Isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus using centrifugal partition chromatography.

Replacement of synthetic colorants with natural ones is a current marketing trend. Nevertheless, the naturally occurring blue color is rare compared to other colours. In this work, centrifugal partition chromatography (CPC) process was developed as a more efficient and sustainable alternative to reversed phase column chromatography (RP-CC) for the preparative-scale purification of portisins. The strategy began with the extraction of anthocyanins from blueberry surplus and hemi-synthesis of respective portisins. Then, the CPC method development started with the biphasic solvent system selection followed by the optimization of the operating parameters and ended up with a comparison with RP-CC. Aiming at maximizing the portisin content, process throughput, efficiency, and minimizing the environmental risk factor, the effect of sample load (100-500 mg/100 mL of column volume), mobile phase flow rate (10-20 mL/min), and rotation speed (1000-1600 rpm) was evaluated. The two-phase solvent system consisted of tert­butyl­methyl ether, n-butanol, acetonitrile, and water (volume ratio 2:2:1:5) acidified with 0.1 vol.% of HCl was selected. The best conditions were 464 mg of sample/100 mL of column volume, 20 mL/min of mobile phase flow rate, and 1600 rpm of rotation speed at reversed phase mode, allowing the purification of portisins by 5-fold. Compared to the RP-CC, the CPC process efficiency was 2.4 times higher, while the CPC process environmental risk factor was 5.5 times lower. Overall, this study suggests that CPC can be considered an effective, and sustainable alternative process for the preparative isolation of portisins. With this purification approach, the blueberry surplus has been valorized and a naturally derived product has been prepared, allowing its subsequent use as a natural blue colorant.

Polymeric Micellar Formulation Enhances Antimicrobial and Anticancer Properties of Salinomycin.

PURPOSE: Salinomycin (SAL) is a polyether compound that exhibits strong antimicrobial as well as anticancer activity. Nanomedicine has been at the forefront of drug delivery research with the aim of increasing the efficacy, specificity and reduce toxicity of drugs. There is an intersection between infection and cancer, and cancer patients are prone to bacterial infections. In this study, polymeric micelles were prepared using Pluronic® F127 (PM) to encapsulate SAL (PM_SAL) with the view of enhancing antimicrobial and anticancer activity. METHODS: A Quality by Design (QbD) approach was utilized to synthesize PM_SAL, and nanoformulation activity was determined against bacterial (S. aureus, MRSA and E. coli). Effects on cancer cell line A549, i.e. cell viability, prevention of P-gp efflux, vimentin expression, effects on migratory ability of A549 cells. Anticancer activity was determined by ability to eradicate cancer stem-like cells. RESULTS: PM_SAL demonstrated only efficacy against MRSA, being even higher than that obtained with SAL. In A549 cells, a 15-fold increase in P-gp's expression as well as a significant decrease of the cell's migration, was observed. CONCLUSIONS: PM_SAL can interfere with the oncogenic protein VIM, involved in the crucial mechanisms EMT, downregulating its expression. Altogether data obtained indicates that this antibiotic and the developed polymeric micelle system is a very promising inhibitor of tumor cell growth.

Evading P-glycoprotein mediated-efflux chemoresistance using Solid Lipid Nanoparticles.

Multidrug resistance (MDR), whereby cancer cells become resistant to the cytotoxic effects of various structurally and mechanistically unrelated chemotherapeutic agents, is a major problem in the clinical treatment of cancer. P-glycoprotein (P-gp) is a transmembrane protein responsible for drug efflux, which decreases drug intracellular bioavailability, consequently decreasing their efficacy against cancer. Solid Lipid Nanoparticles (SLNs) have not only the ability to protect the entrapped drug against proteolytic degradation, but also allow a selective intracellular targeting. Hypothetically, the entrapped drug enter the target cells by different uptake mechanisms, "nanocitose", as compared to the free drug and may evade efflux-transporters, like P-gp. The functional role of P-gp in limiting the permeability of the anticancer drug paclitaxel (Ptx) was assessed in MDA-MB-436 cells. The observed increase in the pharmacologic efficacy of drug entrapped in SLN relatively to the free drug indicates that this system is shielding the drug. Therefore, "blinding" the nanoparticle from the efflux transporters. The effect was confirmed by the decrease expression of P-gp with loaded-SLNs and through the impact on cellular MDR1 expression. Besides the ability to prevent MDR events, functionalization of SLN with a specific antibody against membrane receptors (anti-CD44v6) improves the nanoparticle capability to target selectively malignant cells. This results allow to anticipate that poor clinical outcomes related to tumour P-gp overexpression might be overcome in a near future.

