Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formul...

Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs.

Therapeutic messenger RNA vaccines enable delivery of whole antigens, which can be advantageous over peptide vaccines. However, optimal efficacy requires both intracellular delivery, to allow antigen translation, and appropriate immune activation. Here, we developed a combinatorial library of ionizable lipid-like materials to identify mRNA delivery vehicles that facilitate mRNA delivery in vivo and provide potent and specific immune activation. Using a three-dimensional multi-component reaction system, we synthesized and evaluated the vaccine potential of over 1,000 lipid formulations. The top candidate formulations induced a robust immune response, and were able to inhibit tumor growth and prolong survival in melanoma and human papillomavirus E7 in vivo tumor models. The top-performing lipids share a common structure: an unsaturated lipid tail, a dihydroimidazole linker and cyclic amine head groups. These formulations induce antigen-presenting cell maturation via the intracellular stimulator of interferon genes (STING) pathway, rather than through Toll-like receptors, and result in limited systemic cytokine expression and enhanced anti-tumor efficacy.

Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation.

This review describes recent research developments on biological conversion of hemicellulosic biomass towards production of xylitol by taking advantage of power of biotechnology. Xylitol is a five-carbon sugar alcohol with established commercial uses in different healthcare sectors and especially as an alternative sweetener for diabetic persons. Xylitol can be synthesized either by chemical hydrogenation of xylose or by fermentation. The precursor xylose is produced from biomass by chemical or enzymatic hydrolysis and can be converted to xylitol primarily by yeast strains which offer the possibilities of economic production by reducing required energy when compared to chemical production. Biomass hydrolysis under an acidic environment is the most commonly used practice and is influenced by various process parameters. Several microbial growth inhibitors are produced during chemical hydrolysis that reduce xylitol production from xylose, a detoxification step is therefore essential. Enzymatic hydrolysis has advantages over chemical conversion although more research is necessary to reduce inhibition due to structural variation from different substrates or plant species. Enzymatic xylitol production is mostly an integral process of microbial species belonging to the Candida genus. Extensive research has been performed to screen for xylitol producing microbial strains as well as to understand microbial metabolism, the xylitol metabolic pathway, cofactor requirements, development of robust recombinant strains, optimization of bioconversion parameters and xylitol production strategies using free and immobilized cells. The imperative role of hydrolysis of xylose containing biomass and subsequent process parameters has major impact on economis of bioconversion. The review identifies ways forward for improved enzymatic xylitol production to compete with current chemical processes.

Current trends in biotechnological production of xylitol and future prospects

Conventional, ultrasound-assisted, and accelerated-solvent extractions of anthocyanins from purple sweet potatoes

L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

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Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Success in experiments and/or technology mainly depends on a properly designed process or product. The traditional method of process optimization involves the study of one variable at a time, which requires a number of combinations of experiments that are time, cost and labor intensive. The Taguchi method of design of experiments is a simple statistical tool involving a system of tabulated designs (arrays) that allows a maximum number of main effects to be estimated in an unbiased (orthogonal) fashion with a minimum number of experimental runs. It has been applied to predict the significant contribution of the design variable(s) and the optimum combination of each variable by conducting experiments on a real-time basis. The modeling that is performed essentially relates signal-to-noise ratio to the control variables in a 'main effect only' approach. This approach enables both multiple response and dynamic problems to be studied by handling noise factors. Taguchi principles and concepts have made extensive contributions to industry by bringing focused awareness to robustness, noise and quality. This methodology has been widely applied in many industrial sectors; however, its application in biological sciences has been limited. In the present review, the application and comparison of the Taguchi methodology has been emphasized with specific case studies in the field of biotechnology, particularly in diverse areas like fermentation, food processing, molecular biology, wastewater treatment and bioremediation.

Rao Rs

Papers

The Taguchi methodology as a statistical tool for biotechnological applications: A critical appraisal