Tailoring carbon nanotubes surface chemistry for the purification of antileukemic drugs

Biopharmaceuticals have been used to overcome fatal diseases related with aging. L-aspariginase (LA), in particular, has shown evidences of being efficient for leukemia therapy. The LA production and purification require several steps increasing the cost of the process. In this work, functionalized carbon nanotubes (CNTs) were studied as a cost-effective support to purify LA. It was shown that CNTs have strong affinity for the target biopharmaceutical, meaning that can be a promising alternative for the adsorption and purification of LA.publishe

Multi-walled carbon nanotubes as a platform for Immunoglobulin G attachment

Nanomaterials have been extensively used in different applications due to their peculiar characteristics and nanoscale dimensions. Among nanoparticles, carbon-based nanomaterials are becoming highly attractive for biomedical applications such as diagnosis, tissue engineering, drug delivery, and biosensing. The conjugation of carbon-based nanomaterials with antibodies combines the properties of these materials with the specific and selective recognition ability of the antibodies to antigens. The present work proposes a process intensification approach for immunoglobulin G (IgG present in rabbit serum) attachment on multi-walled carbon nanotubes (MWCNTs) in a single step. The effect of several parameters, namely MWCNTs external diameter, rabbit serum concentration, MWCNTs functionalization and pH value, on the IgG attachment yield was evaluated. The dilution of rabbit serum decreased other protein attachment, namely rabbit serum albumin (RSA), while increasing the IgG yield to 100%. The interaction mechanisms between IgG and MWCNTs were evaluated at pH 5.0 to 8.0. The protonation of IgG amino acids indicates that N-term are the most reactive amino acids in the antibody structure. The identification of the N-term reactivity at pH 8.0 allows to indicate a possible orientation of the antibody over the MWCNTs surface, described as “end-on”. Since the amount of RSA attached to MWNT decreased with the increase in serum dilution, the IgG orientation and amine activity was not affected. This orientation demonstrates that the IgG attachment over the surface of the MWCNTs could be an effective strategy to maintain the antigen recognition by the antibody, and to be used for biomedical applications.publishe

Carbon nanomaterials for the purification of antileukemic drugs

Getting older is the biggest risk factor for most fatal diseases, including cancer, heart disease and Alzheimer. To overcome such age-related society diseases, it is crucial to optimize the production and purification of biopharmaceuticals, such as nucleic acid-based products, antibodies and recombinant proteins and enzymes. Low cost production combined with high purity levels allow their routinely use by a widespread population. Continuous progresses have been made for the development of recombinant therapeutic enzymes. L-asparaginase (LA) is an antileukemic biopharmaceutical enzyme of current high-cost. LA is produced via fermentation and its purification usually comprises several steps that account up to 80% of its total production cost (1). This work aims to develop sustainable technologies to extract and purify LA. Reusable functionalized nanomaterials, namely carbon nanomaterials (CNTs), are used as cost-effective purification techniques for the target enzyme. Initially, the synthesis and modification of CNTs was performed. Different CNTs were obtained and used for the purification of LA. Commercial LA was used for the first purification tests, in order to understand the behaviour of the enzyme in contact with the nanomaterial. Experimental conditions, such as pH, and material/LA ratio, contact time were optimized. LA activity was quantified by Nessler reaction (2). The first results reveal a total adsorption of LA by the CNTs. Depending on the CNT functionalization/ treatment, different values of recovered activity of LA were obtained. The modified CNTs are shown to be very promising nanomaterials for the purification of LA. The LA was easily attached to CNTs by adsorption under mild conditions. CNTs supports can be a real alternative for a single step immobilization/purification of LA.publishe

Unveiling the Influence of Carbon Nanotube Diameter and Surface Modification on the Anchorage of L-Asparaginase