Self-assembly PEGylation assists SLN-paclitaxel delivery inducing cancer cell apoptosis upon internalization.

In past years, a considerable progress has been made in the conversion of conventional chemotherapy into potent and safe nanomedicines. The ultimate goal is to improve the therapeutic window of current chemotherapeutics by reducing systemic toxicities and to deliver higher concentrations of the chemotherapeutic agents to malignant cells. In this work, we report that PEGylation of the nanocarriers increases drug intracellular bioavailability leading therefore to higher therapeutic efficacy. The surface of the already patented solid lipid nanoparticles (SLN) loaded with paclitaxel (SLN-PTX) was coated with a PEG layer (SLN-PTX_PEG) through an innovative process to provide stable and highly effective nanoparticles complying with the predefined pharmaceutical quality target product profile. We observed that PEGylation not only stabilizes the SLN, but also modulates their cellular uptake kinetics. As a consequence, the intracellular concentration of chemotherapeutics delivered by SLN-PTX_PEG increases. This leads to the increase of efficacy and thus it is expected to significantly circumvent cancer cell resistance and increase patient survival and cure.

Experimental design towards an optimal lipid nanosystem: a new opportunity for paclitaxel-based therapeutics.

Lipid based nanoparticles represent a class of nanocarriers that have caused great expectation, particularly due to their suitability to incorporate BCS class II and IV drugs. The use of solid lipid nanoparticles (SLNs) as a nanocarrier for antineoplastic agents has been underexplored when compared to the encapsulation of the same agents in polymeric particles. The preparation and efficacy assessment of a SLN platform as drug delivery carrier for anticancer agents, herein proposed as a strategy to find innovative formulations, could dramatically improve the outcome of cancer therapy. Considering these lipid nanoparticles, despite the great amount of insights described in the literature, it seems that improving their manufacturability could be the missing step to convert this system into a drug product. A way to circumvent that problem would be to select a preparation method that could take advantage of the pharmaceutical industry installed capabilities, thus speeding-up the scale-up translational steps while maintaining both regulatory compliance and flexibility. The High Pressure Homogenization (HPH) has proved to be a reliable process for SLN preparation. However, the use of the high-shear mixer, a well established process to manufacture coarse dispersions at industrial scale, has still not been fully explored to prepare SLN. In this study, we explore the possibility of using the hot emulsification/solidification method to prepare SLN's that complies with the current pharmaceutical quality requirements. Thus, a high-shear based process that consistently accomplishes performance requirements was optimized in order to standardize the nanocarrier production following the identification of some process and formulation critical parameters. A hydrophobic drug, Paclitaxel (Ptx) was successfully incorporated using the proposed developed method. The particles physicochemical characteristics changes caused by the drug entrapment as well as the particles stability were also evaluated. In addition the ability of SLN to travel across biological barriers due to its matrix lipid nature was explored upon comparing the efficacy of the drug loaded SLN with the conventional marketed drug product (Taxol®). The cellular uptake studies showed that the developed Ptx loaded SLN were in fact internalized and demonstrated higher efficacy in the cancer cells death process than Taxol. The experimental data demonstrated that the hot homogenization technique using a high-shear mechanical homogenizer allows the preparation of suitable size (around 150 nm) SLN. Overall, the results obtained can be particularly impactful in the forthcoming SLN research.

Lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles.

UNLABELLED: Lymphatic drainage plays an important role in the uptake of particulates in the respiratory system, being also associated to the spreading of lung cancer through metastasis development. In recent years solid lipid nanoparticles (SLN) have been proposed as carriers of anti-tumoural drugs, for their low toxicity and surface characteristics make them suitable for either imaging (gamma-scintigraphy) or therapy upon encapsulation of cytotoxic drugs. Assessment of inhaled radiolabelled SLN biodistribution is described in the present work. METHODS: Nanoparticles (200 nm) were radiolabelled with 99mTc using the lipophilic chelator D,L-hexamehylpropyleneamine oxime (HMPAO). Biodistribution studies were carried out following aerosolisation and administration of a 99mTc-HMPAO-SLN suspension to a group of adult male Wistar rats. A 60 min dynamic image acquisition was performed in a gamma-camera, followed by static image collection at 30 min intervals up to 4 h postinhalation. Radiation counting was performed in organ samples, collected after the animals were sacrificed. RESULTS: The data show an important and significant uptake of the radiolabelled SLN into the lymphatics after inhalation, and a high rate of distribution in periaortic, axillar and inguinal lymph nodes. CONCLUSION: Results indicate that SLN could be effective colloidal carriers for lymphoscintigraphy or therapy upon pulmonary delivery.