Synthesis and characterization of L-asparaginase encapsulated Poly-l-lysine-graft-poly(ethylene) glycol polymer nanoparticles for potential therapeutic delivery applications

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Keri Joanna Goff (Creator)
Western Carolina University (WCU )
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Rangika Hikkaduwa Koralege

Abstract: L-asparaginase (L-ASNase) is a therapeutic enzyme that is widely used for the treatment of hematopoietic diseases such as acute lymphoblastic leukemia and lymphomas since 1970. L-ASNase can destroy asparagine dependent tumors by degrading circulating L-asparagine and destroying malignant cells. Being a therapeutic enzyme and essentially a protein drug, L-ASNase has intrinsic drawbacks such as low stability, short circulating lifetime, and low catalytic activity under physiological conditions. Due to the bacterial origin of L-ASNase, immunogenicity is another major problem with high frequency of hypersensitivity reactions. L-ASNase delivered via pegylated-L-ASNase (PEG (poly(ethylene)glycol)-ASNase) form has been shown to improve clinical outcome of this therapy. The focus of this project is to successfully encapsulate a catalytically active, stable, therapeutic protein in PLL-g-PEG NPs.The goals of this project include (1) synthesis and characterization a PLL-g-PEG co-polymer, (2) synthesis of protein-polymer nanoparticles by encapsulating a model protein, bovine serum albumin (BSA) in PLL-g-PEG , (3) synthesis of protein-polymer nanoparticles by encapsulating therapeutic protein L-ASNase in PLL-g-PEG , (4) perform physicochemical characterization including nanoparticle size and surface charge, (5) evaluate encapsulation efficiency of L-ASNase in NPs, (6) evaluate stability of NPs under physiological conditions, and (7) evaluate enzymatic activity of encapsulated L-ASNase. In the present study, we have successfully synthesized and characterized a maleimide functionalized PLL-g-PEG co-polymer. Nanoparticles were formed through electrostatic interactions of the cationic backbone of the PLL-g-PEG copolymer and negatively charged L-ASNase. L-ASNase nanoparticles had an average hydrodynamic diameter of 114.5 ± 5.66 nm and a near neutral zeta potential of 0.436 ± 0.258 mV. The extent of L-ASNase encapsulation was determined to be 100% according to the SDS-PAGE data. Additionally, SDS-PAGE data provides conclusive results that these therapeutic nanoparticles are stable in solution at physiological pH conditions for more than 6 months and long-term particle stability studies are in progress. Encapsulated L-ASNase showed an average of 10.5 nmole/min/mL activity and that is 53% as a percentage compared to the free L-ASNase positive control. These particles were stable in 10% FBS for more than 17 hours at 37 ?C. In conclusion, we were able to successfully encapsulate a catalytically active, stable, therapeutic protein in PLL-g-PEG NPs.

Additional Information

Language: English
Date: 2022

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