Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30-40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.publishe

Building better biobetters: from fundamentals to industrial application

Biological drugs or biopharmaceuticals off patent open a large market for biosimilars and biobetters, follow-on biologics. Biobetters, in particular, are new drugs designed from existing ones with improved properties such as higher selectivity, stability, half-life and/or lower toxicity/immunogenicity. Glycosylation is one of the most used strategies to improve biological drugs, nonetheless bioconjugation is an additional alternative and refers to the covalent attachment of polymers to biological drugs. Extensive research on novel polymers is underway, nonetheless PEGylation is still the best alternative with the longest clinical track record. Innovative trends based on genetic engineering techniques such as fusion proteins and PASylation are also promising. In this review, all these alternatives wereexplored as well as current market trends, legislation and future perspectives.publishe

Potentiation of combined p19Arf and interferon-beta cancer gene therapy through its association with doxorubicin chemotherapy

Balancing safety and efficacy is a major consideration for cancer treatments, especially when combining cancer immunotherapy with other treatment modalities such as chemotherapy. Approaches that induce immunogenic cell death (ICD) are expected to eliminate cancer cells by direct cell killing as well as activation of an antitumor immune response. We have developed a gene therapy approach based on p19Arf and interferon-β gene transfer that, similar to conventional inducers of ICD, results in the release of DAMPS and immune activation. Here, aiming to potentiate this response, we explore whether association between our approach and treatment with doxorubicin (Dox), a known inducer of ICD, could further potentiate treatment efficacy without inducing cardiotoxicity, a critical side effect of Dox. Using central composite rotational design analysis, we show that cooperation between gene transfer and chemotherapy killed MCA205 and B16F10 cells and permitted the application of reduced viral and drug doses. The treatments also cooperated to induce elevated levels of ICD markers in MCA205, which correlated with improved efficacy of immunotherapy in vivo. Treatment of subcutaneous MCA205 tumors associating gene transfer and low dose (10 mg/kg) chemotherapy resulted in inhibition of tumor progression. Moreover, the reduced dose did not cause cardiotoxicity as compared to the therapeutic dose of Dox (20 mg/kg). The association of p19Arf/interferon-β gene transfer and Dox chemotherapy potentiated antitumor response and minimized cardiotoxicity

Bioconversion of α-chitin into N-acetyl-glucosamine using chitinases produced by marine-derived Aeromonas caviae isolates

11 páginas.-- 5 figuras.-- 3 tablas.-- 57 referenciasbstract: N-Acetyl-d-glucosamine (GlcNAc) is a monosaccharide with great application potential in the food, cosmetic, pharmaceutical, and biomaterial areas. GlcNAc is currently produced by chemical hydrolysis of chitin, but the current processes are environmentally unfriendly, have low yield and high cost. This study demonstrates the potential to produce GlcNAc from α-chitin using chitinases of ten marine-derived Aeromonas isolates as a sustainable alternative to the current chemical process. The isolates were characterized as Aeromonas caviae by multilocus sequence analysis (MLSA) using six housekeeping genes (gltA, groL, gyrB, metG, ppsA, and recA), not presented the virulence genes verified (alt, act, ast, ahh1, aer, aerA, hlyA, ascV and ascFG), but showed hemolytic activity on blood agar. GlcNAc was produced at 37 °C, pH 5.0, 2% (w/v) colloidal chitin and crude chitinase extracts (0.5 U mL−1) by all the isolates with yields from 14 to 85% at 6 h, 17–89% at 12 h and 19–93% after 24 h. The highest yield of GlcNAc was observed by A. caviae CH129 (93%). This study demonstrates one of the most efficient chitin enzymatic hydrolysis procedures and A. caviae isolates with great potential for chitinases expression and GlcNAc production. Graphical abstract: [Figure not available: see fulltext.].The authors also thank the financial support provided from Sao Paulo Research Foundation (FAPESP), Grants 2012/16824-0 and 2013/18773-6.Peer reviewe

Development and characterization of miltefosine-loaded polymeric micelles for cancer treatment

Miltefosine presents antineoplastic activity but high hemolytic potential. Its use in cancer has been limited to treating cutaneous metastasis of breast cancer. To decrease hemolytic potential, we developed a formulation of miltefosine-loaded polymeric micelles (PM) of the copolymer Pluronic-F127. A central composite design was applied and the analysis of variance showed that the optimum level of hydrodynamic diameter and polydispersity index predicted by the model and experimentally confirmed were 29 nm and 0.105, respectively. Thermal analyses confirmed that miltefosine was molecularly dispersed within PM. Pluronic-F127 PM with miltefosine 80 μM presented a significant reduction of hemolytic effect (80%, p < 0.05) in comparison to free drug. In vitro assays against HeLa carcinoma cells demonstrated similar cytotoxicity to free miltefosine and PM. Our results suggest that, by lowering hemolytic potential, miltefosine-loaded Pluronic-F127 PM a promising alternative to broaden this drug use in cancer therapy, as well as of other alkylphosphocholines.Fil: Valenzuela Oses, Johanna K.. Universidade de Sao Paulo; BrasilFil: García, Mónica Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; ArgentinaFil: Feitosa, Valker A.. Universidade de Sao Paulo; Brasil. Institute for Technological Research; BrasilFil: Pachioni Vasconcelos, Juliana A.. Universidade de Sao Paulo; BrasilFil: Gomes-Filho, Sandro M.. Universidade de Sao Paulo; BrasilFil: Lourenço, Felipe R.. Universidade de Sao Paulo; BrasilFil: Cerize, Natalia N.P.. Institute for Technological Research; BrasilFil: Bassères, Daniela S.. Universidade de Sao Paulo; BrasilFil: Rangel-Yagui, Carlota O.. Universidade de Sao Paulo; Brasi

