PEG-PCL Nanocapsules Containing Curcumin: Validation of HPLC Method for Analyzing Drug-loading Efficiency, Stability Testing and Cytotoxicity on NIH-3T3 Cell Line

Abstract Curcumin (CUR) shows potential use for treating cancer. However, CUR has low solubility and reduced bioavailability, which limit its clinical effect. Therefore, the development of nanocarriers can overcome these problems and can ensure the desired pharmacological effect. In addition, it is mandatory to prove the quality, the efficacy, and the safety for a novel nanomedicine to be approved. In that sense, this paper aimed (a) to prepare CUR-loaded polyethylene glycol-poly(ε-caprolactone) nanocapsules; (b) to validate an analytical method by high performance liquid chromatography (HPLC) for quantifying CUR in these nanoformulations; (c) to evaluate the physicochemical stability of these formulations; and to investigate their cytotoxicity on NIH-3T3 mouse fibroblast cells. The HPLC method was specific to CUR in the loaded nanocapsules, linear (r = 0.9994) in a range of 10.0 to 90.0 µg.mL-1 with limits of detection and quantification of 0.160 and 0.480 µg.mL-1, respectively. Precision was demonstrated by a relative standard deviation lower than 5%. Suitable accuracy (102.37 ± 0.92%) was obtained. Values of pH, particle size, polydispersity index, and zeta potential presented no statistical difference (p > 0.05) for CUR-loaded nanoparticles. No cytotoxicity was observed against NIH-3T3 mouse embryo fibroblast cell line using both the tetrazolium salt and sulforhodamine B assays. In conclusion, a simple and inexpensive HPLC method was validated for the CUR quantification in the suspensions of nanocapsules. The obtained polymeric nanocapsules containing CUR showed suitable results for all the performed assays and can be further investigated as a feasible novel approach for cancer treatment.

Preparation of mupirocin-loaded polymeric nanocapsules using essential oil of rosemary

Abstract The purpose of this study was to prepare and characterize mupirocin-loaded polymeric nanocapsules using two different oils and to develop and validate an analytical method for quantitative determination by high performance liquid chromatography. The mean size of the nanoparticles was 233.05 nm and 275.03 nm for nanocapsules with a rosemary oil like oily core and caprylic/capric triglyceride, respectively, and a good polydispersity index below 0.25 for both formulations. The nanocapsules showed good stability when stored at 40 ºC and room temperature for 30 days. The quantitative method was performed with a mobile phase consisting of ammonium ammonium acetate (0.05 M adjusted to pH 5.0 with acetic acid) and acetonitrile 60:40 (v/v); the flow rate was 0.8 mL/min, UV detection at 230 nm. The analytical method was linear in the range of 5.0-15.0 µg/mL, specific for both oils, accurate, precise (intermediate precision RSD = 1.68% and repeatability RSD = 0.81%) and robust under the evaluated conditions. Therefore, this method can be performed for quantification of mupirocin in polymeric nanocapsules containing both oils.

Efeitos das nanocápsulas de núcleo lipídico contendo acetileugenol em melanomas: estudos in vivo e in vitro / Effects of lipid-core nanocapsules with acetyleugenol in melanomas: in vivo and in vitro studies

