WO2020259018A1 - 一种自组装过氧化氢酶纳米颗粒及其制备方法和应用 - Google Patents

一种自组装过氧化氢酶纳米颗粒及其制备方法和应用 Download PDF

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WO2020259018A1
WO2020259018A1 PCT/CN2020/085054 CN2020085054W WO2020259018A1 WO 2020259018 A1 WO2020259018 A1 WO 2020259018A1 CN 2020085054 W CN2020085054 W CN 2020085054W WO 2020259018 A1 WO2020259018 A1 WO 2020259018A1
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catalase
assembled
self
nanoparticles
nanoparticle
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饶平凡
柯李晶
周建武
余兆硕
罗思浩
高观祯
汪惠勤
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浙江工商大学
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    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
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    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
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  • the invention belongs to the technical field of biology, food and nanomaterials, and particularly relates to a self-assembled catalase nanoparticle and a preparation method and application thereof.
  • Catalase is an enzyme that catalyzes the decomposition of hydrogen peroxide into oxygen and water. It exists in the peroxide body of the cell. It is an important redox balance component in the body and can eliminate excessive excess in the body. Free radicals such as hydrogen oxide form an important barrier to the body's immunity.
  • exogenous catalase has been widely used in the treatment of immune-related tumor diseases, and there is an urgent need to further develop biological materials based on catalase with higher physiological activity.
  • domestic and foreign researchers have developed many catalase mimics with scavenging free radicals, but the preparation route of these mimics is complicated, and the safety in vivo is not clear.
  • nanotechnology has attracted much attention in many fields because of its huge advantages brought by the nanoscale, such as greatly enhancing the original functional properties of active ingredients at the molecular scale, easier entry into cells, good targeting, and high efficiency.
  • many studies have focused on the research and development of catalase nanoparticles in order to obtain nano-biomaterials with more significant physiological properties, such as the use of shear force combined with organic reagents, calcium carbonate coating methods, etc., to improve catalase It is inherently stable, but these methods have many problems such as residual chemical reagents, high safety risks, complex and uncontrollable preparations.
  • the invention utilizes the principle that the hydrophobic interaction between proteins is increased under heat treatment conditions, and it is easier to form larger-scale supramolecular polymers.
  • Catalase derived from bovine liver is used as the raw material, and thermal processing incubation is adopted to control temperature and pH. Prepare active catalase nanoparticles in green.
  • the purpose of the present invention is to provide a self-assembled catalase nanoparticle and its preparation method and application.
  • the preparation method of the present invention is green and safe, and the operation is simple, and the obtained self-assembled catalase nano particles have a controllable particle size, and have higher activity and stability.
  • a method for preparing self-assembled catalase nanoparticles includes dissolving lyophilized catalase powder to obtain a catalase solution, adjusting the pH of the catalase solution, and then centrifuging or filtering to obtain a supernatant or The filtrate is obtained by further incubating the supernatant or filtrate with heat.
  • the concentration of the catalase solution is 0.2-5 mg/mL; preferably, the concentration of the catalase solution is 0.2-1.6 mg/mL.
  • the pH value of the catalase solution is adjusted to be 6.5-9.5; preferably, the pH value is 7.0-8.0.
  • the speed of the centrifugation is 2000-5000 rpm/min.
  • the filtration is membrane filtration, and the membrane filtration has a pore size of 0.22 ⁇ m-0.45 ⁇ m.
  • the thermal incubation is to heat the supernatant or the filtrate at a temperature of 45-90° C. for 15 s-15 min; the higher the heating temperature in the present invention, the shorter the heating time required. For example, it only needs to be heated for 15-60s at a temperature of 65-90°C.
  • the present invention also provides self-assembled catalase nanoparticles obtained by the above preparation method, and the self-assembled catalase nanoparticles have a particle size of 30-200 nm.
  • the enzyme activity of the self-assembled catalase nanoparticles is ⁇ 3100 U/mg.
  • the invention also provides an application of the self-assembled catalase nanoparticle in food or medicine.
