Nanoarmored Multi-Enzyme Cascade Catalysis.

This chapter reports a single-step preparation of nanoarmored bi-enzyme systems assembled on 1-D and 2-D nanomaterials, with glucose oxidase and peroxidase enzymes as model systems for cascade bio-catalysis. This is a simple and facile method to both exfoliate the bulk 1D (carbon nanotubes, CNT) and 2D nanomaterials (α-Zirconium phosphate, α-ZrP) and bind the enzymes in a single step. Exfoliation of the bulk material enhances the accessible surface area of the materials for the enzyme binding, and it also boosts the diffusion of reagents from the bulk phase to the active sites of the bio-catalysts. For example, a mixture of horseradish peroxidase, glucose oxidase, and bovine serum albumin (BSA) were adsorbed on the surfaces of the α-ZrP nanoplates or carbon nanotubes (CNT) as the bulk materials are exfoliated simultaneously, in a one-step process. The resulting bio-catalysts were thoroughly characterized by powder X-ray diffraction, electron microscopy, biochemical and biophysical methods, while enzyme activity studies proved successful binding of enzymes with retention of activities or even enhancements in their specific activities. For example, GOx/HRP/BSA/CNT displayed 6 times the activity of a mixture of GOx/HRP/BSA, under otherwise identical conditions. Similarly, GOx/HRP/BSA/ZrP had 3.5 times the activity of the corresponding mixture of GOx/HRP/BSA, in the absence of the nanoplates. These robust nano-dispersions worked extraordinarily well as active bio-catalysts. These two kinds of fabricated biocatalyst dispersions are also highly stable.

One-step preparation of bioactive enzyme/inorganic materials.

Simultaneous exfoliation of crystalline α-zirconium phosphate (α-ZrP) nanosheets and enzyme binding, induced by shearing, without the addition of any toxic additives is reported here for the first time. These materials were thoroughly characterized and used for applications. The bulk α-ZrP material (20 mg mL-1) was exfoliated with low concentrations of a protein such as bovine serum albumin (BSA, 3 mg mL-1) in a shear reactor at 10k rpm for <80 minutes. Exfoliation was monitored by powder X-ray diffraction with samples displaying a gradual but complete loss of the 7.6 Å (002) peak, which is characteristic of bulk α-ZrP. The fully exfoliated sample loaded with the protein was characterized by transmission and scanning electron microscopy in addition to other biophysical methods. Lysozyme, glucose oxidase, met-hemoglobin, and ovalbumin also induced exfoliation and directly produced enzyme/ZrP biocatalysts. Thus, exfoliation, biophilization and enzyme binding are accomplished in a single step. Several factors contributed to the exfoliation kinetics, and the rate increased with α-ZrP and BSA concentrations and decreased with pH. However, the exfoliation efficiency inversely depended on the isoelectric point of the protein with ovalbumin (pI = 4.5) being the best and lysozyme (pI = 11.1) being the worst. A strong correlation between the protein size and exfoliation efficiency was noted, and the latter suggests the role of hydrodynamic factors in the process. Exfoliation was also achieved by simple stirring using a magnetic stirrer, under low volumes, and model enzymes, indicating 60-90% retention of bound enzymatic activities. The addition of BSA to enzymes as the diluent and stabilizing agent also prevents enzymes from the denaturing effect caused by stirring. This new method requires no pre-treatment of α-ZrP with toxic exfoliating agents such as tetrabutyl ammonium hydroxide and provides bioactive enzyme/inorganic materials in a single step. These protein-loaded biocompatible nanosheets may be useful for biocatalysis and biomedical applications.

Exfoliated and water dispersible biocarbon nanotubes for enzymology applications.

