The surface functionalization approach produced an innovative formulation having a much higher anti-proliferative effect than the bromelain solution, even though both in vitro proteolytic activity were similar, opening up a great opportunity for further studies in nanomedicine.

Methotrexate is a folic acid antagonist and its incorporation into nanoformulations is a promising strategy to increase the drug antiproliferative effect on human breast cancer cells by overexpressing folate receptors. To evaluate the efficiency and selectivity of nanoformulations containing methotrexate and its diethyl ester derivative, using two mechanisms of drug incorporation (encapsulation and surface functionalization) in the in vitro cellular uptake and antiproliferative activity in non-tumoral immortalized human keratinocytes (HaCaT) and in human breast carcinoma cells (MCF-7). Methotrexate and its diethyl ester derivative were incorporated into multiwall lipid-core nanocapsules with hydrodynamic diameters lower than 160 nm and higher drug incorporation efficiency. The nanoformulations were applied to semiconfluent HaCaT or MCF-7 cells. After 24 h, the nanocapsules were internalized into HaCaT and MCF-7 cells; however, no significant difference was observed between the nanoformulations in HaCaT (low expression of folate receptors), while they showed significantly higher cellular uptakes than the blank-nanoformulation in MCF-7, which was the highest uptakes observed for the drug functionalized-nanocapsules. No antiproliferative activity was observed in HaCaT culture, whereas drug-containing nanoformulations showed antiproliferative activity against MCF-7 cells. The effect was higher for drug-surface functionalized nanocapsules. In conclusion, methotrexate-functionalized-nanocapsules showed enhanced and selective antiproliferative activity to human breast cancer cells (MCF-7) being promising products for further in vivo pre-clinical evaluations.

PurposeThe low penetration of drugs across the blood–brain barrier (BBB) compromises the delivery of chemotherapeutic agents to the brain parenchyma and contributes to the poor prognosis of glioblastoma multiforme (GBM). We investigated the efficacy of methotrexate-loaded lipid-core nanocapsules (MTX-LNC) administered by the oral route to treat murine GBM, its ability to cross the BBB, and the mechanisms of MTX-LNC uptake by cultured GL261 glioma and BV2 microglia cells.Materials and methodsFemale C57B/6 mice were used in intravital microscopy assays to investigate the penetrance of rhodamine B-label MTX-LNC (RhoB/MTX-LNC) in the brain after oral or IV administration, and to evaluate the BBB integrity. Intracranial implantation of GL261 cells was undertaken to induce a murine GBM model, and the effectiveness of oral MTX or MTX-LNC treatments (started on Day 10 of GBM, every 2 days for 12 days) was quantified by tumor size, body weight, and leukogram. Pharmacological blockade of endocytic pathways was done to investigate the mechanisms of MTX-LNC uptake by cultured GL261 and microglia BV2 cells by using fluorescence microscopy. The effect of MTX-LNC or MTX on GL261 and BV2 proliferation was evaluated to compare the cytotoxicity of such compounds.ResultsRhoB/MTX-LNC was detected in brain parenchyma of mice after IV or oral administration, without any damage on BBB. Oral treatment with MTX-LNC reduced tumor volume and prevented weight loss and leukopenia in comparison to MTX-treated mice. MTX-LNC uptake by GL261 is caveolae-dependent, whereas endocytosis of MTX-LNC by BV2 occurs via phagocytosis and macropinocytosis. Both MTX-LNC and MTX reduced GL261 and BV2 proliferation; however, MTX-LNC showed higher efficacy in the inhibition of glioma proliferation.ConclusionTogether, we infer that the higher ability of MTX-LNC to cross the BBB and be captured by cancer and immune brain cells by different mechanisms is responsible for the higher efficacy of oral MTX-LNC treatment in GBM.

Background The analysis of plant material from Cannabis sativa L. has long been targeted on its main psychologically active metabolite, Δ9-tetrahydrocannabinol (THC). In addition to the diverse plant composition and medicinal interest in several cannabinoids, these compounds may also be related to the different characteristics of samples sold illegally. Currently, it is indisputable that other cannabinoids should also be considered on cannabis assays. Mass spectrometry has been used to identify and characterize substances in the most different scenarios, and knowing the analyte fragmentation profile is essential for characterizing samples of unknown origin. Objective In this work, a flow injection analysis–tandem mass spectrometry (FIA–ESI-MS/MS) with electrospray ionization in positive and negative modes was used to evaluate the fragmentation profiles of eight cannabinoids commonly found in cannabis samples: THC, tetrahydrocannabinolic acid (THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA) and cannabinol (CBN). Methods By exploring the fragmentation data from mass spectrometry, the samples were classified using a chemometric model of partial least squares discriminant analysis (PLS-DA). Results When ESI in negative mode is used with adequate collision energies, it is possible to identify differences in the fragmentation of isomers. Based on that, chemometric tools were employed to classify different samples. The PLS-DA applied to FIA–ESI-MS/MS data yielded satisfactory classification. Conclusion Thus, the results presented can be applied as a preliminary tool in the analysis of unknown samples, guiding for more accurate investigations in terms of chemical composition. Highlights Study of the cannabinoid fragmentation pattern by flow injection mass spectrometry. Cannabinoids distinction by fragment spectra after negative electrospray ionization. Multivariate data analysis (PLS-DA) allowed to classify different cannabis samples.

Cannabis sativa L is one of the most used drugs in the world. Information about the plant’s age and storage can help forensic scientists to identify and to track samples. The ratio between the cannabinoids tetrahydrocannabinol (THC) and cannabinol (CBN) has been related to the degradation of cannabis with time. Thus, this study aimed to test Multivariate Image Analysis (MIA) to evaluate cannabis extracts concerning its colors. Initially, 52 samples of Cannabis sativa L. extracts were analyzed by Gas Chromatography coupled to Flame Ionization Detector (GC/FID) to quantify THC and CBN. Afterwards, the extract samples were photographed and analyzed by two different multivariate analysis tools: ChemoStat®, a free chemometrics software, and PhotoMetrix PRO®, an app for mobile devices. Using exploratory analysis of principal component analysis (PCA) and hierarchical cluster analysis (HCA). It was observed that the more intense the color for an extract, the higher concentration of THC and CBN it has, while the lighter color extracts correspond to samples with no THC. The results suggest to propose a simple method for previous clustering of samples that may precede chromatographic analyzes, assist in chemical profile studies or simply aggregate samples of similar profiles for analyzed together.