  • the present invention uses centrifugation and filtration to remove the excessive polymer impurities in the catalase freeze-dried powder, and then under heat treatment conditions, the hydrophobic interaction between catalase proteins is increased, thereby easily forming larger scale super Molecular polymers, self-assemble to form self-assembled catalase nanoparticles with larger nanometer size.
  • the preparation of self-assembled catalase nanoparticles of the present invention is green and safe, does not require chemical cross-linking and structure, and is simple to operate.
  • the self-assembled catalase nanoparticles of the present invention can accurately prepare self-assembled catalase nanoparticles of different particle sizes by adjusting the pH of the system, thereby producing different physiological effects, and the resulting self-assembled nanoparticles
  • the particle size distribution of catalase is concentrated and uniform.
  • the self-assembled catalase nanoparticles of the present invention have greatly improved stability.
  • Figure 1 The effect of different pH on the formation of self-assembled catalase nanoparticles: a) average particle size diagram; b) Zeta-potential potential diagram.
  • Figure 2 Particle size distribution of self-assembled catalase nanoparticles: a), b), and c) are catalase nanoparticles formed under the conditions of pH 7.0, 7.2, and 8.0, respectively.
  • FIG. 3 SDS-PAGE diagram of self-assembled catalase nanoparticles: a) Non-reducing electrophoresis, from left to right are natural CAT and CAT dissolved in different pH conditions (pH 7.0, 7.2, 7.4, 7.6, 7.8, 8.0) Nanoparticles (pH 7.0, 7.2, 7.4, 7.6, 7.8, 8.0); b) Reduction electrophoresis (sequence as before).
  • FIG. 4 SEM observation image (a) and circular dichrograph (b) of self-assembled catalase nanoparticles.
  • Figure 5 Enzyme activity of self-assembled catalase nanoparticles prepared at different pH.
  • Figure 6 The effect of self-assembled catalase nanoparticles prepared at different pH on the survival rate of macrophages.
  • the size distribution of self-assembled catalase nanoparticles is shown in Figure 2; the SDS-PAGE electrophoresis image is shown in Figure 3; the SEM image and CD spectrum are shown in Figure 4.
  • the enzyme activity of self-assembled catalase nanoparticles is 7732.50 ⁇ 235.74U/mg, see Figure 5.
  • the size distribution of the self-assembled catalase nanoparticles is shown in Figure 2; the SDS-PAGE electrophoresis image is shown in Figure 3; the SEM image and the CD spectrum are shown in Figure 4.
  • the enzyme activity of the self-assembled catalase nanoparticles is 7382.50 ⁇ 221.77U/mg, see Figure 5.
  • the size distribution of self-assembled catalase nanoparticles is shown in Figure 2; and its SDS -PAGE electrophoresis diagram is shown in Figure 3; SEM electron microscope image, CD spectrum diagram is shown in Figure 4, and the enzyme activity of the nanoparticles is ⁇ 3100U/mg, shown in Figure 5.
  • the self-assembled catalase can be adjusted by controlling the pH value of the system.
  • the size of the nanoparticles can be used according to different needs.
  • the absolute value of the storage stability standard ⁇ -potential is significantly improved.
  • self-assembled catalase nanoparticles can withstand SDS hydrolysis and improve chemical stability Sex.
