Co-delivery of gemcitabine and salinomycin in PEGylated liposomes for enhanced anticancer efficacy against colorectal cancer.

Colorectal cancer remains one of the major causes of morbidity and mortality in both developed and emerging countries. Cancer stem cells (CSCs) are a subpopulation of cells within the tumor mass harboring stem cell characteristics, considered responsible for tumor initiation, growth, relapse, and treatment failure. Lately, it has become clear that both CSCs and non-CSCs have to be eliminated for the successful eradication of cancer. Drug delivery systems have been extensively employed to enhance drug efficacy. In this study, salinomycin (SAL), a selective anti-CSC drug, and gemcitabine (GEM), a conventional anticancer drug, were co-loaded in liposomes and tested for optimal therapeutic efficacy. We employed the Design of Experiments approach to develop and optimize a liposomal delivery system for GEM and SAL. The antiproliferative effect of the liposomes was evaluated in SW-620 human colorectal cancer cells. The GEM and SAL-loaded liposomes exhibited adequate size, polydispersity, zeta potential, and drug content. The in vitro release study showed a sustained release of GEM and SAL from the liposomes over 72 h. Moreover, no sign of liposome aggregation was seen over 1 month and in a biological medium (FBS). The in vitro cytotoxic effects of the co-loaded liposomes were superior to that of single GEM either in free or liposomal form. The combination therapy using GEM and SAL co-loaded in liposomes could be a promising strategy for tackling colorectal cancer.

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.

Liposomal Curcumin is Better than Curcumin to Alleviate Complications in Experimental Diabetic Mellitus.

Curcumin (CC) is known to have anti-inflammatory and anti-oxidative properties and has already been tested for its efficiency in different diseases including diabetes mellitus (DM). New formulations and route administration were designed to obtain products with higher bioavailability. Our study aimed to test the effect of intraperitoneal (i.p.) administration of liposomal curcumin (lCC) as pre-treatment in streptozotocin(STZ)-induced DM in rats on oxidative stress, liver, and pancreatic functional parameters. Forty-two Wistar-Bratislava rats were randomly divided into six groups (seven animals/group): control (no diabetes), control-STZ (STZ-induced DM -60 mg/100g body weight a single dose intraperitoneal administration, and no CC pre-treatment), two groups with DM and CC pre-treatment (1mg/100g bw-STZ + CC1, 2 mg/100g bw-STZ + CC2), and two groups with DM and lCC pre-treatment (1 mg/100g bw-STZ + lCC1, 2 mg/100g bw-STZ + lCC1). Intraperitoneal administration of Curcumin in diabetic rats showed a significant reduction of nitric oxide, malondialdehyde, total oxidative stress, and catalase for both evaluated formulations (CC and lCC) compared to control group (p < 0.005), with higher efficacy of lCC formulation compared to CC solution (p < 0.002, excepting catalase for STZ + CC2vs. STZ + lCC1when p = 0.0845). The CC and lCC showed hepatoprotective and hypoglycemic effects, a decrease in oxidative stress and improvement in anti-oxidative capacity status against STZ-induced DM in rats (p < 0.002). The lCC also proved better efficacy on MMP-2, and -9 plasma levels as compared to CC (p < 0.003, excepting STZ + CC2 vs. STZ + lCC1 comparison with p = 0.0553). The lCC demonstrated significantly better efficacy as compared to curcumin solution on all serum levels of the investigated markers, sustaining its possible use as adjuvant therapy in DM.

A quality by design approach to optimise disulfide-linked hyaluronic acid hydrogels.

In this study, the disulfide-linked hyaluronic acid (HA) hydrogels were optimised for potential application as a scaffold in tissue engineering through the Quality by Design (QbD) approach. For this purpose, HA was first modified by incorporating the cysteine moiety into the HA backbone, which promoted the formation of disulfide cross-linked HA hydrogel at physiological pH. Utilising a Design of Experiments (DoE) methodology, the critical factors to achieve stable biomaterials, i.e. the degree of HA substitution, HA molecular weight, and coupling agent ratio, were explored. To establish a design space, the DoE was performed with 65 kDa, 138 kDa and 200 kDa HA and variable concentrations of coupling agent to optimise conditions to obtain HA hydrogel with improved rheological properties. Thus, HA hydrogel with a 12 % degree of modification, storage modulus of ≈2321 Pa and loss modulus of ≈15 Pa, was achieved with the optimum ratio of coupling agent. Furthermore, biocompatibility assessments in C28/I2 chondrocyte cells demonstrated the non-toxic nature of the hydrogel, underscoring its potential for tissue regeneration. Our findings highlight the efficacy of the QbD approach in designing HA hydrogels with tailored properties for biomedical applications.

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.

The effect of intravenous administration of liposomal curcumin in addition to sumatriptan treatment in an experimental migraine model in rats.

BACKGROUND: Curcumin has antioxidative properties that could be useful in various diseases due to its ability to act on multiple targets of various cellular pathways. We aimed to assess the efficacy of liposomal curcumin compared with curcumin solution, when in addition to sumatriptan (ST) treatment, in an experimental migraine model induced with nitroglycerin (NTG) in rats. METHODS: Seven groups of 9 rats each were investigated: control group without migraine (1 mL saline solution intraperitoneal injection [ip]), control group with induced migraine, NTG+ST group (ST), NTG+ST+curcumin1 (CC1) group - 1 mg/100 g body weight (bw), NTG+ST+CC2 - 2 mg/100 g bw, NTG+ST+liposomal curcumin1 (lCC1) group - 1 mg/100 g bw, and NTG+ST+lCC2 (lCC2) group - 2 mg/100 g bw. NTG and ST were administered as 1 mL ip NTG | 1 mg/100 g bw and 1 mL ip ST | 1 mg/100 g bw, respectively. Plasma total oxidative stress (TOS), malondialdehyde (MDA), nitric oxide (NOx), thiol levels, as well as total antioxidative capacity (TAC) were assessed. The nociception process was assessed by counting the number of flinches and shakes after the formalin test. RESULTS: The plasma TOS, MDA, and NOx levels, as oxidative stress parameters, were significantly decreased in the curcumin-treated groups, especially where curcumin was in liposomal form. The thiol and TAC were also improved by the curcumin treatment, with the best results obtained for the liposomal curcumin. The closest number of flinches and shakes to the control group was obtained for the group treated with liposomal curcumin at a dose of 2 mg/100 g bw. CONCLUSION: Liposomal curcumin in a dose of 2 mg/100 g bw when in addition to ST treatment could be an optimum therapeutic strategy for migraine attacks and could represent a base for future clinical research and application.

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.

Simultaneous quantification of l-α-phosphatidylcholine and cholesterol in liposomes using near infrared spectrometry and chemometry.

This paper describes the development and validation of a multivariate method based on transmittance NIR spectroscopy for simultaneous quantification of l-α-phosphatidylcholine (LPC) and cholesterol (CHO). Method development was based on a D-optimal experimental design consisting of 16 LPC-CHO mixtures. Calibration models were generated by partial least-squares (PLS) and principal component regression (PCR) method followed by leave-one-out cross-validation. Among the spectra pretreatment methods tested, Norris Gap first derivative was the best for both LPC and CHO quantification, combined with PLS multivariate method. The method was validated (trueness, precision, accuracy) for the concentration range 50-150% of the expected concentration in liposomes. This method was successfully applied for the characterization of liposomes prepared using the two excipients.