In this chapter, we report a simple and facile method to armor enzymes with carbon nanotubes (CNTs) which are exfoliated, and debundled using bovine serum albumin (BSA). The fabricated CNT/BSA dispersions are biofriendly, biocompatible, defect-free, and highly stable solutions. BSA gives maximum exfoliation efficiency, exceeding the 4mg/mL of CNT concentration compared to any previous reports. Further, the produced bioCNT dispersions were characterized by UV-visible, Raman, circular dichroism spectroscopy, and scanning electron microscopy (SEM). Exfoliation and debundling of the bioCNT dispersions is possible due to the π-π interaction, hydrogen bonding, hydrophobic interaction, and electrostatic attractive forces driving the adsorption of BSA on CNTs surface. Protein adsorption then makes a highly stable suspension in water that can be stored for a prolonged period. CNT dispersions are stable over a wide range of pH from 3 to 10 and at 4°C or 25°C for more than 2 months. Here, we also report the facile, inexpensive and green-chemistry method to fabricate a buckypaper (CNT paper), composed of the high packing density, self-assembled and randomly oriented bioCNTs, and these assemblies could be used in many emerging applications like air and water purification, nanocomposites, energy storage, and biosensing. Moreover, the CNT dispersions stabilized by BSA were successfully used in enzyme binding and kinetic studies and bound enzyme retained substantial catalytic activity. The current approach may facilitate bulk production of water dispersed CNTs in both academic and industrial laboratories. This is done by a simple method of stirring, which provides new opportunities for a wider range of CNT applications.

Engineering functional inorganic nanobiomaterials: controlling interactions between 2D-nanosheets and enzymes.

A better understanding of the enzyme-nanosheet interface is imperative for the design of functional, robust inorganic nanobiomaterials and biodevices, now more than ever, for use in a broad spectrum of applications. This feature article discusses recent advances in controlling the enzyme-nanosheet interface with regards to α-zirconium(iv) phosphate (α-ZrP), graphene oxide (GO), graphene, and MoS2 nanosheets. Specific focus will be placed on understanding the mechanisms with which these materials interact with enzymes and elaborate on particular ways to engineer and control these interactions. Our main discoveries include: (1) upon adsorption to the nanosheet surface, a decrease in the entropy of the enzyme's denatured state enhances stability; (2) proteins are used to create biophilic landing pads for increased enzyme stability on many different types of nanosheets; (3) proteins and enzymes are used as exfoliants by shear force to produce biofunctionalized nanosheet suspensions; and (4) bionfunctionalized nanosheets exhibit no acute toxicity. Recognizing proper methods to engineer the interface between enzymes and 2D-nanosheets, therefore, is an important step towards making green, sustainable, and environmentally conscious inorganic bionanomaterials for sensing, catalysis and drug delivery applications, as well as towards the successful manipulation of enzymes for advanced applications.

Real-world outcomes and management strategies for venetoclax-treated chronic lymphocytic leukemia patients in the United States.

Venetoclax is a BCL2 inhibitor approved for 17p-deleted relapsed/refractory chronic lymphocytic leukemia with activity following kinase inhibitors. We conducted a multicenter retrospective cohort analysis of patients with chronic lymphocytic leukemia treated with venetoclax to describe outcomes, toxicities, and treatment selection following venetoclax discontinuation. A total of 141 chronic lymphocytic leukemia patients were included (98% relapsed/refractory). Median age at venetoclax initiation was 67 years (range 37-91), median prior therapies was 3 (0-11), 81% unmutated IGHV, 45% del(17p), and 26.8% complex karyotype (≥ 3 abnormalities). Prior to venetoclax initiation, 89% received a B-cell receptor antagonist. For tumor lysis syndrome prophylaxis, 93% received allopurinol, 92% normal saline, and 45% rasburicase. Dose escalation to the maximum recommended dose of 400 mg daily was achieved in 85% of patients. Adverse events of interest included neutropenia in 47.4%, thrombocytopenia in 36%, tumor lysis syndrome in 13.4%, neutropenic fever in 11.6%, and diarrhea in 7.3%. The overall response rate to venetoclax was 72% (19.4% complete remission). With a median follow up of 7 months, median progression free survival and overall survival for the entire cohort have not been reached. To date, 41 venetoclax treated patients have discontinued therapy and 24 have received a subsequent therapy, most commonly ibrutinib. In the largest clinical experience of venetoclax-treated chronic lymphocytic leukemia patients, the majority successfully completed and maintained a maximum recommended dose. Response rates and duration of response appear comparable to clinical trial data. Venetoclax was active in patients with mutations known to confer ibrutinib resistance. Optimal sequencing of newer chronic lymphocytic leukemia therapies requires further study.