Validation of a UV-spectrophotometric analytical method for determination of LPSF/AC04 from inclusion complex and liposomes

The aim of this study was to develop and validate a UV spectrophotometric method for determination of LPSF/AC04 from inclusion complex and encapsulated into liposomes. The validation parameters were determined according to the International Conference on Harmonisation (ICH) and National Health Surveillance Agency (ANVISA) guidelines. LPSF/AC04 was determined at 250 nm in methanol by a UV spectrophotometric method, exhibiting linearity in the range from 0.3 to 2 µg.mL−1 (Absorbance=0.18068 x [LPSF/AC04 µg.mL-1] + 0.00348), (r2=0.9995). The limits of detection and quantification were 0.047µg.mL−1 and 0.143µg.mL−1, respectively. The method was accurate, precise, reproducible and robust since all the samples analyzed had coefficient of variation of less than 5% and no statistically significant difference between theoretical and practical concentrations was detected. Thus, a rapid, simple, low cost and sensitive spectrophotometric method was developed and validated for determining the content of inclusion complex and liposomes containing LPSF/AC04.

O objetivo deste estudo foi desenvolver e validar um método espectrofotométrico para determinação do LPSF/AC04 em complexo de inclusão e encapsulado em lipossomas. Os parâmetros de validação foram determinados de acordo com o International Conference on Harmonisation (ICH) e Agência Nacional de Vigilância Sanitária (ANVISA). OLPSF/AC04 foi determinado a 250 nm em metanol pelo método espectrofotométrico UV, que apresenta linearidade na faixa de 0,3 a 2 µg/mL (Absorbância = 0,18068 x [LPSF/AC04 µg/mL] + 0,00348), (r2 = 0,9995). Os limites de detecção e quantificação foi 0,047 µg/mL e 0,143 µg/mL, respectivamente. O método foi exato, preciso, reprodutível e robusto e todas as amostras analisadas apresentaram coeficiente de variação menor que 5% e não houve diferença estatisticamente significante entre a concentração teórica e a prática. Assim, um método espectrofotométrico rápido, simples, sensível e de baixo custo foi desenvolvido e validado para determinar o conteúdo do LPSF/AC04 em complexos de inclusão e encapsulados em lipossomas.

Marcado con 99m de liposomas convencionales y su evaluación biológica / 99mTc-labeling and biological evaluation of conventional liposomes

Introducción. Los liposomas son sistemas supramoleculares autoensamblables preparados ad hoc, compuestos de fosfolípidos y colesterol, diseñados para transporte de fármacos o radionucleidos. El 99mTc es el radionucleido más empleado por sus propiedades físicas apropiadas para la adquisición de imágenes y estudios en pacientes en el área de medicina nuclear (emisor gamma puro con E = 140 KeV , t1/2 = 6 horas). Objetivo. Evaluar la potencialidad de liposomas convencionales marcados con 99mTc como agente de diagnóstico de procesos tumorales. Método. La composición estudiada es: fosfatidilcolina, dioleilfosfatidiletanolamina y colesterol (1.1:0.4:1 relación molar). Se utilizó como agente reductor SnF2, en diferentes cantidades para optimizar el marcado. La pureza radioquímica y eficiencia de marcado se evaluaron con sistemas cromatográficos ITLC-SG FM NaCl 0,9 por ciento, ITLC-SG FM Piridina:ácido acético:agua (3:5:1.5 v/v). Se adquirieron imágenes a 1 h post inyección de los liposomas en ratones sanos y portadores de tumor mamario espontáneo. Resultados. El liposoma marcado mostró estabilidad durante 24 h, siendo la cantidad óptima de reductor 138 ug. La mejor captación en tumor fue a 1 h post administración del radiofármaco en los estudios centellográficos. Conclusiones. El método optimizado permite obtener liposomas marcados con 99mTc en forma sencilla, eficiente y estable. Estos radiofármacos mostraron un comportamiento fiscoquímico y biológico adecuado como agentes de diagnóstico tumoral requiriéndose estudios posteriores para su confirmación.

