Albumin/asparaginase capsules prepared by ultrasound to retain ammonia

Abstract

Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2 % of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.

BSA/ASN/Pol407 nanoparticles for acute lymphoblastic leukemia treatment

“…To characterize the immobilization process for the different formulations, two parameters were evaluated: nanoparticles formation efficiency and nanoparticles encapsulation efficiency [11]. For both parameters, only the protein in the aqueous phase, obtained after nanoparticles centrifugation, was used for quantification.…”

“…For the Z-potential analysis, the same conditions were used with the nanoparticles dispersed in ultra-pure water. The parameters were determined by photon correlation spectroscopy, using dynamic light scattering (DLS) (Malvern Instruments, Nano-ZS) [11]. The values for viscosity and refractive index were 0.8872 cP and 1.330, respectively.…”

“…When the reaction was performed in ultra-pure water, the enzyme was inactivated on ice and removed from the medium by ultra-centrifugation (10,000 RCF, 30 min) in microtubes with a membrane cut-off of 5000 Da. After ultra-centrifugation, the membrane was washed with ultra-pure water to prevent amino acid retention in the membrane, and quantification of ammonia and aspartic acid was performed [11]. Ammonia quantification was determined by a stopped assay using Nessler's reagent.…”

“…Previously, we reported the ability of albumin/asparaginase capsules prepared by ultrasound to retain ammonia [11]. Despite the positive results, the nanoparticles physical properties were not suitable for an intravenous administration and thus an improvement was essential regarding the intended application.…”

Nanopartículas Poliméricas Degradáveis Para Carreamento De Proteínas: Com Foco Na Enzima L-Asparaginase

“…Estas nanocápsulas contendo L-asparaginase não causaram efeito citotóxico significativo na linhagem de macrófagos leucêmicos de camundongos RAW 264.7, as quais são positivas para a expressão de asparagina sintase. (Tinoco et al, 2016) A encapsulação de L-asparaginase em nanovesículas de quitosana modificada e lipídios levou ao aumento da atividade enzimática e ao aumento da estabilidade com variação de temperatura e pH, além de diminuir a taxa de proteólise. Além disso, após o encapsulamento, a enzima exibiu maior atividade anti-câncer de pulmão do que a enzima livre.…”