Solubilização micelar do ibuprofeno: influência do grupo polar dos tensoativos no grau de solubilização

An important property of micelles with particular significance in pharmacy is their ability to increase the solubility of poorly soluble drugs in water, thus increasing their bioavailability. In this work, the solubilization of ibuprofen (IBU) was studied in micellar solutions of three surfactants possessing the same hydrocarbon tail but different hydrophilic head groups, namely sodium dodecyl sulphate (SDS), dodecyltrimethylammonium bromide (DTAB), and n-dodecyl octa(ethylene oxide) (C12EO8). The results showed that, irrespective of the surfactant type, the solubility of IBU increased linearly with increasing surfactant concentration, as a consequence of the association between the drug and the micelles. The 80 mM DTAB and the 80 mM C12EO8 micellar solutions resulted in a 16-fold increase in solubility of IBU when compared to the buffer solution, whereas the 80 mM SDS micellar solution resulted in a 5.5-fold increase in IBU solubility. The highest value of molar solubilization capacity (chi) was obtained with DTAB, chi = 0.97, followed by C12EO8 ,chi = 0.72, and finally SDS, chi = 0.23. However, due to the stronger tendency of the nonionic surfactant in forming micelles in solution, at the same surfactant concentration, we obtained the same solubility of IBU in both DTAB and C12EO8.Uma propriedade importante das micelas, do ponto de vista farmacêutico, refere-se ao potencial destas em solubilizar fármacos pouco solúveis em água, aumentando sua biodisponibilidade. No presente trabalho, estudou-se a solubilização de ibuprofeno (IBU) em soluções micelares constituídas de três tensoativos apresentando a mesma cauda apolar, porém diferentes grupos hidrofílicos. Os tensoativos estudados foram dodecil sulfato de sódio (SDS), brometo de dodeciltrimetilamônio (DTAB) e óxido de n-dodecil octaetileno (C12EO8). De acordo com os resultados obtidos, a solubilidade do IBU aumentou linearmente com o aumento da concentração de todos os tensoativos estudados, devido às interações entre as micelas e o fármaco. O fármaco IBU apresentou um aumento de 16 vezes em sua solubilidade na presença de DTAB 80 mM e de C12EO8 80 mM. Por outro lado, na presença de SDS 80 mM a solubilidade do IBU aumentou apenas 5,5 vezes. O maior valor para o parâmetro capacidade molar de solubilização (chi) foi observado com o tensoativo DTAB, chi = 0,97, seguido pelo C12EO8, chi = 0,72 e, finalmente, o SDS, chi = 0,23. Entretanto, devido à grande tendência do C12EO8 em formar micelas, o perfil de solubilidade do IBU foi semelhante em DTAB e C12EO8

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

Can affinity interactions influence the partitioning of glucose-6-phosphate dehydrogenase in two-phase aqueous micellar systems?

In this work, we provide an investigation of the role and strength of affinity interactions on the partitioning of the glucose-6-phosphate dehydrogenase in aqueous two-phase micellar systems. These systems are constituted of micellar surfactant solutions and offer both hydrophobic and hydrophilic environments, providing selectivity to biomolecules. We studied G6PD partitioning in systems composed of the nonionic surfactants, separately, in the presence and absence of affinity ligands. We observed that G6PD partitions to the micelle-poor phase, owing to the strength of excluded-volume interactions in these systems that drive the protein to the micelle-poor phase, where there is more free volume available

Micellar solubilization of ibuprofen: influence of surfactant head groups on the extent of solubilization Solubilização micelar do ibuprofeno: influência do grupo polar dos tensoativos no grau de solubilização

