Improved pharmacokinetics and reduced side effects of doxorubicin therapy by liposomal co-encapsulation with curcumin.

The goal of the current study was to investigate the pharmacokinetic profile, tissue distribution and adverse effects of long-circulating liposomes (LCL) with curcumin (CURC) and doxorubicin (DOX), in order to provide further evidence for previously demonstrated enhanced antitumor efficacy in colon cancer models. The pharmacokinetic studies were carried out in healthy rats, following the i.v. injection of a single dose of LCL-CURC-DOX (1 mg/kg DOX). For the tissue distribution study, DOX concentration in tumours, heart and liver were measured after the administration of two i.v. doses of LCL-CURC-DOX (2.5 mg/kg DOX and 5 mg/kg CURC) to Balb/c mice bearing C26 colon tumours. Markers of murine cardiac and hepatic oxidative status were determined to provide additional insights into the benefit of co-encapsulating CURC and DOX in LCL over DOX-induced adverse effects in these organs. The current study demonstrated that the liposomal association of CURC and DOX effectively improved the pharmacokinetics and biodistribution of DOX, limiting its side effects, via CURC-dependent antioxidant effects.

Optimization of prednisolone-loaded long-circulating liposomes via application of Quality by Design (QbD) approach.

Quality by design principles (QbD) were used to assist the formulation of prednisolone-loaded long-circulating liposomes (LCL-PLP) in order to gain a more comprehensive understanding of the preparation process. This approach enables us to improve the final product quality in terms of liposomal drug concentration, encapsulation efficiency and size, and to minimize preparation variability. A 19-run D-optimal experimental design was used to study the impact of the highest risk factors on PLP liposomal concentration (Y1- µg/ml), encapsulation efficiency (Y2-%) and size (Y3-nm). Out of six investigated factors, four of them were identified as critical parameters affecting the studied responses. PLP molar concentration and the molar ratio of DPPC to MPEG-2000-DSPE had a positive impact on both Y1 and Y2, while the rotation speed at the formation of the lipid film had a negative impact. Y3 was highly influenced by prednisolone molar concentration and extrusion temperature. The accuracy and robustness of the model was further on confirmed. The developed model was used to optimize the formulation of LCL-PLP for efficient accumulation of the drug to tumor tissue. The cytotoxicity of the optimized LCL-PLP on C26 murine colon carcinoma cells was assessed. LCL-PLP exerted significant anti-angiogenic and anti-inflammatory effects on M2 macrophages, affecting indirectly the C26 colon carcinoma cell proliferation and development.

A step forward towards the development of stable freeze-dried liposomes: a quality by design approach (QbD).

This study highlights the advantages of using a Quality by Design (QbD) approach in order to gain a more comprehensive understanding of the freeze-drying process of pravastatin-loaded long-circulating liposomes (LCL-PRAV). Within the QbD paradigm, the present study aimed to establish the design space for the optimization of freeze-dried LCL-PRAV by means of Design of Experiment (DOE). The encapsulated solute retention (ESR), the average particle size, and zeta potential after freeze-drying, the residual moisture content, the macroscopic cake appearance, the glass transition temperature (Tg) of the freeze-dried cake, and the primary drying time were defined as critical quality attributes (CQAs) for the freeze-dried final product. Further on, the influence of lyoprotectant type, freezing rate, shelf temperature during primary drying, and the presence of an annealing step on the CQAs was investigated through a 21-run D-optimal experimental design. Three-dimensional response surfaces were generated to complete the statistical analysis and for a better understanding of the influence of variables and their interactions on the responses. The developed model was then used to build the design space for the freeze-dried liposomes, within which the product quality was assured and the process variability was minimized.

Co-delivery of curcumin and doxorubicin in PEGylated liposomes favored the antineoplastic C26 murine colon carcinoma microenvironment.

