Enzymatic Active Release of Violacein Present in Nanostructured Lipid Carrier by Lipase Encapsulated in 3D-Bioprinted Chitosan-Hydroxypropyl Methylcellulose Matrix With Anticancer Activity

Abstract

Violacein (Viol) is a bacterial purple water-insoluble pigment synthesized by Chromobacterium violaceum and other microorganisms that display many beneficial therapeutic properties including anticancer activity. Viol was produced, purified in our laboratory, and encapsulated in a nanostructured lipid carrier (NLC). The NLC is composed of the solid lipid myristyl myristate, an oily lipid mixture composed of capric and caprylic acids, and the surfactant poloxamer P188. Dormant lipase from Rhizomucor miehei was incorporated into the NLC-Viol to develop an active release system. The NLC particle size determined by dynamic light scattering brings around 150 nm particle size and ζ≈ −9.0 mV with or without lipase, but the incorporation of lipase increase the PdI from 0.241 to 0.319 (≈32%). For scaffold development, a 2.5 hydroxypropyl methylcellulose/chitosan ratio was obtained after optimization of a composite for extrusion in a 3D-bioprinter developed and constructed in our laboratory. Final Viol encapsulation efficiency in the printings was over 90%. Kinetic release of the biodye at pH = 7.4 from the mesh containing NLC-lipase showed roughly 20% Viol fast release than without the enzyme. However, both Viol kinetic releases displayed similar profiles at pH = 5.0, where the lipase is inactive. The kinetic release of Viol from the NLC-matrices was modeled and the best correlation was found with the Korsmeyer-Peppas model (R2 = 0.95) with n < 0.5 suggesting a Fickian release of Viol from the matrices. Scanning Electron Microscope (SEM) images of the NLC-meshes showed significant differences before and after Viol’s release. Also, the presence of lipase dramatically increased the gaps in the interchain mesh. XRD and Fourier Transform Infrared (FTIR) analyses of the NLC-meshes showed a decrease in the crystalline structure of the composites with the incorporation of the NLC, and the decrease of myristyl myristate in the mesh can be attributed to the lipase activity. TGA profiles of the NLC-meshes showed high thermal stability than the individual components. Cytotoxic studies in A549 and HCT-116 cancer cell lines revealed high anticancer activity of the matrix mediated by mucoadhesive chitosan, plus the biological synergistic activities of violacein and lipase.

Biocatalysts Based on Immobilized Lipases for the Production of Fatty Acid Ethyl Esters: Enhancement of Activity through Ionic Additives and Ion Exchange Supports

“…This heightened activity persists both after immobilization and even in the absence of the additive SDS. This phenomenon, referred to verbatim as "bio-imprinting" by certain authors [72,73], appears to be inapplicable to derivatives obtained here in the absence of SDS. Similar observations were obtained for TLL immobilized on sulfopropyl-Sepharose ® (SP), a cation exchanger, in the presence of CTAB at relatively high concentrations (0.3%) [25].…”

Potential biocide roles of violacein

“…Despite the different surface characteristics of the three tested viruses, all shared hydrophobic moieties on their surfaces that could interact with water-insoluble molecules, such as violacein. Since the interaction of violacein with molecules with hydrophobic motifs (i.e., non-ionic surfactants, cyclodextrins, aromatic ionic liquids, lipid carriers) were previously reported (de Azevedo et al, 2000;Rivero Berti et al, 2019;Rivero Berti et al, 2020;Rivero Berti et al, 2022). It is expected that hydrophobic interactions appear to be the unspecific major mechanism of interaction between the virus and violacein.…”

“…More recently, violacein was encapsulated in a nanostructured lipid carrier with an active release by lipase and 3D printed using a hydroxypropyl cellulose-chitosan matrix. The formulation was tested against the A549 and HCT-116 cancer cell lines (Rivero Berti et al, 2022).…”

“…Violacein has recently attracted the attention of researchers owing to its wide variety of biological activities. During the last two decades, several reports have described numerous biological activities of this pigment, including immunomodulatory, antimicrobial, antiparasitic, antifungal, anticancer, and antiviral activities (Duran et al, 2021a;Duran et al, 2021b;Duran et al, 2022). The relevance of the antioxidant properties of violacein must be analyzed in the context of COVID-19 pathology, as acute infections of SARS-CoV-2 can produce cell death and long-term neurological pathologies.…”

Application of three-dimensional (3D) bioprinting in anti-cancer therapy

“…HTP 3D printing manufacturing platforms enable rapid and efficient preparation of various types of 3D cell models that are tailored to specific research purposes by adjusting the composition, geometry, architecture, and function of bioprinted structures [ [56] , [57] , [58] ]. In addition, 3D bioprinting allows the integration of multiple cell types and bioactive bioinks to create more complex and dynamic 3D cell models encapsulating epithelial-mesenchymal transition (EMT) [ 59 , 60 ]. During the 3D bioprinting process, the choice of bioinks is very important.…”