Background. Liposomes are self-assembled supramolecular systems, composed by phospholipids and cholesterol, designed for the transportation of drugs or radionuclides. Physical properties of 99mTc (pure gamma emitter with E = 140 KeV, t1/2= 6 hours) makes it useful for scintigraphic imaging. Purpose. The goal of this study was to evaluate 99mTc labeled conventional liposomes as potential diagnostic agents for malignant lesions. Methods. The studied liposome composition was hosphatidylcholine: dioleilphosphatidylcholine: cholesterol (1.1:0.4:1molar rate). In order to optimize the labeling, SnF2 was used as reducing agent. Radiochemical purity and labeling efficiency were evaluated using ascending thin layer chromatography. Scintigraphy images were obtained at 1 hour post-injection of labeled liposomes to healthy mice and with spontaneous breast tumors. Results. Labeled liposomes showed stability during 24 hours, being 138 ug the optimal amount of reducing agent for the technique used. Conclusions. The described method allows the production of 99mTc labeled liposomes in a simple, efficient and stable way. The radiopharmaceutical showed a physicochemical and biological behavior that allows its potential use as a tumor imaging agent, although further studies are required to confirm this issue.

Evaluation of the clearance characteristics of liposomes in the human nose by gamma-scintigraphy

The nasal cavity possesses many advantages as a site for drug delivery, such as, ease of administration, applicability for long term treatments and a large surface area for absorption. One important limiting factor for nasal drug delivery is the limited time available for absorption within the nasal cavity due to mucociliary clearance. Several drug delivery systems including different kinds of microspheres and liposomes have been tried for encapsulation of drugs and increasing the residence time in nasal cavity. In this study the clearance rate of three kinds of liposomes: neutral [phosphatidylcholin [PC] and cholesterol [Chol]], cationic [PC, Chol and stearylamine] and fusogenic [PC, Chol, dioleoyl phosphatidylethanol amine] was determined by gamma scintigraphy with lactose powder being used as negative control. Liposomes were prepared by dehydration-rehydration method. 99mTc labeled liposomes were prepared using technetium pertechnetate in the presence of a potent reducing agent, stannus chloride. The labeling procedure was set in a manner that each 150 ml of liposome suspensions contained 2 MBq of radioactivity. Labeling efficiency was calculated by paper chromatography using acetone as mobile phase. Each delivery system containing 2 MBq of activity was sprayed into right nostril of four healthy volunteers and one-minute static views were repeated each half hour until 4 hours. Clearance rates were compared using two Regions of Interest [ROIs]; the initial site of deposition of particles, and all of nasopharynx region. The clearance rate of each one of liposomes was calculated after applying the physical decay corrections. The mean labeling efficiencies for neutral, cationic and fusogenic liposomes were calculated as 91%, 20% and 69%, respectively. The cleared percent of preparations from nasopharynx region after 4 hours was determined as follows: neutral liposomes 18 +/- 2.9%; fusogenic liposomes 53.5 +/- 1.2%; cationic liposomes 69.7 +/- 4.2%; lactose powder 74.5 +/- 4.9%. Neutral liposomes showed the lowest clearance rate compared to lactose powder [P<0.0001], followed by fusogenic liposomes [P<0.01] and cationic liposomes [P<0.05]. The clearance profiles of formulations from deposition ROI and nasopharynx ROI were identical. This study shows the neutral liposomes have the highest mucoadhesion properties and are suitable nasal delivery systems. Furthermore, this study proves that limiting step for the nasal clearance of nasally administered particulate systems is their dislocation from the initial site of deposition, and their following interactions with mucus layer in the rest of nasal passage does not significantly affect the clearance time

Preparation of urea-containing stable plurilamellar liposomes and studying the effect of cholesterol on their encapsulation efficiency and release rate