Our recent studies have demonstrated that the antitumor efficacy of doxorubicin (DOX), administered in long-circulating liposomes (LCL), could be considerably improved after its co-encapsulation with curcumin (CURC). Thus, the question addressed within this article is whether LCL-CURC-DOX can be exploited more efficiently than liposomal DOX for future colorectal cancer therapy. Therefore, we investigated the physicochemical and biological properties of LCL-CURC-DOX and the mechanisms of its antitumor activity in C26 murine colon carcinoma in vivo. Our results proved that the developed nanoformulation based on the co-encapsulation of CURC and DOX met the requirements of a modern drug delivery system for future cancer therapy, demonstrating enhanced antitumor activity on C26 colon carcinoma in vivo. The antitumor efficacy of LCL-CURC-DOX relied on suppressive effects on main protumor processes such as angiogenesis, inflammation, oxidative stress, invasion and resistance to apoptosis, and on the dysregulation of Th1/Th2 cell axis which favored the antineoplastic phenotype of cells in tumor microenvironment (TME). The development of multitargeted strategies aiming at stimulating antitumor effects within the tumor milieu and counteracting the escape mechanisms of cancer cells would be beneficial in the management of colon cancer in the future.

A Quality by Design (QbD) approach to the development of a gradient high-performance liquid chromatography for the simultaneous assay of curcuminoids and doxorubicin from long-circulating liposomes.

The present study highlights the advantages of using an Analytical Quality by Design (AQbD) approach to the optimization of a high-performance liquid chromatography method for the simultaneous assay of curcumin (CUR), demetoxycurcumin (DMC), bisdemetoxycurcumin (BDMC) and doxorubicin (DOX) co-encapsulated in long circulating liposomes. Within the QbD paradigm, the present study aimed to establish the method operable design region (MODR) for the optimization of the high-performance liquid chromatography-fluorescence (HPLC-FLD) assay by means of Design of Experiments (DOE) and response surface methodology, in order to achieve a good separation and quantification of all analyzed compounds along to an acceptable analysis time. A deep understanding of the quality target product profile (QTPP) and of the analytical target profile (ATP), followed by a risk assessment for variables that affect the efficiency of the method led to the development of a precise, accurate and cost-effective method. The assay was linear over the range of 2-20 µg/ml for all investigated compounds. The intra-and inter-day precision were less than 2%, with accuracies between 97-104% of the true values. The method was successfully applied to the quantification of curcuminoids and DOX from long-circulating liposomes.

Anti-angiogenic and anti-inflammatory effects of long-circulating liposomes co-encapsulating curcumin and doxorubicin on C26 murine colon cancer cells.

BACKGROUND: Emerging treatment options for colon cancer are needed to overcome the limitations regarding the side effects of current chemotherapeutics and drug resistance. The goal of this study was to assess the antitumor actions of PEGylated long-circulating liposomes (LCL) co-delivering curcumin (CURC) and doxorubicin (DOX) on murine colon carcinoma cells (C26). METHODS: The cytotoxicity of CURC and DOX, administered alone or in combination, either in free or LCL form, was evaluated with regard to antiproliferative effects on C26 cells and to protumor processes that might be affected. RESULTS: Our results indicated that PEGylated LCL-CURC-DOX exerted strong antiproliferative effects on C26 cells, slightly exceeding those induced by free CURC-DOX, but higher than either agent administered alone in their free form. These effects of LCL-CURC-DOX were due to the inhibition of the production of angiogenic/inflammatory proteins in a NF-κB-dependent manner, but were independent of ROS production or AP-1 c-Jun activation. Notable, the anti-angiogenic actions of LCL-CURC-DOX appeared to be much stronger than those induced by the co-administration of CURC and DOX in their free form, on C26 colon cancer cells. CONCLUSION: LCL-CURC-DOX demonstrated enhanced cytotoxicity on C26 murine colon cancer cells by inhibiting the production of the majority of factors involved in tumor-associated angiogenesis and inflammation and is now being evaluated in vivo regarding its efficacy towards tumor growth in colon cancer.

Formulation Optimization of Freeze-Dried Long-Circulating Liposomes and In-Line Monitoring of the Freeze-Drying Process Using an NIR Spectroscopy Tool.

