الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
Summary
L-asparaginase enzyme (EC 3.5.1.1) is a potent weapon in the treatment of childhood acute lymphoblastic leukemia (ALL) and non-Hodgkin’s lymphoma. As a large amount of L-Asn is required for the proliferation of these cancerous cells, depleting the L-Asn by L-ASNase results in starvation and selective apoptosis of the leukemic blasts. Since ALL severely lacks ASNS expression, being the most dependent subtype on blood Asn, the treatment of ALL gained the most effective outcomes among other blood cancers. Recent evidence proved the clinical potential of L-ASNase in the treatment of other leukemia subtypes and certain solid tumors. However, severe toxicity profiles were introduced by this enzyme upon administration among adult cancer patients, including aggressive immunological side effects as well as non-immune related toxicities. Nanotechnology can help overcome the limitations accompanying conventional L-ASNase therapies, and improve its delivery by providing different strategies for encapsulation or immobilization. Consequently, the aim of the current study was to formulate and biochemically characterize rD. chrysanthemi L-ASNase and to evaluate its anticancer activity.
Recombinant D. chrysanthemi L-ASNase gene was subcloned into the pET-28a (+) expression vector and transformed to E. coli JM 109 (λDE3) cells. The successful transformants were further cultivated and then induced in a large-scale manner to produce the enzyme. The cell lysate was analyzed for successful induction and purification using SDS-PAGE, and a sharp band appeared at 36 kDa approximately.
The rD. chrysanthemi SLNs formulation was prepared using the high shear homogenization/ultrasonication method but the enzyme lost its activity due to stress conditions. Also, the double emulsion method was performed and the best DLS analysis was obtained upon decreasing the lipid concentration at room temperature however, the catalytic activity was lost. The
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w/o/w method was also used for the preparation of liposomes encapsulating rD. chrysanthemi L-ASNase up to 56.6 ± 1.2% (EE%) but, with relatively high DLS and PDI values and low zeta potential, though, activity was lost upon increasing the lipid and surfactant concentrations.
Bovine serum albumin (BSA) NPs were prepared using the desolvation method that comprised a droplet size ranging from 90.67 ± 2.97 to 290.8 ± 64.9 nm and PDI values between 0.159 ± 0.03 and 0.473 ± 0.03. However, TEM results were respectively smaller as 192.2 ± 10.7 nm and 170.1 ± 7.9 nm in case of the BSA NPs and enzyme-immobilized NPs. BSA NPs showed good zetapotential values between -7.82 ± 7.24 and -33.5 ± 5.62 mV indicating good stability profile. The UV-visible spectrum confirmed the formation of the BSA NPs and the binding of the enzyme through the aromatic amino acids. The FT-IR analysis revealed the presence of the enzyme’s characteristic peaks (at 1076.3 and 988.4 cm-1) in the spectrum of the rD. chrysanthemi L-ASNase-BSA NPs. The optimum rD. chrysanthemi L-ASNase immobilized formulation was monitored for the in-vivo release profile that continued sustainably up to 24 h compared to the free enzyme. Nevertheless, further biochemical investigations and anticancer assessment are required.
Magnetic NPs (Fe3O4 NPs) were used as solid support for the immobilization of rD. chrysanthemi L-ASNase, providing an incredibly large surface area for interaction resulting in activity enhancement by 122.7%. The pure and immobilized Fe3O4 NPs were characterized using TEM, the resulting mean sizes were 12.6 ± 0.07 and 48.33 ± 7.5 nm, respectively. The XRD confirmed the cubic pure Fe3O4 NPs crystals pattern according to ICSD card No. 79-0416. Moreover, the FT-IR spectroscopy was performed for the free and immobilized NPs which revealed the characteristic bands of Fe3O4 NPs formation between 544 cm-1 and 630 cm-1. As well as, the presence of L-ASNase characteristic band in the rD. chrysanthemi L-ASNase-Fe3O4 NPs spectrum at 1077 cm-1 supporting the successful immobilization of L-ASNase. A drug release experiment
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was developed accounting for a remarked sustained release rate with only 12.1% released drug within 24 h, in addition to, 41.4% enzyme activity was retained in the NPs after 24 h. Recombinant D. chrysanthemi L-ASNase-Fe3O4 NPs managed to enter 5 successive cycles while retaining 95.3 and 92.9% residual activity after the second and third cycles and 20.8% after the fifth cycle. The rD. chrysanthemi L-ASNase-Fe3O4 NPs showed a better short stability profile than the free enzyme to record 75.5% and 18.1% residual activity, respectively after 35 days. The immobilized enzyme remained active (29.8%) after 93 days, unlike the free enzyme which lost its activity after 35 days.
The rD. chrysanthemi L-ASNase-Fe3O4 NPs showed interesting biochemical properties compared to the naked enzyme; the optimum temperature of was 55°C and 60°C for the free and immobilized enzymes, respectively. However, the active operational region of the immobilized enzyme has widened between 37-60°C. The optimum pH was found to be 8 and 7 for the free and immobilized enzyme, respectively. Even though, the immobilized enzyme was more stable at a much wider range of pH (7-10) than the free enzyme. The thermostability profile at 37, 45, 50, and 55°C of the immobilized enzyme was improved at higher temperatures, and retained 75.9, 67.6, 57.7 and 18.6% residual activity after 1 h, while the free enzyme retained 93.1, 60.3, 29.2 and 15.1%. The kinetic parameters were significantly improved as the Km value of the immobilized enzyme decreased by 4.4 folds when compared to the free enzyme indicating higher enzyme substrate affinity (P< 0.0005).
The MTT assay revealed that almost all treatments with rD. chrysanthemi L-ASNase, Fe3O4 NPs and rD. chrysanthemi L-ASNase-Fe3O4 NPs inhibited the cell viability of THP1, K562, and A549 cell lines in a concentration-dependent manner. THP1, K562, and A549 (RPMI) displayed higher sensitivity to all treatments. The rD. chrysanthemi L-ASNase-Fe3O4 NPs showed a remarkably higher cytotoxic effect on almost all cell line attempts compared to the free enzyme and Fe3O4 NPs.
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