric acid in 1 mL of deionized water under stirring ionized water to obtain solution two; at room temperature, add 2ml of concentrated ammonia water to solution one, stir evenly, heat to 90-95 ° C, add solution two, react for 3-4 hours, cool to room temperature, wash with water, and centrifuge to obtain CeO 2 clusters; (2)按比例，配制80-100mg/mL的CeO2团簇水悬浮液，将10-20mg的MW≈58000的聚乙烯吡咯烷酮和10-40mg K2PtCl4加入到5-10mL水中，得到溶液三；将1mL所述CeO2团簇水悬浮液加入到溶液三中，90-95℃反应15-20分钟，再滴入0.8mL新配制的50-100mM的NaBH4水溶液；在90-95℃反应20-30分钟；冷却至室温，水洗，离心，得到单原子纳米酶；(2) Prepare 80-100 mg/mL CeO 2 cluster water suspension in proportion, add 10-20 mg of MW≈58000 polyvinylpyrrolidone and 10-40 mg K 2 PtCl 4 to 5-10 mL of water to obtain a solution Three; add 1 mL of the CeO 2 cluster aqueous suspension to solution three, react at 90-95°C for 15-20 minutes, and then dropwise add 0.8 mL of a freshly prepared 50-100mM NaBH 4 aqueous solution; at 90-95° C. React for 20-30 minutes; cool to room temperature, wash with water, and centrifuge to obtain single-atom nanozyme; (3)将单原子纳米酶的水溶液滴在活化亲水碳布上，或涂覆在活化亲水碳布上，或将活化亲水碳布浸于单原子纳米酶的水溶液中并取出；干燥，得到含有单原子纳米酶的活化亲水碳布；(3) drop the aqueous solution of the single-atom nanozyme on the activated hydrophilic carbon cloth, or coat it on the activated hydrophilic carbon cloth, or immerse the activated hydrophilic carbon cloth in the aqueous solution of the single-atom nanozyme and take it out; dry , to obtain activated hydrophilic carbon cloth containing single-atom nanozyme; (4)在医用胶带上表面依次设置所述含有单原子纳米酶的活化亲水碳布和隔离纸，得到用于皮肤创伤的单原子纳米酶贴剂。(4) Disposing the activated hydrophilic carbon cloth containing single-atom nanozyme and release paper in sequence on the upper surface of the medical tape to obtain a single-atom nanozyme patch for skin wounds. 2.权利要求1的方法制备的用于皮肤创伤的单原子纳米酶贴剂。2. The single-atom nanozyme patch for skin wound prepared by the method of claim 1.
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CN108993134A (en) * 2018-07-16 2018-12-14 天津大学 The method of the monatomic catalyst degradation esters VOCs of palladium of low-temperature microwave enhanced oxidation cerium load

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CN106975100A (en) * 2016-01-15 2017-07-25 浙江大学 A kind of nano composite material of cerium oxide/mesoporous silicon and its preparation method and application
CN108855069A (en) * 2018-06-19 2018-11-23 华侨大学 A kind of nano bar-shape Pt/CeO2The preparation method of loaded catalyst and the application in CO catalysis reaction
CN108993134A (en) * 2018-07-16 2018-12-14 天津大学 The method of the monatomic catalyst degradation esters VOCs of palladium of low-temperature microwave enhanced oxidation cerium load

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