The effect of lyoprotectant type and concentration on the stability of freeze-dried prednisolone sodium phosphate-loaded long-circulating liposomes was investigated. Trehalose at a 5:1 carbohydrate to lipid molar ratio proved to be superior in maintaining the structural integrity and the permeability properties of the liposome bilayers, assuring the desired characteristics of the final product: a cake with a porous structure and easy to reconstitute, a similar size to the liposomes before freeze-drying, a high percent of encapsulated drug, and a low residual moisture content. Further on, the study demonstrated the possibility of near-infrared spectroscopy to provide valuable insights for detecting critical changes in acyl chain packing of the liposome bilayer. By visualizing the spectra after principal component analysis, one can predict if any harm has occurred to liposome integrity during the process. Moreover, near-infrared spectroscopy enabled us to determine the end points of primary and secondary drying without disturbing the normal freeze-drying procedure, which allowed us to gain a better understanding of the process and to improve process efficiency by optimizing the primary and secondary drying time.

Development of antiproliferative long-circulating liposomes co-encapsulating doxorubicin and curcumin, through the use of a quality-by-design approach.

The aim of this work was to use the quality-by-design (QbD) approach in the development of long-circulating liposomes co-loaded with curcumin (CUR) and doxorubicin (DOX) and to evaluate the cytotoxic potential of these liposomes in vitro using C26 murine colon carcinoma cell line. Based on a risk assessment, six parameters, namely the phospholipid, CUR and DOX concentrations, the phospholipid:cholesterol molar ratio, the temperature during the evaporation and hydration steps and the pH of the phosphate buffer, were identified as potential risk factors for the quality of the final product. The influence of these variables on the critical quality attributes of the co-loaded liposomal CUR and DOX was investigated: particle size, zeta potential, drug loading and entrapment efficiency. For this, a 26-2 factorial design was employed to establish a proper regression model and to generate the contour plots for the responses. The obtained data served to establish the design space for which different combinations of variables yielded liposomes with characteristics within predefined specifications. The validation of the model was carried out by preparing two liposomal formulations corresponding to the robust set point from within the design space and one outside the design space and calculating the percentage bias between the predicted and actual experimental results. The in vitro antiproliferative test showed that at higher CUR concentrations, the liposomes co-encapsulating CUR and DOX had a greater cytotoxic effect than DOX-loaded liposomes. Overall, this study showed that QbD is a useful instrument for controlling and optimizing the manufacturing process of liposomes co-loaded with CUR and DOX and that this nanoparticulate system possesses a great potential for use in colon cancer therapy.

Liposomal prednisolone phosphate potentiates the antitumor activity of liposomal 5-fluorouracil in C26 murine colon carcinoma in vivo.

The antitumor efficacy of 5-fluorouracil (5-FU) in advanced colorectal cancer (CRC) is hindered not only by the low therapeutic index, but also by tumor cell resistance to this cytotoxic drug. Therefore, to enhance the 5-FU antitumor activity, the present research used a novel tumor-targeted therapy based on the co-administration of 5-FU encapsulated in long-circulating liposomes (LCL-5-FU) together with liposomal prednisolone phosphate (LCL-PLP), a formulation with known anti-angiogenic actions on C26 murine colon carcinoma cells. Thus, we assessed the in vivo effects of the combined liposomal drug therapy on C26 carcinoma growth as well as on the production of molecular markers with key roles in tumor development such as angiogenic, inflammatory, and oxidative stress molecules. To get further insight into the polarization state of tumor microenvironment after the treatment, we determined the IL-10/IL-12p70 ratio in tumors. Our results showed that combined liposomal drug therapy inhibited almost totally tumor growth and was superior as antitumor activity to both single liposomal drug therapies tested. The antitumor efficacy of the combined therapy was mainly related to the anti-angiogenic and anti-inflammatory actions on C26 carcinoma milieu, being favored by its controlling effect on intratumor oxidative stress and the skewing of polarization of tumor microenvironmental cells toward their antineoplastic phenotypes. Thus, our study unveils a promising treatment strategy for CRC that should be furthermore considered.