L-asparaginase (ASNase, EC 3.5.1.1) is an amidohydrolase enzyme known for its anti-cancer properties, with an ever-increasing commercial value. Immobilization has been studied to improve the enzyme’s efficiency, enabling its recovery and reuse, enhancing its stability and half-life time. In this work, the effect of pH, contact time and enzyme concentration during the ASNase physical adsorption onto pristine and functionalized multi-walled carbon nanotubes (MWCNTs and f-MWCNTs, respectively) with different size diameters was investigated by maximizing ASNase relative recovered activity (RRA) and immobilization yield (IY). Immobilized ASNase reusability and kinetic parameters were also evaluated. The ASNase immobilization onto f-MWCNTs offered higher loading capacities, enhanced reusability, and improved enzyme affinity to the substrate, attaining RRA and IY of 100 and 99%, respectively, at the best immobilization conditions (0.4 mg/mL of ASNase, pH 8, 30 min of contact time). In addition, MWCNTs diameter proved to play a critical role in determining the enzyme binding affinity, as evidenced by the best results attained with f-MWCNTs with diameters of 10–20 nm and 20–40 nm. This study provided essential information on the impact of MWCNTs diameter and their surface functionalization on ASNase efficiency, which may be helpful for the development of innovative biomedical devices or food pre-treatment solutionspublishe

Immobilization of L-asparaginase towards surface-modified carbon nanotubes

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes L-asparagine hydrolysis into L-aspartic acid and ammonia and is mainly applied in pharmaceutical and food industries [1]. The ASNase currently commercialized for pharmaceutical purposes is produced from two main bacterial sources: recombinant Escherichia coli and Erwinia chrysanthemi. However, some disadvantages are associated with its free form, such as the shorter half-life [2]. Immobilization of ASNase has been proposed as an efficient approach to overcome this limitation [3]. In this work, a straightforward method, including the functionalization of multi-walled carbon nanotubes (MWCNTs) through a hydrothermal oxidation treatment with nitric acid, and the immobilization of ASNase by adsorption over pristine and modified MWCNTs was investigated. Different operation conditions, including pH, contact time, ASNase/MWCNT mass ratio, and the operational stability of the immobilized ASNase were evaluated. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95%, under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5´10–3 g.mL-1of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), proving its suitability for application in industrial processes.publishe

Immobilization of L-asparaginase towards surface-modified carbon nanotubes

L-asparaginase (LA) is an enzyme that catalyzes L-asparagine hydrolysis into L-aspartic acid and ammonia and is mainly applied in pharmaceutical and food industries. The LA currently commercialized for pharmaceutical purposes is produced from two main bacterial sources: recombinant Escherichia coli and Erwinia chrysanthemi. However, some disadvantages are associated with its free form, such as the shorter half-life. Immobilization of LA has been proposed as an efficient approach to overcome this limitation. In this work, a straightforward method, including the functionalization of multi-walled carbon nanotubes (MWCNTs) through a hydrothermal oxidation treatment and the immobilization of LA by adsorption over pristine and modified MWCNTs was investigated. Different operation conditions, including pH, contact time, ASNase/MWCNT mass ratio, and the operational stability of the immobilized LA, were evaluated. The characterization of the LA-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric analysis (TGA), and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial LA above 95%, under the optimized adsorption conditions (pH 8, 60 min of contact, and 1.510–3 g.mL-1 of LA). The LA-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), proving its suitability for application in industrial processes.publishe

Superior operational stability of immobilized L-asparaginase over surface-modified carbon nanotubes

L-asparaginase (ASNase, EC 3.5.1.1) is an enzyme that catalyzes the L-asparagine hydrolysis into L-aspartic acid and ammonia, being mainly applied in pharmaceutical and food industries. However, some disadvantages are associated with its free form, such as the ASNase short half-life, which may be overcome by enzyme immobilization. In this work, the immobilization of ASNase by adsorption over pristine and modified multi-walled carbon nanotubes (MWCNTs) was investigated, the latter corresponding to functionalized MWCNTs through a hydrothermal oxidation treatment. Different operating conditions, including pH, contact time and ASNase/MWCNT mass ratio, as well as the operational stability of the immobilized ASNase, were evaluated. For comparison purposes, data regarding the ASNase immobilization with pristine MWCNT was detailed. The characterization of the ASNase-MWCNT bioconjugate was addressed using different techniques, namely Transmission Electron Microscopy (TEM), Thermogravimetric Analysis (TGA) and Raman spectroscopy. Functionalized MWCNTs showed promising results, with an immobilization yield and a relative recovered activity of commercial ASNase above 95% under the optimized adsorption conditions (pH 8, 60 min of contact and 1.5 × 10-3 g mL-1 of ASNase). The ASNase-MWCNT bioconjugate also showed improved enzyme operational stability (6 consecutive reaction cycles without activity loss), paving the way for its use in industrial processes.publishe