O melanoma é uma neoplasia de pele invasivo, com maior taxa de morte, sem tratamento efetivo. Nanocápsulas poliméricas de núcleo lipídico (LNC) tem sido empregadas com sucesso como carreadores de fármacos hidrofóbicos. Como o eugenol é um composto hidrofóbico com atividades antiproliferativas e pró-apoptóticas em células cancerosas, visamos avaliar os efeitos dos tratamentos com acetileugenol (AC), LNC ou LNC contendo acetileugenol (LNC-AC) em modelo de melanoma in vivo em camundongos C57B6, e a citotoxicidade dos mesmos em células endoteliais (HUVEC) e de melanoma (SK-Mel-28) in vitro. Os resultados obtidos mostraram que: 1) tratamentos i.p. com as LNC ou com LNC-AC (50 mg/kg, 3-10 dia de indução do tumor) induziram toxicidade sistêmica e, somente o tratamento com LNC inibiu o desenvolvimento do melanoma. O tratamento com LNC, mas não com a mistura de triglicerídeos de cadeia média, por via oral, inibiu o desenvolvimento tumoral, sem toxicidade. Adicionalmente, os tratamentos com AC, LNC ou LNC-AC não foram eficazes quando administrados em fase tardia de evolução tumoral (50 mg/kg, 7-17 dia de indução do tumor, via oral); 2) os tratamentos agudos com AC, LNC ou LNC-AC (20 mg/kg, 200 µL, e.v.) não alteraram o número de leucócitos circulantes, mas os tratamentos com LNC ou com LNC-AC reduziram o comportamento de rolling dos leucócitos em vênulas póscapilares do músculo cremaster e causaram hemólise, sendo que este último efeito também foi observado após tratamento in vitro em hemácias murinas; 3) Os estudos in vitro mostraram que as LNC e LNC-AC foram captadas pelas células HUVEC e SK-Mel-28 após 1 hora de incubação; que a incubação com LNC-AC induziu apoptose tardia e necrose com maior eficácia em SK-Mel-28 do que em HUVEC; que as incubações com LNC ou LNC-AC exerceram efeitos antiproliferativos, induzindo parada na fase G2/M do ciclo celular das duas linhagens de células avaliadas; que somente a incubação com AC ou LNC-AC inibiu a adesão ao Matrigel® com maior eficácia na linhagem SK-Mel-28 do que HUVEC; que somente a incubação com as LNC reduziram a expressão de VCAM-1 em HUVEC e que as incubações com LNC ou LNC-AC reduziram a expressão de ß3 integrina em SK-Mel- 28; que nenhum dos tratamentos alterou a migração celular das HUVEC ou SK-Mel- 28; que somente a incubação com LNC-AC reduziu os níveis de espécies reativas de oxigênio em HUVEC e SK-Mel-28; que a incubação com LNC ou LNC-AC aumentou a produção de óxido nítrico (NO) pelas duas linhagens de células avaliadas; que o tratamento com L-NAME reverteu os níveis de NO e a inibição sobre a proliferação celular induzida pela incubação com LNC ou LNC-AC e; que o tratamento de células de melanoma murino com LNC ou LNC-AC parece alterar a polarizar os neutrófilos para o fenótipo N1. Associados, os resultados obtidos mostram o tratamento oral com LNC inibe o crescimento do melanoma sem induzir efeitos tóxicos, e que este efeito benéfico pode ser dependente, pelo menos em parte, da nanoencapsulação dos triglicerídios de cadeia média e da supraestrutura da formulação, com toxicidade direta sobre as células de melanoma e possível modulação do microambiente tumoral

Melanoma is the most invasive skin cancer, with high rates of death without effective treatment. Polymeric lipid-core nanocapsules (LNC) has been successfully used as carriers of hydrophobic drugs. As eugenol is an hydrophobic compound with antiproliferative and pro-apoptotic activity in cancer cells, here we aimed to evaluate the effects of treatments with acetyleugenol (AC), LNC or LNC containing acetyleugenol (LNC-AC) in an in vivo melanoma model in C57BL6 mice and the cytotoxicity of the treatments in vitro, using endothelial (HUVEC) and melanoma (SK-Mel- 28) cells. The results obtained showed that: 1) i.p. treatments with LNC or LNCAC (50 mg/kg, 3-10 days of tumor injection) induced systemic toxicity and, only the treatment with LNC inhibited the melanoma development. Treatment with LNC, but not with mix of triglycerides of medium chain, by oral route, inhibited the tumor development, without toxicity. In addition, the treatments with AC, LNC or LNC-AC were not effective when administered in the late stage of tumor evolution (50 mg/kg, 10-20 days of tumor induction, oral route); 2) the acute treatments with AC, LNC or LNC-AC (20 mg/kg, 200 µL, intravenous route) did not altered the number of circulating leukocytes, but the treatments with LNC or LNC-AC reduced the rolling behavior of leukocytes in postcapillary venules of the cremaster muscle and induced hemolysis. The latter effect was also observed after in vitro treatment using murine erythrocytes; 3) In vitro studies showed that the LNC and LNC-AC suffered uptake by HUVEC and SK-Mel-28 cells after 1 hour of incubation; that the incubation with LNC-AC induced late apoptosis and necrosis more effectively in SK-Mel-28 than in HUVEC cells; that the incubation with LNC or LNC-AC presented antiproliferative effects, by inducing G2M arrest in cell cycle in both cells lines evaluated; that only the incubation with AC or LNC-AC inhibited the adhesion in Matrigel® with more efficaccy in SK-Mel-28 than in HUVEC cells; that only incubtion with LNC reduced the VCAM-1 expression in HUVEC and the incubation with LNC or LNC-AC reduced the ß3 integrin expression in SK-Mel-28 cells; that any treatment affected the HUVEC or SK-Mel- 28 migration; that only the incubation with LNC-AC reduced the levels of reactive species of oxygen in HUVEC and SK-Mel-28 cells; that the incubation with LNC or LNC-AC increased the nitric oxide (NO) production by both cell lines used; that the treatment with L-NAME reversed the NO levels and the inhibition on cell proliferation induced by incubation with LNC or LNC-AC and; that the in vitro treatment of murine with LNC or LNC-AC altered the neutrophil polarization to N1 phenotype. Together, results obtained show that the oral treatment with LNC inhibit the melanoma growth without any toxic effect, and that the beneficial effect could be dependent, at least in part, of nanoencapsulation of medium chain triglycerides and the supraestrucuture of the formulation, with direct toxicity on melanoma cells and possible modulation of tumor microenvironment

Derivados porfirínicos nanoencapsulados como fotossensibilizadores em terapia fotodinâmica / Nanoencapsulated porphyrin derivatives as photosensitizers in photodynamic therapy

Uma série de oito derivados porfirínicos monocatiônicos, na forma de base-livre e metalados com Zn(II), e tendo grupos metila ou [Ru(bipy)2Cl]+ ligados ao átomo de nitrogênio do substituinte piridil em uma das posições meso do anel, foram sintetizadas e caracterizadas, visando sua aplicação como fotossensibilizadores no tratamento de tumores de pele por TFD. Além de possuírem características anfifílicas que favorecem a sua interação com membranas biológicas, os compostos apresentaram um elevado rendimento quântico de formação de oxigênio singlete, principalmente os derivados metilados das porfirinas base-livre. A baixa solubilidade em meio aquoso, fator limitante da utilização como fotossensibilizadores, foi superada por meio do encapsulamento dos mesmos em micro e nanocápsulas poliméricas, pelo método de coacervação. Duas formulações, uma baseada em hidroxietilcelulose (HEC) e outra na mistura atelocolágeno marinho/goma xantana (ACM), foram preparadas e suas eficiências fotodinâmicas e citotoxicidade frente à células HeLa determinadas. As formulações de ACM mostraram ser não-tóxicas, enquanto os preparados com HEC apresentaram uma pequena, mas significativa citotoxicidade no escuro. Por outro lado, todas as formulações se tornaram tóxicas quando irradiadas com laser de 650 nm ou luz branca proveniente de lâmpada de mercúrio. Dentre as formulações preparadas, as mais ativas foram aquelas preparadas com os derivados porfirínicos base-livre metilados em ACM, provavelmente em virtude da maior eficiência de entrega do composto fotoativo em relação ao sistema polimérico de HEC, aliado aos elevados rendimentos quânticos de formação de oxigênio singlete em relação aos demais compostos. Além disso, o encapsulamento influenciou significativamentente a interação e citolocalização dos fotossensibilizadores, e consequentemente a taxa e o mecanismo de morte celular. Os estudos por microscopia de fluorescência confocal, evidenciaram que as nanocápsulas de ACM são capazes de penetrar na membrana das células, alcançando o citoplasma e liberando gradativamente o composto fotoativo no seu interior. Quando não encapsulada, em solução de dmso, a porfirina se acumula preferencialmente na membrana celular. Consequentemente, neste caso a irradiação levou ao comprometimento da integridade da mesma e o desencadeamento da necrose celular, indesejável na terapia por ocasionar inflamação nos tecidos. Em contrapartida, o tratamento com formulações de fotossensibilizadores nanoencapsulados promoveram menos danos mas favoreceram a apoptose, um mecanismo de morte programada que geralmente leva a regeneração rápida e total dos tecidos, e ausência de processos inflamatórios