Biomoléculas extraídas por SBAs: ejemplos prácticos

The actual biotechnology industry demands fast and economic upstream and downstream processes to purify biomolecules. In this context, different purification techniques, that offer both high recovery and purity to the final product, have been assayed by different research groups. Liquid-liquid extraction with aqueous two-phase systems is one of the most studied methodologies for bio-separation. This technique presents several advantages such as mild conditions of working, cost-effectiveness, short-time consumption and high recovery percentage of the final product. With the aim to present a comparison of liquid-liquid extractions with other techniques, several aqueous two-phase extraction processes of biomolecules are presented in this review. We presented the advantages and disadvantages of them as of the compared systems. In general, the highest final product purities are achieved when different methodologies are combine, being the chromatographic ones the most applied in the last stages for the high purification factor obtained after them. Alternative methodologies, such as aqueous two-phase systems (ATPS), i.e., PEG/salts or ionic liquids; aqueous two-phase micellar systems, using solvents and surfactants; and extractive fermentation with ATPS, are relevant for both cost-effectiveness and time-saving of the purification process.La industria biotecnologica actual exige procesos rápidos y económicos para la producción y la purificación de biomoléculas. En este contexto, diferentes técnicas separativas que ofrezcan un alto rendimiento y una alta pureza del producto final han sido evaluadas por diferentes grupos de investigación. La extracción líquido-líquido con sistemas bifasicos acuosos (SBAs) es una de las metodologías más estudiadas para bio-separación. Esta técnica presenta varias ventajas, tales como condiciones suaves de trabajo, alta relación costo-beneficio, cortos tiempos de consumo y alto porcentaje de recuperación del producto final. Con el objetivo de comparar extracciones líquido-líquido con otras técnicas de separación, diferentes procesos de extracción de biomoléculas seran presentados en este trabajo de revisión. Adicionalmente, se presentaran las ventajas y desventajas de cada uno de ellos. En general, los más altos grados de pureza del producto final fueron obtenidos al combinar diferentes metodologías, siendo las cromatográficas las más aplicados en las últimas etapas debido a los elevados factores de purificación obtenidos después de ellas. Metodologías alternativas como por ejemplo, SBAs basados en mezclas de PEG/sal o liquídos iónicos/sal, sistemas micelares de dos fases acuosas (SMDFAs) formados por solventes o surfactantes; fermentación extractiva utilizando SBA, también resultan ser relevantes debido a sus bajos costos y cortos tiempo en sus procesos de purificación

Biomolecules extracted by ATPs: practical examples

The actual biotechnology industry demands fast and economic upstream and downstream processes to purify biomolecules. In this context, different purification techniques, that offer both high recovery and purity to the final product, have been assayed by different research groups. Liquid-liquid extraction with aqueous two-phase systems is one of the most studied methodologies for bio-separation. This technique presents several advantages such as mild conditions of working, cost-effectiveness, short-time consumption and high recovery percentage of the final product. With the aim to present a comparison of liquid-liquid extractions with other techniques, several aqueous two-phase extraction processes of biomolecules are presented in this review. We presented the advantages and disadvantages of them as of the compared systems. In general, the highest final product purities are achieved when different methodologies are combine, being the chromatographic ones the most applied in the last stages for the high purification factor obtained after them. Alternative methodologies, such as aqueous two-phase systems (ATPS), i.e., PEG/salts or ionic liquids; aqueous two-phase micellar systems, using solvents and surfactants; and extractive fermentation with ATPS, are relevant for both cost-effectiveness and time-saving of the purification process.La industria biotecnologica actual exige procesos rápidos y económicos para la producción y la purificación de biomoléculas. En este contexto, diferentes técnicas separativas que ofrezcan un alto rendimiento y una alta pureza del producto final han sido evaluadas por diferentes grupos de investigación. La extracción líquido-líquido con sistemas bifasicos acuosos (SBAs) es una de las metodologías más estudiadas para bio-separación. Esta técnica presenta varias ventajas, tales como condiciones suaves de trabajo, alta relación costo-beneficio, cortos tiempos de consumo y alto porcentaje de recuperación del producto final. Con el objetivo de comparar extracciones líquido-líquido con otras técnicas de separación, diferentes procesos de extracción de biomoléculas seran presentados en este trabajo de revisión. Adicionalmente, se presentaran las ventajas y desventajas de cada uno de ellos. En general, los más altos grados de pureza del producto final fueron obtenidos al combinar diferentes metodologías, siendo las cromatográficas las más aplicados en las últimas etapas debido a los elevados factores de purificación obtenidos después de ellas. Metodologías alternativas como por ejemplo, SBAs basados en mezclas de PEG/sal o liquídos iónicos/sal, sistemas micelares de dos fases acuosas (SMDFAs) formados por solventes o surfactantes; fermentación extractiva utilizando SBA, también resultan ser relevantes debido a sus bajos costos y cortos tiempo en sus procesos de purificación.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq