Introduction:
Inflammatory processes are a typical response of the organism to various injuries, providing elimination of the causes of damage and tissue repair. In the course of cell damage, reactive oxygen species (ROS) are formed, which stimulate further cell destruction. One way to stop the secondary inflammatory process is to use antioxidant enzymes, such as superoxide dismutase, which can neutralize active radicals. However, native enzymes in the body are quickly proteolyzed and eliminated. One of the most effective ways to protect an enzyme in the body is to coat it with polymer complexes. The aim of this study is to synthesize and characterize nanoparticles based on superoxide dismutase (SOD) enzyme encapsulated in cationic block copolymers poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) and N₃-poly(ethylene glycol)-poly{N’-[N-(2-aminoethyl)-2-aminoethyl]aspartamide (N₃-PEG-pAsp-DET) using the crosslinking agent bis-sulfosuccinimidyl suberate (BS3). It is also planned to study the targeting delivery of nanoparticles using nanoparticle conjugates with E-selectin antibody (CD62E HAE 1-f).

Methods:
The nanoparticles were characterized using Nanoparticle Tracking Analysis and Dynamic Light Scattering techniques. The activity of the protein was determined using the pyrogallol autooxidation inhibition assay. The cytotoxicity of the nanoparticles in Hek293, SH-SY5Y and HaCaT cell lines was estimated using the MTT assay. The amount of protein was determined colorimetrically using BCA assay.

Results:
The PEG-PLL-based nanoparticles were synthesized and characterized, the measured size was D_h = 162 nm, PdI 0.197. The ζ-potential of the PEG-PLL-based nanoparticles was estimated as -4.76 mV. The activity of the encapsulated SOD was 3.05×10⁶ U/mL, which corresponds to 28% of the native enzyme activity. The protein concentration estimated via A₂₆₀/A₂₈₀ absorption was 1.01 mg/mL. No evidence of toxicity in Hek293 cell line was observed for nanoparticles concentrations with protein activity range of 10– 60 kU/mL. Tert-butyl peroxide was used as inflammation inducer, the IC₅₀ value was estimated as 50 μM in Hek293 cell line. The cells demonstrate complete survival after coincubation with nanoparticles (60 kU/mL) in the presence of 50 μM TbuOOH. In the case of N₃-PEG-pAsp(DET)-based nanoparticles, the measured size was 165 nm (D_h), PdI 0.242. The residue enzyme activity was 2.9×10⁵ U/mL, which corresponds to 1.7% of those for native enzyme. The protein concentration determined by spectrophotometric method was 2.01 mg/mL. The targeted anti-E-selectin antibody was conjugated to free PEG ends using popargyl-PEG₅-NHS linker via Cu(I)-catalyzed azide-alkyne cycloaddition. The protein concentration in anti-E-selectin-conjugated nanoparticles was estimated as 0.49 mg/mL, respectively. The residue activity of SOD was 6.27% (anti-E-selectin-conjugated NP).

Conclusions:
Block ionomer complexes of SOD and block copolymer (PEG-PLL or PEG-pAsp(DET)) were synthesized and characterized. The antioxidant activity of SOD-based nanoparticles was demonstrated in tBuOOH-induced inflammation model of Hek293 cell line. E-selectin-conjugated nanoparticles were synthesized and characterized. Thus, E-Selectin targeted SOD-based nanoparticles are promising anti-inflammatory therapeutic agent.

Conflicts of interest:
The authors declare that they have no conflict of interest in the publication of this article. The authors have no conflicts of interest to report in this work. Abstract was not submitted elsewhere and was first published here.

Funding:
This study was supported in part by Russian Science Foundation (Grant: 22-13-00261), MSU State Topic (121041500039-8 and 123032300028-0) and MSU Program of Development.