  • Example 5 Macrophages applied to the mucosa of the digestive tract

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Abstract

本发明公开了一种自组装过氧化氢酶纳米颗粒及其制备方法和应用。本发明的自组装过氧化氢酶纳米颗粒通过将过氧化氢酶冻干粉溶解获得过氧化氢酶溶液，调整过氧化氢酶溶液的pH，然后经离心或过滤获得上清液或过滤液，将上清液或过滤液进一步热孵育而得。本发明的自组装过氧化氢酶纳米颗粒能够应用促进免疫细胞生长，调节机体免疫的药品或食品中。

Description

一种自组装过氧化氢酶纳米颗粒及其制备方法和应用 技术领域
本发明属于生物、食品与纳米材料技术领域，特别是涉及一种自组装过氧化氢酶纳米颗粒及其制备方法和应用。
背景技术
过氧化氢酶(Catalase，CAT)是催化过氧化氢分解成氧和水的酶，存在于细胞的过氧化物体内，是机体内重要的氧化还原平衡的组成，可消除机体内如过多过氧化氢等自由基，形成对机体免疫的重要屏障。近年来，外源性过氧化氢酶已广泛应用于免疫相关的肿瘤疾病治疗，亟需进一步开发基于过氧化氢酶具有更高生理活性的生物材料。目前国内外研究者开发了众多具有清除自由基的过氧化氢酶模拟药物，但这些模拟物制备路线复杂，且在体内的安全性尚不明确。
当前，纳米科技在诸多领域备受关注，因其具有纳米尺度所带来的巨大优势，如可大大增强分子尺度下活性成分原本的功能性质，更易进入细胞、靶向性好、作用效率高等。基于此，众多研究聚焦在了过氧化氢酶纳米颗粒的研发上以期得到生理性能更显著的纳米生物材料，如利用剪切力结合有机试剂法，碳酸钙包裹法等，提高了过氧化氢酶本身的稳定性，然而这些方法均存在残留化学试剂，安全风险高，制备复杂不可控等诸多问题。
目前国内外尚无自组装而成的过氧化氢酶纳米颗粒的制备与研究的相关报道。本发明利用在热处理条件下，蛋白质之间疏水作用加大，更易形成更大尺度超分子聚合物的原理，以牛肝来源的过氧化氢酶为原料，采用热加工孵育方式，控制温度以及pH以绿色制备有活性的过氧化氢酶纳米颗粒。
发明内容
本发明的目的在于提供一种自组装过氧化氢酶纳米颗粒及其制备方法和应 用。本发明的制备方法绿色安全，操作简单，得到的自组装过氧化氢酶纳米颗粒粒径可控，且具有较高的活性和稳定性。
为了达到上述的目的，本发明采取以下技术方案：
一种自组装过氧化氢酶纳米颗粒的制备方法，包括将过氧化氢酶冻干粉溶解获得过氧化氢酶溶液，调整过氧化氢酶溶液的pH，然后经离心或过滤获得上清液或过滤液，将上清液或过滤液进一步热孵育而得。
进一步地，所述制备方法中，所述过氧化氢酶溶液的浓度为0.2-5mg/mL；优选的，所述过氧化氢酶溶液的浓度为0.2-1.6mg/mL。
进一步地，所述制备方法中，调整过氧化氢酶溶液的pH值为6.5-9.5；优选的，所述pH值为7.0-8.0。
进一步地，所述制备方法中，所述离心的速度为2000-5000rpm/min。
进一步地，所述制备方法中，所述过滤为膜过滤，膜过滤孔径为0.22μm-0.45μm。
进一步地，所述制备方法中，所述热孵育为将上清液或过滤液在45-90℃的温度下加热15s-15min；本发明加热温度越高，所需的加热时间越短。例如在65-90℃的温度下仅需加热15-60s。
本发明还提供上述制备方法获得的自组装过氧化氢酶纳米颗粒，所述自组装过氧化氢酶纳米颗粒的粒径为30-200nm。
进一步地，所述自组装过氧化氢酶纳米颗粒的酶活力≥3100U/mg。
本发明还提供一种上述自组装过氧化氢酶纳米颗粒在食品或药品中的应用。
进一步地，所述自组装过氧化氢酶纳米颗粒在具有免疫调节功能的食品或药品中的应用。
本发明的原理：