A series of eight monocationic porphyrins derivatives as free-base and Zn(II) complexes, with methyl or [Ru(bipy)2Cl]+ groups bond to the nitrogen atom of the meso-pyridyl substituent, were synthesized and characterized aiming their application as photosensitizers in the treatment of skin tumors by Photodynamic Therapy (PDT). Those compounds, especially the methylated derivatives of the free-base porphyrins, are potentially useful because showed higher quantum yields for the photoinduced formation of singlet oxygen and amphiphilic character, that enhanced their interaction with biological membranes. The low solubility in aqueous media, a limiting factor for use as photosensitizers, was overcome by encapsulating them in polymeric micro-and nanocapsules using the coacervation method. Two formulations, one based on hydroxyethyl cellulose (HEC) and another in the marine atelocollagen/xanthan gum (MAC) mixture were prepared and their photodynamic efficiencies and cytotoxicity against HeLa cells determined. The MAC formulations were shown to be non-toxic, while the HEC presented a small but significant cytotoxicity in the dark. On the other hand, all formulations become toxic when irradiated with 650 nm laser or white light from a mercury lamp source. The most actives, among all formulations, were those prepared using the methylated free-base porphyrin derivatives and MAC, probably due to the higher delivery efficiency of the photosensitizers in relation to the formulations prepared with HEC, associated with their higher quantum yields of photoinduced formation of singlet oxygen of those species as compared with the other compounds. In addition, the encapsulation influenced the interaction and cytolocalization of the photosensitizers, and consequently the rate and mechanism of cell death. The MAC nanocapsules were shown to penetrate the cell membrane by confocal fluorescence microscopy, reaching the cytoplasm where gradually released the photoactive compound. However, the porphyrin derivatives preferentially accumulated in the cell membrane when in DMSO solution. Consequently, the irradiation compromised the membrane integrity leading to necrosis, an undesirable process since cause tissue inflammation. In contrast, the treatment with encapsulated photosensitizer formulations promoted a much lower level of photoinduced damage and apoptosis, a more desirable death mechanism characterized by a complete and rapid regeneration of tissues and absence of inflammatory processes

Development and validation of a fluorimetric method to determine curcumin in lipid and polymeric nanocapsule suspensions

A simple, rapid, and sensitive fluorimetric method was developed and validated to quantify curcumin in lipid and polymeric nanocapsule suspensions, using acetonitrile as a solvent. The excitation and emission wavelengths were set at 397 nm and 508 nm, respectively. The calibration graph was linear from 0.1 to 0.6 µg/mL with a correlation coefficient of 0.9982. The detection and quantitation limits were 0.03 and 0.10 µg/mL, respectively. The validation results confirmed that the developed method is specific, linear, accurate, and precise for its intended use. The current method was successfully applied to the evaluation of curcumin content in lipid and polymeric nanocapsule suspensions during the early stage of formulation development.

Um método fluorimétrico simples, rápido e sensível foi desenvolvido e validado para quantificação da curcumina em suspensões de nanocápsulas lipídicas e poliméricas, usando acetonitrila como solvente. Os comprimentos de onda de excitação e emissão foram 397 nm e 508 nm, respectivamente. Nas condições testadas, a curva de calibração demonstrou-se linear na faixa de 0,1 a 0,6 µg/mL, exibindo coeficiente de correlação de 0,9982. Os limites de detecção e quantificação foram 0,03 e 0,10 µg/mL, respectivamente. Os resultados da validação confirmaram que o método desenvolvido é específico, linear, exato e preciso para o uso proposto. O presente método foi aplicado com sucesso para a avaliação do teor de curcumina nas suspensões de nanocápsulas lipídicas e poliméricas durante o estágio inicial do desenvolvimento da formulação.