Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 9;9(1):340.
doi: 10.1186/1556-276X-9-340. eCollection 2014.

Preparation and nanoencapsulation of l-asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study

Elham Bahreini  1 , Khosrow Aghaiypour  2 , Roghayeh Abbasalipourkabir  1 , Ali Rezaei Mokarram  2 , Mohammad Taghi Goodarzi  1 , Massoud Saidijam  1
Affiliations

Preparation and nanoencapsulation of l-asparaginase II in chitosan-tripolyphosphate nanoparticles and in vitro release study

Elham Bahreini et al. Nanoscale Res Lett. .

Abstract

This paper describes the production, purification, and immobilization of l-asparaginase II (ASNase II) in chitosan nanoparticles (CSNPs). ASNase II is an effective antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy. Cloned ASNase II gene (ansB) in pAED4 plasmid was transformed into Escherichia coli BL21pLysS (DE3) competent cells and expressed under optimal conditions. The lyophilized enzyme was loaded into CSNPs by ionotropic gelation method. In order to get optimal entrapment efficiency, CSNP preparation, chitosan/tripolyphosphate (CS/TPP) ratio, and protein loading were investigated. ASNase II loading into CSNPs was confirmed by Fourier transform infrared (FTIR) spectroscopy, and morphological observation was carried out by transmission electron microscopy. Three absolute CS/TPP ratios were studied. Entrapment efficiency and loading capacity increased with increasing CS and TPP concentration. The best ratio was applied for obtaining optimal ASNase II-loaded CSNPs with the highest entrapment efficiency. Size, zeta potential, entrapment efficiency, and loading capacity of the optimal ASNase II-CSNPs were 340 ± 12 nm, 21.2 ± 3 mV, 76.2% and 47.6%, respectively. The immobilized enzyme showed an increased in vitro half-life in comparison with the free enzyme. The pH and thermostability of the immobilized enzyme was comparable with the free enzyme. This study leads to a better understanding of how to prepare CSNPs, how to achieve high encapsulation efficiency for a high molecular weight protein, and how to prolong the release of protein from CSNPs. A conceptual understanding of biological responses to ASNase II-loaded CSNPs is needed for the development of novel methods of drug delivery.

Keywords: Cross-linking; Enzyme immobilization; Half-life; Ionotropic gelation; Nanoparticle; Optimization.

PubMed Disclaimer

Figures

Figure 1
Figure 1
SDS-PAGE (15%) analysis of ASNase II purification using DEAE-Sepharose. Lane 1: protein marker. Lane 2: Crude extract of E. coli by alkaline lysis. Lanes 3 to 11: purified ASNase II eluted from the DEAE-Sepharose column in selected fractions.
Figure 2
Figure 2
FTIR spectra of (A) ASNase II, (B) CS, (C) CSNPs, and (d) ASNase II-loaded CSNPs.
Figure 3
Figure 3
TEM images of CSNPs (A) and ASNase II-loaded CSNPs (B).
Figure 4
Figure 4
ASNase II release profiles from the ASNase II-loaded CSNPs in three solutions. (a) Glycerol (5%)-PBS solution (pH 7.4), (b) PBS solution (pH 7.4), and (c) DDW containing 5% glycerol (pH 7.0). CS/TPP of 0.4/0.095 loaded with 4 mg protein.
Figure 5
Figure 5
Effect of pH on the activity (A) and stability (B) of free and immobilized ASNase II. Activity was measured at standard conditions and compared with untreated control.
Figure 6
Figure 6
Effect of temperature on the stability of free and immobilized ASNase II. After the enzymes were incubated in the buffer solutions (pH 8.5) for 60 min at varying temperatures, the remaining activities were measured at 37°C.
Figure 7
Figure 7
The in vitro half-life of the free (A) and immobilized ASNase II (B) in PBS containing 5% glycerol (pH 7.4).
Figure 8
Figure 8
The in vitro half-life of the free (A) and immobilized ASNase II (B) in DDW containing 5% glycerol (pH 7.0).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Narta UK, Kanwar SS, Azmi W. Pharmacological and clinical evaluation of L-asparaginase in the treatment of leukemia. Crit Rev Oncol Hematol. 2007;9:208–221. doi: 10.1016/j.critrevonc.2006.07.009. - DOI - PubMed
    1. Pasut G, Sergi M, Veronese FM. Anti-cancer PEG-enzymes: 30 years old, but still a current approach. Adv Drug Deliv Rev. 2008;9:69–78. doi: 10.1016/j.addr.2007.04.018. - DOI - PubMed
    1. Wolf M, Wirth M, Pittner F, Gabor F. Stabilisation and determination of the biological activity of L-asparaginase in poly(D, L-lactide-co-glycolide) nanospheres. Int J Pharm. 2003;9:141–152. doi: 10.1016/S0378-5173(03)00071-1. - DOI - PubMed
    1. Matsushima A, Nishimura H, Ashihara Y, Yokota Y, Inada Y. Modification of E. coli asparaginase with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-S-triazine(activated PEG2); disappearance of binding ability towards anti-serum and retention of enzymic activity. Chem Lett. 1980;9:773–776.
    1. Uren JR, Hargis BJ, Beardsley P. Immunological and pharmacological characterization of poly-DL-alanyl-modified Erwinia carotovora L-asparaginase. Cancer Res. 1982;9:4068–4071. - PubMed