本发明利用离心和过滤的手段除去过氧化氢酶冻干粉中的过大的聚合物杂质，然后在热处理条件下，过氧化氢酶蛋白质之间疏水作用加大，从而易形成更大尺度超分子聚合物，自组装形成具有更大纳米尺寸的自组装过氧化氢酶纳 米颗粒。
本发明具有以下技术特点：
(1)本发明涉及的自组装过氧化氢酶纳米颗粒制备绿色安全，无需化学交联与构造，操作简单。
(2)本发明涉及的自组装过氧化氢酶纳米颗粒可通过调整体系的pH精确制备不同粒径大小的自组装过氧化氢酶纳米颗粒，由此产生不同的生理效应，且所得自组装纳米过氧化氢酶粒径分布集中，均一。
(3)本发明涉及的自组装过氧化氢酶纳米颗粒相比原来天然过氧化氢酶，稳定性大大提高。
附图说明
图1不同pH对自组装过氧化氢酶纳米颗粒形成的影响：a)平均粒径图；b)Zeta-potential电位图。
图2自组装过氧化氢酶纳米颗粒的粒径分布:a)，b)，c)分别为pH 7.0，7.2，8.0条件下形成的过氧化氢酶纳米颗粒。
图3自组装过氧化氢酶纳米颗粒SDS-PAGE图:a)非还原型电泳，从左至右为不同pH条件(pH 7.0，7.2，7.4，7.6，7.8，8.0)溶解的天然CAT与CAT纳米颗粒(pH 7.0，7.2，7.4，7.6，7.8，8.0)；b)还原型电泳(顺序如前)。
图4自组装过氧化氢酶纳米颗粒的SEM电镜观察图(a)及圆二色谱图(b)。
图5不同pH下制备的自组装过氧化氢酶纳米颗粒的酶活。
图6不同pH下制备的自组装过氧化氢酶纳米颗粒对巨噬细胞生存率的影响。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例的技术方案进行清楚、完整的描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普 通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。
除非另作定义，本公开所使用的技术术语或者科学术语应当为本发明所属领域内有一般技能的人士所理解的通常意义。
实施例1
称取过氧化氢酶冻干粉0.8mg，溶解于1mL pH 7.0，0.02M的磷酸盐缓冲液中，振荡使之充分溶解，5000rpm/min离心转速下离心10min后选取上清液，至于1.5mL离心管中。然后将离心管至干热孵育器中，60℃下保温孵育10min，即得到自组装过氧化氢酶纳米颗粒。用激光粒度仪测定其粒径和表面电位，测定粒径为134.90±3.18nm，表面电荷范围在-11.40±1.07mV，见图1，图1中的天然是指未经处理的过氧化氢酶；自组装过氧化氢酶纳米颗粒粒度分布见图2；其SDS-PAGE电泳图见图3；SEM电镜图，CD光谱图见图4，自组装过氧化氢酶纳米颗粒其酶活力为7732.50±235.74U/mg，见图5。
实施例2
称取过氧化氢酶冻干粉0.8mg，溶解于1mL pH 7.2，0.02M的磷酸盐缓冲液中，振荡使之充分溶解，5000rpm/min离心转速下离心10min后选取上清液，至于1.5mL离心管中。然后将离心管至干热孵育器中，60℃下保温孵育10min，即为自组装过氧化氢酶纳米颗粒。用激光粒度仪测定其粒径和表面电位，测定粒径为63.40±1.23nm，表面电荷范围在-27.0±2.21mV，见图1；自组装过氧化氢酶纳米颗粒纳米颗粒粒度分布见图2；自组装过氧化氢酶纳米颗粒纳米颗粒粒度分布见图2；其SDS-PAGE电泳图见图3；SEM电镜图，CD光谱图见图4，自组装过氧化氢酶纳米颗粒其酶活力为7382.50±221.77U/mg，见图5。
实施例3
