In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic Enzyme

Abstract

Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.

Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles

“…We recently described how at an optimized initial pH of 6.0, CS acquires a helical conformation that favoured a more controlled ionic gelation [ 10 ]. Interestingly, the increasing amounts of each HA in the absence of TPP ( Figure 1 ; CS:TPP mass ratio of 1:0) followed a similar behaviour to the previously reported TPP loading.…”

“…In this case, the increasing amount of negative charges from CM-β-CD was able to improve the control in the colloidal system even in absence of TPP, (decreasing PI up to ~0.1; Figure 2 A). With a DS of three and taking into account that at the final formulation pH of 6.5 the carboxyl moieties are negatively charged, each CM-β-CD is actually contributing with 21 negative charges (opposed to the TPP triple negative charge at pH of 6.5) [ 10 ]. Moreover, particularly at the optimized CS:TPP mass ratio of 5:1, the addition of CM-β-CD maintained the NP mean size at lower values, with a decreased surface charge.…”

“…The previous optimization of CS NP regarding concentration and protonation status proved to be critical in the formulation process [ 10 ]. With similar CS to CD ratios, other authors were only able to produce NP from HP-β-CD with a mean size ≥300 nm and a PI ~0.3 and, from CM-β-CD, a mean size ≥230 nm was obtained [ 19 ].…”

“…The process of opsonization is described as mainly dependent from random Brownian motions, but when NP comes into contact with complement components, any of several attractive forces including van der Walls, electrostatic, ionic, hydrophobic/hydrophilic and others can be involved in the binding and activation of opsonins at their surface [ 34 ]. Thus, the observed mild increase is a direct consequence of the previous optimized ionic balance between CS and TPP, which rendered CS amines at the final formulation pH (close to neutralization) less reactive [ 10 ].…”

“…Recently, we reported the rational design of a DDS based on the ionic gelation of CS with tripolyphosphate (TPP) for the formulation of the labile 200 kDa homotetrameric human phenylalanine hydroxylase (hPAH), which is a structurally and functionally complex enzyme [ 10 ]. Impaired hPAH activity, due to mutations in the encoding gene, is responsible for phenylketonuria (PKU), which is the most common genetic disorder of amino acid metabolism [ 11 ].…”