An important property of micelles with particular significance in pharmacy is their ability to increase the solubility of poorly soluble drugs in water, thus increasing their bioavailability. In this work, the solubilization of ibuprofen (IBU) was studied in micellar solutions of three surfactants possessing the same hydrocarbon tail but different hydrophilic head groups, namely sodium dodecyl sulphate (SDS), dodecyltrimethylammonium bromide (DTAB), and n-dodecyl octa(ethylene oxide) (C12EO8). The results showed that, irrespective of the surfactant type, the solubility of IBU increased linearly with increasing surfactant concentration, as a consequence of the association between the drug and the micelles. The 80 mM DTAB and the 80 mM C12EO8 micellar solutions resulted in a 16-fold increase in solubility of IBU when compared to the buffer solution, whereas the 80 mM SDS micellar solution resulted in a 5.5-fold increase in IBU solubility. The highest value of molar solubilization capacity (chi) was obtained with DTAB, chi = 0.97, followed by C12EO8 ,chi = 0.72, and finally SDS, chi = 0.23. However, due to the stronger tendency of the nonionic surfactant in forming micelles in solution, at the same surfactant concentration, we obtained the same solubility of IBU in both DTAB and C12EO8.<br>Uma propriedade importante das micelas, do ponto de vista farmacêutico, refere-se ao potencial destas em solubilizar fármacos pouco solúveis em água, aumentando sua biodisponibilidade. No presente trabalho, estudou-se a solubilização de ibuprofeno (IBU) em soluções micelares constituídas de três tensoativos apresentando a mesma cauda apolar, porém diferentes grupos hidrofílicos. Os tensoativos estudados foram dodecil sulfato de sódio (SDS), brometo de dodeciltrimetilamônio (DTAB) e óxido de n-dodecil octaetileno (C12EO8). De acordo com os resultados obtidos, a solubilidade do IBU aumentou linearmente com o aumento da concentração de todos os tensoativos estudados, devido às interações entre as micelas e o fármaco. O fármaco IBU apresentou um aumento de 16 vezes em sua solubilidade na presença de DTAB 80 mM e de C12EO8 80 mM. Por outro lado, na presença de SDS 80 mM a solubilidade do IBU aumentou apenas 5,5 vezes. O maior valor para o parâmetro capacidade molar de solubilização (chi) foi observado com o tensoativo DTAB, chi = 0,97, seguido pelo C12EO8, chi = 0,72 e, finalmente, o SDS, chi = 0,23. Entretanto, devido à grande tendência do C12EO8 em formar micelas, o perfil de solubilidade do IBU foi semelhante em DTAB e C12EO8

Multistep purification of cytochrome c PEGylated forms using polymer-based aqueous biphasic systems

Chemical PEGylation of proteins has been used to improve their physicochemical properties and kinetics. However, the PEGylation reactions lead to a heterogeneous mixture of PEGylated conjugates and unreacted protein, which is a challenge for the design of an efficient downstream process. The purification of PEGylated proteins should address the two main issues: the separation of PEGylated conjugates from the unreacted protein and the fractionation of the PEGylated conjugates on the basis of their degree of PEGylation. The present study aims at the development of liquid-liquid extraction processes for the purification of PEGylated conjugates. An initial study of the partition behavior of cytochrome c and their PEGylated conjugates (Cyt-c-PEG-4 and Cyt-c-PEG-8) on polyethylene-glycol (PEG) + potassium phosphate buffer (pH = 7) aqueous biphasic systems (ABS) shows that PEGs with intermediate molecular weights (PEG MW = 1000-2000) allow the separation of the PEGylated conjugates from the unreacted protein in a single step. It is further shown that the PEGylated conjugates can be efficiently separated using ABS based on PEGs with high molecular weight (PEG MW = 6000-8000) and a study of the protein stability after purification was carried using circular dichroism. A downstream process to separate Cyt-c, Cyt-c-PEG-4 and Cyt-c-PEG-8 with high purities (96.5% Cyt-c, 85.8% Cyt-c-PEG-4, and 99.0% Cyt-c-PEG-8) was developed. The process proposed addresses not only the efficient separation of each of the protein forms but also the recycling of the unreacted protein purified and the ABS phases, which was successfully used in a new step of PEGylationpublishe

Imidazolium-based ionic liquids as adjuvants to form Polyethylene Glycol with Salt Buffer Aqueous Biphasic Systems

