Challenges in estimating the encapsulation efficiency of proteins in polymersomes - Which is the best method?

Abstract Polymersomes are nanometric vesicles that can encapsulate large and hydrophilic biomolecules, such as proteins, in the aqueous core. Data in literature show large variation in encapsulation efficiency (%EE) values depending on the method used for calculation. We investigated different approaches (direct and indirect) to quantify the %EE of different proteins (catalase, bovine serum albumin-BSA, L-asparaginase and lysozyme) in Pluronic L-121 polymersomes. Direct methods allow quantification of the actual payload of the polymersomes and indirect methods are based on the quantification of the remaining non-encapsulated protein. The protein-loaded polymersomes produced presented approximately 152 nm of diameter (PDI ~ 0.4). Higher %EE values were obtained with the indirect method (up to 25%), attributed to partial entanglement of free protein in the polymersomes poly(Ethylene Glycol) corona. For the direct methods, vesicles were disrupted with chloroform or proteins precipitated with solvents. Reasonable agreement was found between the two protocols, with values up to 8%, 6%, 17.6% and 0.9% for catalase, BSA, L-asparaginase and lysozyme, respectively. We believe direct determination is the best alternative to quantify the %EE and the combination of both protocols would make results more reliable. Finally, no clear correlation was observed between protein size and encapsulation efficiency.

Biopharmaceuticals from microorganisms: from production to purification

ABSTRACT The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

Biopharmaceuticals from microorganisms: from production to purification

ABSTRACT The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN , , and ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

Polymeric micelles and molecular modeling applied to the development of radiopharmaceuticals

Micelles composed of amphiphilic copolymers linked to a radioactive element are used in nuclear medicine predominantly as a diagnostic application. A relevant advantage of polymeric micelles in aqueous solution is their resulting particle size, which can vary from 10 to 100 nm in diameter. In this review, polymeric micelles labeled with radioisotopes including technetium (99mTc) and indium (111In), and their clinical applications for several diagnostic techniques, such as single photon emission computed tomography (SPECT), gamma-scintigraphy, and nuclear magnetic resonance (NMR), were discussed. Also, micelle use primarily for the diagnosis of lymphatic ducts and sentinel lymph nodes received special attention. Notably, the employment of these diagnostic techniques can be considered a significant tool for functionally exploring body systems as well as investigating molecular pathways involved in the disease process. The use of molecular modeling methodologies and computer-aided drug design strategies can also yield valuable information for the rational design and development of novel radiopharmaceuticals.

Micelas poliméricas compostas de copolímeros ligadas a um elemento radioativo são utilizadas em Medicina Nuclear com aplicação predominantemente diagnóstica. A vantagem relevante da utilização de micelas poliméricas em solução aquosa é o tamanho de suas partículas, as quais podem variar de 10 a 100 nm de diâmetro. Neste trabalho de revisão são apresentadas micelas poliméricas marcadas com radioisotopos, como tecnécio-99m (99mTc) e índio-111 (111In), assim como suas aplicações clínicas em técnicas de diagnóstico como Tomografia por emissão de Fóton Único (Single photon Emission Computed Tomography - SPECT), cintilografia, e Ressonância Magnética Nuclear (RMN). Neste contexto, sua aplicação em diagnóstico de sistema linfático e linfonodo sentinela recebe atenção especial. O emprego de técnicas de diagnóstico pode ser considerado uma ferramenta importante para a exploração de sistemas no organismo humano assim como para a investigação de caminhos moleculares envolvidos nos processos de diversas doenças. O uso de metodologias de modelagem molecular e estratégias de desenvolvimento de fármacos assistidas computacionalmente também pode fornecer informações valiosas para o planejamento e o desenvolvimento racional de novos radiofármacos.

A 2D-QSPR approach to predict blood-brain barrier penetration of drugs acting on the central nervous system

Drugs acting on the central nervous system (CNS) have to cross the blood-brain barrier (BBB) in order to perform their pharmacological actions. Passive BBB diffusion can be partially expressed by the blood/brain partition coefficient (logBB). As the experimental evaluation of logBB is time and cost consuming, theoretical methods such as quantitative structure-property relationships (QSPR) can be useful to predict logBB values. In this study, a 2D-QSPR approach was applied to a set of 28 drugs acting on the CNS, using the logBB property as biological data. The best QSPR model [n = 21, r = 0.94 (r² = 0.88), s = 0.28, and Q² = 0.82] presented three molecular descriptors: calculated n-octanol/water partition coefficient (ClogP), polar surface area (PSA), and polarizability (α). Six out of the seven compounds from the test set were well predicted, which corresponds to good external predictability (85.7%). These findings can be helpful to guide future approaches regarding those molecular descriptors which must be considered for estimating the logBB property, and also for predicting the BBB crossing ability for molecules structurally related to the investigated set.

Fármacos que atuam no sistema nervoso central (SNC) devem atravessar a barreira hematoencefálica (BHE) para exercerem suas ações farmacológicas. A difusão passiva através da BHE pode ser parcialmente expressa pelo coeficiente de partição entre os compartimentos encefálico e sanguíneo (logBB, brain/blood partition coefficient). Considerando-se que a avaliação experimental de logBB é dispendiosa e demorada, métodos teóricos como estudos das relações entre estrutura química e propriedade (QSPR, Quantitative Structure-Property Relationships) podem ser utilizados na previsão dos valores de logBB. Neste estudo, uma abordagem de QSPR-2D foi aplicada a um conjunto de 28 moléculas com ação central, usando logBB como propriedade biológica. O melhor modelo de QSPR [n = 21, r = 0,94 (r² = 0,88), s = 0,28 e Q² = 0,82] apresentou três descritores moleculares: o coeficiente calculado de partição n-octanol/água (ClogP), área de superfície polar (PSA) e polarizabilidade (α). Seis dos sete compostos do conjunto de avaliação foram bem previstos pelo modelo, o que corresponde a um bom poder de previsão externa (85,7%). Os resultados obtidos podem auxiliar de forma relevante em estudos futuros, orientando quais descritores moleculares devem ser considerados para estimar logBB e prever a passagem através da BHE de moléculas estruturalmente relacionadas às do conjunto investigado.

Sistemas micelares de duas fases aquosas aplicados à solubilização de fármacos / Two-phase aqueous micellar systems applied to the purification of enzymes

A partição das enzimas glicose-6-fosfato desidrogenase (G6PD) e uroquinase (UK) em sistemas micelares de duas fases aquosas foi investigada, teórica e experimentalmente. Inicialmente, a partição de G6PD em sistema micelar de duas fases aquosas composto pelo tensoativo não-iônico `C IND. 10'`E IND. 4' (óxido de n-deciltetraetileno) foi estudada. Observou-se uma partição governada primariamente por interações repulsivas estéricas, do tipo "volume de exclusão", sendo a G6PD recuperada preferencialmente na fase inferior do sistema, pobre em micelas, resultando em valores de `K IND. G6PDï inferiores a 1,0. Os tensoativos catiônicos `C IND. nïTAB (brometos de alquiltrimetilamonio, n = 8, 10 e 12) foram adicionados ao tensoativo `C IND. 10ï`E IND. 4ï, de modo a formar sistemas micelares mistos (não-iônico/catiônico) de duas fases aquosas e atrair a G6PD de carga efetiva negativa para a fase superior, rica em micelas positivamente carregadas...