称取过氧化氢酶冻干粉0.8mg，溶解于1mL pH(7.4～8.0)，0.02M的磷酸盐缓冲液中，振荡使之充分溶解，5000rpm/min离心转速下离心10min后选取上清液，至于1.5mL离心管中。然后将离心管至干热孵育器中，60℃下保温孵育10min，即为自组装过氧化氢酶纳米颗粒。用激光粒度仪测定其粒径和表面 电位，测定粒径约为40nm，表面电荷范围在-15.6～-19.5mV，见图1；自组装过氧化氢酶纳米颗粒粒度分布见图2；其SDS-PAGE电泳图见图3；SEM电镜图，CD光谱图见图4，纳米颗粒其酶活力≥3100U/mg，见图5。
由实施例1-3可以看出，随着pH值的增大，自组装过氧化氢酶纳米颗粒的粒径逐渐减小，因此，可以通过控制体系的pH值来调整过自组装氧化氢酶纳米颗粒的尺寸，从而根据不同需求场合应用。并且，在稳定性方面，过氧化氢酶自组装形成纳米尺寸后，贮藏稳定性标准ζ-电位绝对值显著提升，同时，自组装过氧化氢酶纳米颗粒可以耐受SDS水解，提升了化学稳定性。
实施例4
称取过氧化氢酶冻干粉0.8mg，溶解于1mL pH 7.2，0.02M的磷酸盐缓冲液中，振荡使之充分溶解，经0.45μm或0.22μm孔径的膜滤，得到滤液，至于1.5mL离心管中。然后将离心管至干热孵育器中，75℃下保温孵育30s，即得到自组装过氧化氢酶纳米颗粒，用激光粒度仪测定其粒径和表面电位，测定粒径约为110nm，纳米颗粒其酶活力≥3300U/mg。
实施例5：应用于消化道黏膜的巨噬细胞
以SD大鼠消化道黏膜的巨噬细胞为模型，以存活率为指标，探讨不同pH(pH 7.0,pH 7.2,pH 8.0)条件下形成的自组装过氧化氢酶纳米颗粒以及天然过氧化氢酶在不同浓度下对巨噬细胞的影响，生化分析结果见图6。
结果显示，在一定范围内自组装过氧化氢酶纳米颗粒对促进巨噬细胞生长的能力强于天然过氧化氢酶，且最高促进作用＞77％。由此看出，作为纳米药物，纳米抗氧化酶作用于机体时，不单单需要考虑纳米抗氧化酶的本身酶活，也要考虑形成纳米结构的粒径大小以最大化发挥纳米抗氧化酶的生理活性。
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求保护范围内。

Claims (10)

  1. 一种自组装过氧化氢酶纳米颗粒的制备方法，其特征在于，包括将过氧化氢酶冻干粉溶解获得过氧化氢酶溶液，调整过氧化氢酶溶液的pH，然后经离心或过滤获得上清液或过滤液，将上清液或过滤液进一步热孵育而得。
  2. 根据权利要求1所述的一种自组装过氧化氢酶纳米颗粒的制备方法，其特征在于，所述过氧化氢酶溶液的浓度为0.2-5.0mg/mL。
  3. 根据权利要求1所述的一种自组装过氧化氢酶纳米颗粒的制备方法，其特征在于，调整过氧化氢酶溶液的pH值为6.5-9.5。
  4. 根据权利要求1所述的一种自组装过氧化氢酶纳米颗粒的制备方法，其特征在于，所述离心的速度为2000-5000rpm/min。
  5. 根据权利要求1所述的一种自组装过氧化氢酶纳米颗粒的制备方法，其特征在于，所述过滤为膜过滤，膜过滤孔径为0.22μm-0.45μm。
  6. 根据权利要求1所述的一种自组装过氧化氢酶纳米颗粒的制备方法，其特征在于，所述热孵育为将上清液或过滤液在45-90℃的温度下加热15s-15min。
  7. 一种如权利要求1-6所述的制备方法获得的自组装过氧化氢酶纳米颗粒，其特征在于，所述过氧化氢酶纳米颗粒的粒径为30-200nm。
  8. 根据权利要求1所述的自组装过氧化氢酶纳米颗粒，其特征在于，所述过氧化氢酶纳米颗粒的酶活力≥3100U/mg。
  9. 一种如权利要求1-8所述的自组装过氧化氢酶纳米颗粒在食品或药品中的应用。
  10. 根据权利要求9所述的自组装过氧化氢酶纳米颗粒在食品或药品中的应用，其特征在于，所述自组装过氧化氢酶纳米颗粒在具有免疫调节功能的食品或药品中的应用。
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