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Nano preparation for improving stability of enzyme drugs and preparation method and application of nano preparation

A nano-formulation and stability technology, applied in the field of biopharmaceuticals, can solve the problems of uncontrollable sources of poly-lysine and application limitations.

Pending Publication Date: 2019-03-05
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some studies use poly-L-lysine (poly-L-lysines, PLL) as a carrier to load CAT into macrophages through electrostatic adsorption, and use the tropism of macrophages in vivo to achieve targeted drug delivery; among them , PLL coating can significantly improve the stability of CAT, ensure that it can maintain activity to a certain extent after being phagocytized by macrophages, and enter neuron cells through the interaction between cells and neurons to play an antioxidant role, but polylysine The source is uncontrollable, so its application is greatly limited

Method used

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  • Nano preparation for improving stability of enzyme drugs and preparation method and application of nano preparation
  • Nano preparation for improving stability of enzyme drugs and preparation method and application of nano preparation
  • Nano preparation for improving stability of enzyme drugs and preparation method and application of nano preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Synthetic PEG-DGL

[0036] The amino group on the surface of DGL reacts specifically with the NHS at one end of the functional PEG. According to the ratio of DGL:PEG=1:6 (mol / mol), NHS-PEG was dissolved in PBS 7.4 buffer solution, and then added dropwise to the stirred DGL solution (5mg / ml, PBS, pH=7.4) , and the reaction was stirred at room temperature for 1 h. Transfer to a 5kDa ultrafiltration tube, add PBS, centrifuge at 6000rpm for 20min, repeat twice, and purify to obtain PEG-DGL;

[0037] Preparation of PEG-DGL-PDP

[0038] The amino groups on the surface of DGL react specifically with the NHS on the surface of the crosslinker N-succinimidyl 3-(2-pyridyldithio)-propionate, SPDP. SPDP and PEG-DGL (DGL:SPDP=1:2mol / mol) were mixed in PBS solution (100mM Na 3 PO 4 , 1mM EDTA, pH=7.4), and the reaction was stirred at room temperature for 1-3h. Part of the amino groups on the surface of DGL are replaced by PDP groups to obtain PEG-DGL-PDP;

[0039] Preparation o...

Embodiment 2

[0053] Example 2 In vitro stability study of CAT in nanoparticles

[0054] The freshly prepared PEG-DGL / CAT-Aco nanoparticles (0.5 mg / ml CAT) were added with trypsin (10 -5 M) Mixed and incubated at 37°C for 3 hours. Using CAT-Aco as a control, the PEG-DGL / CAT-Aco nanoparticles (mass ratio of 1.25, 2.50, 5.00, 7.50, 10.00) were degraded by trypsin according to the above method. The residual CAT activity was used to investigate the stability of nanoparticles;

[0055] like Figure 4 As shown, when the mass ratio of nanoparticles (DGL:CAT) was in the range of 1.25-10.00, embedding CAT-Aco in PEG-DGL significantly increased the stability of the enzyme, and with the increase in the mass ratio of DGL to CAT, the PEG-DGL The stability of / CAT-Aco nanoparticles gradually increased, and when it reached a certain level, the stability of nanoparticles remained within a certain range, indicating that nanoparticles can reduce the degradation of CAT by trypsin, and the PEG-DGL / There wa...

Embodiment 3

[0056] Example 3 Investigation experiment of intracellular stability of CAT in nanoparticles

[0057] HL-60 cells were mixed with catalase and different types of nanoparticles in serum-free medium, and incubated at 37°C for 1 h. The cell suspension was collected and centrifuged at 2000 rpm for 5 min. Add HBSS to resuspend, centrifuge at 2000 rpm for 5 min, repeat twice, add fresh culture medium, and culture in a 30°C incubator. At different time points, collect cells and culture medium, and use catalase detection kit to measure the intracellular catalase activity and the enzyme activity released from HL-60 cells according to the instructions;

[0058]For the determination of intracellular catalase activity at different time points, a certain amount of cell lysate (containing 1% protease inhibitor) needs to be added to the collected cells, and after incubation on ice for 15 minutes, the cells are crushed with an ultrasonic pulverizer (power 100w , ultrasonication for 3 s), ce...

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Abstract

The invention belongs to the technical field of biopharmacy, and relates to a nano preparation method for improving stability of enzyme drugs. The nano preparation is nanoparticles PEG-DGL / En-Aco prepared from cis-acotonic anhydride (Aco), dendritic poly-l-lysine (DGL) and functional polyethylene glycol (NHS-PEG). The entrapment efficiency of the prepared nanoparticles is 71.46+ / - 0.31%, the appearance of the nanoparticles is round, and the nanoparticles are spherical; by the nanoparticles, degradation of trypsin to CAT can be relieved, in 72 h, the enzymatic activity of PEG-DGL / CAT-Aco nanoparticles in HL-60 cells is remarkably higher than free CAT, and in an extracellular matrix, the enzymatic activity of crosslinked nanoparticles is gradually increased along with time; by the prepared nanoparticles, the area of cerebral infarction of an animal pattern can be remarkably reduced; and ischemia reperfusion injury can be inhibited obviously, and the nano preparation has potential nerve and blood vessel protection effect. The prepared nanoparticles can be used for preparing drugs for treating stroke.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a nano preparation for improving the stability of enzyme drugs, in particular to a nano preparation prepared by a charge adsorption method and its use in protecting the stability of enzyme drugs. Background technique [0002] With the rapid development of bioengineering technology, biological macromolecular drugs have become one of the most promising fields in drug research and development in the 21st century. The prior art discloses that macromolecular drugs such as polypeptides and proteins have obvious advantages in the treatment of the central nervous system (Central nervous system, CNS) due to their high specificity, and are increasingly used in preclinical and clinical research, Reports show that more than hundreds of protein and polypeptide drugs have been used in clinical practice, and macromolecular drugs have become a research hotspot in the current related tec...

Claims

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Application Information

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IPC IPC(8): A61K9/51A61K47/22A61K47/10A61K38/44A61P9/10
CPCA61K9/5123A61K9/5146A61K38/44C12Y111/01006
Inventor 秦晶张春王建新王丽敏
Owner FUDAN UNIV
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