Aqueous biphasic systems (ABS) are biocompatible systems applied in the extraction of biomolecules. Despite the biocompatibility of polymers and, particularly polyethylene glycol (PEG), to form ABS, their limitation in terms of phase separation is recognized. A new approach was recently proposed based on the use of ionic liquids (ILs) as adjuvants in ABS, enlarging the polarity range of these systems. Up to now, the effects of ILs in PEG-salt ABS have been poorly described. To overcome this limitation, the phase diagrams of imidazolium-based ILs acting as adjuvants in ABS based in PEG with potassium salt buffers (pH = 7), that is potassium citrate (C6H5K3O7/C6H8O7) and potassium phosphate (K2HPO4/KH2PO4) buffers, are herein addressed. Imidazolium-based ILs were focused in this work, since they have been applied on the purification of several biomolecules with success, even as adjuvants or electrolytes. The phase diagrams were mapped out for PEG/salt ABS without adjuvants. In this work, systems composed of PEG (1000, 1500, 2000, 3350, 4000, 6000, and 8000) with potassium phosphate buffer and PEG (2000, 6000, 10 000, and 20 000) with potassium citrate buffer were tested. Moreover, the presence of 5 wt % of imidazolium-based ILs (varying the anion moiety) for the system PEG 1500 with potassium phosphate buffer was also investigated. Imidazolium-based ILs with different anions were tested to investigate a large range of polarities attributed to the adjuvant. Moreover, the effect of the adjuvant content (5, 10, and 20 wt %) in the PEG 2000 with potassium citrate buffer system was studied for two distinct ILs, namely [C4mim][CF3SO3] and [C4mim][(CH3O)2PO2], with lower and higher energy of intramolecular hydrogen bond, EHB, respectively, a parameter representing the ions' hydration. A correlation between the anion moiety of imidazolium-based IL and the ability to form two phases was observed, being this related to the ILs' anion EHB value. The concentration of the adjuvant confirmed the effects of enhancing or decreasing the ability to form two phases for ILs with lower and higher EHB value, respectively.publishe

Extraction of clavulanic acid using aqueous two-phase micellar system

An investigation of clavulanic acid behavior in an aqueous two-phase micellar system employing the surfactants n-decyltetraethylene oxide (C(10)E(4)) and dodecyldimethylamine oxide (DDAO) was carried out. According to the results, clavulanic acid partitions evenly between the two phases of DDAO micellar system, mixed DDAO C(10)E(4) micellar system, as well as C10E4 micellar system. Therefore, electrostatic interactions between positively charged DDAO-containing micelles and negatively charged drug were not strong enough to influence the partitioning. Nevertheless, clavulanic acid extraction from Streptomyces clavuligerus fermentation broth in C(10)E(4) micellar system employing a previous protein denaturation step provided recovery of 52% clavulanic acid with removal of 70% of the contaminant proteins, which is already promising as a purification strategy. (C) 2011 International Union of Biochemistry and Molecular Biology, Inc. Volume 58, Number 2, March/April 2011, Pages 103-108. E-mail: [email protected] Council for Scientific and Technological Development (CNPq-Brazil)State of Sao Paulo Research Support Foundation (Fapesp-Brazil

In situpurification of periplasmatic L-asparaginase by aqueous two phase systems with ionic liquids (ILs) as adjuvants

(ALL) and lymphosarcoma. Considering its main use in cancer therapy, the most important request for ASNase production is the need for a highly pure biopharmaceutical obtained in the final of the downstream process, which is considered as the crucial step in its production. RESULTS: This work proposes the use of polymer–salt aqueous two-phase systems (ATPS) based on polyethylene glycol and citrate buffer, with ionic liquids (ILs) as adjuvants, combined with the permeabilization of cell membrane using n-dodecane and glycine for the in situ purification of periplasmatic ASNase from Escherichia coli cells. The process proposed was optimized (polymer molecular weight, pH, tie-line length/mixture point, presence, nature and concentration of the adjuvant). The results show that ASNase partitions mostly to the PEG-rich phase, due to hydrophobic interactions between both PEG and enzyme. Remarkably, the addition of 5 wt% of 1-butyl-3-methylimidazolium methanesulfonate [C4mim][CH3SO3] as adjuvant lead to high recoveries [87.94 ± 0.03 (%)], purification factors (20.09 ± 0.35), and a final specific activity SA = 3.61 ± 0.38 U mg-1 protein, from a crude enzyme extract with a SA = 0.18 ± 0.05 U mg-1 protein. Moreover, better results were achieved when a prepurification step consisting of an ammonium sulfate precipitation was combined with the optimized ATPS, achieving an increased SA = 22.01 ± 1.36 U mg-1 protein and PF = 173.8. CONCLUSIONS: A novel integrated downstream process was successfully implemented for the in situ purification of ASNase from fermentation broth.publishe