Liposomes have attracted much attention as a novel drug delivery system for controlled and/or targeted release of drugs. In reverse-phase evaporation, which is a well-known method of preparation for LUVs and SPLVs, the phospholipid concentration affects the preparation process, as well as characteristics of the resulting liposomes. Drug release rate from liposomes depends on permeability of the liposomal membranes. Cholesterol [CH] is quite often included in liposomal membranes to reduce their permeability to water-soluble molecules. In this study, the required concentration of the phospholipid Ovotin' 160 [O160] for the preparation of urea-containing stable plurilamellar vesicles, and the effect of different percentages of cholesterol on the encapsulation parameters and release rate of urea as a water-soluble model drug were investigated. The results show that there is a critical concentration of the phospholipid, under which the capability for the formation of a stable emulsion [in the emulsification part of the preparation process] sharply decreases. The release rate and encapsulation parameters increased when the molar ratio of cholesterol to O160 was 5% and decreased with the ratios of 50% and 100%. Therefore, in preparation of the optimum samples a balance between the encapsulation parameters as well as the release pattern should be considered carefully

Aspectos farmacotécnicos dos lipossomas: produçäo, caracterizaçäo e estabilidade / Pharmaceutical aspects of liposomes: manufacture, characterization and stability

O tamanho, a carga e a rigidez dos lipossomas dependem da composição da bicamada lipídica e do método empregado na preparação das vesículas. De acordo com suas características, lipossomas podem permanecer na circulação sangüínea por um curto espaço de tempo (alguns minutos) ou por muitas horas (alguns dias) caso sejam estáveis e não sejam reconhecidos por macrófagos. Para sua utilização como veiculadores de fármacos, os lipossomas devem ser preparados de forma a apresentar reprodutibilidade e homogeneidade na produção lote-a-lote, na estabilidade e nas características de liberação do fármaco. Nesta revisão, estão descritos os métodos mais comumente empregados na preparação das vesículas e são discutidos os principais aspectos relacionados à estabilidade e aos processos industriais

Lipossomas como transportadores de fármacos. Parte I. Aplicaçöes farmacêuticas, composiçäo, propriedades e farmacocinética - Revisäo / Liposomes as drug carriers. Part I. Pharmaceutical applications, composition, properties and pharmacokinetics - Review

A capacidade dos lipossomas de incorporar, transportar e liberar um grande número de agentes terapêuticos favorece sua utilizaçäo nas mais variadas áreas. As principais aplicaçöes farmacêuticas estäo relacionadas ao uso dos lipossomas como veículo näo-tóxico para liberaçäo de inúmeros fármacos com diferentes características de hidrofilia e lipofilia. Outro importante aspecto é o emprego das vesículas como sistema reservatório para liberaçäo prolongada de fármacos. Ainda, os lipossomas podem ser utilizados para direcionar agentes terapêuticos para sítios intracelulares e outros tecidos específicos ou, mesmo, para evitar que o fármaco alcance determinados locais no organismo. Nesta revisäo, algumas das aplicaçöes farmacêuticas, composiçäo e propriedades das vesículas, e algumas consideraçöes farmacocinéticas säo, resumidamente, abordadas.

Lipossomas: estratégia biotecnológica para liberaçäo controlada e direcionamento intracelular de fármacos com efeito antimicobacteriano

Este texto relata o estudo da pesquisa relacionada com a aplicaçäo dos lipossomas no controle da liberaçäo e direcionamento de fármacos para doenças bacterianas intracelulares, tais como a tuberculose. Lipossomas possuem muitas aplicaçöes farmacêuticas, e este artigo aborda primariamente o potencial deste sistema coloidal para encapsulaçäo de fármacos, especialmente de compostos antimicobacterianos. Säo apresentados casos nos quais os lipossomas säo usados com sucesso para implementar o efeito antibiótico. Mecanismos envolvidos na liberaçäo intracelular dos fármaco, possibilidades de aplicaçöes, pesquisas e esforços no desenvolvimento para se atingir esses objetivos säo discutidos neste trabalho.