288 research outputs found
Ultra-Filtration of Human Serum for Improved Quantitative Analysis of Low Molecular Weight Biomarkers using ATR-IR Spectroscopy
Infrared spectroscopy is a reliable, rapid and cost effective characterisation technique, delivering a molecular finger print of the sample. It is expected that its sensitivity would enable detection of small chemical variations in biological samples associated with disease. ATR-IR is particularly suitable for liquid sample analysis and, although air drying is commonly performed before data collection, just a drop of human serum is enough for screening and early diagnosis. However, the dynamic range of constituent biochemical concentrations in the serum composition remains a limiting factor to the reliability of the technique. Using glucose as a model spike in human serum, it has been demonstrated in the present study that fractionating the serum prior to spectroscopic analysis can considerably improve the precision and accuracy of quantitative models based on the Partial Least Squares Regression algorithm. By depleting the abundant high molecular weight proteins, which otherwise dominate the spectral signatures collected, the ability to monitor changes in the concentrations of the low molecular weight constituents is enhanced. The Root Mean Square Error for the Validation set (RMSEV) has been improved by a factor of 5 following human serum processing with an average relative error in the predictive values below 1% is achieved. Moreover, the approach is easily transferable to different bodily fluids, which would support the development of more efficient and suitable clinical protocols for exploration of vibrational spectroscopy based ex-vivo diagnostic tools
Peptides to Overcome the Limitations of Current Anticancer and Antimicrobial Nanotherapies
Biomedical research devotes a huge effort to the development of efficient non-viral nanovectors (NV) to improve the effectiveness of standard therapies. NVs should be stable, sustainable and biocompatible and enable controlled and targeted delivery of drugs. With the aim to foster the advancements of such devices, this review reports some recent results applicable to treat two types of pathologies, cancer and microbial infections, aiming to provide guidance in the overall design of personalized nanomedicines and highlight the key role played by peptides in this field. Additionally, future challenges and potential perspectives are illustrated, in the hope of accelerating the translational advances of nanomedicine
Quantitative and qualitative effect of gH625 on the nanoliposome-mediated delivery of mitoxantrone anticancer drug to HeLa cells
The present work investigates in vitro the delivery of the anticancer drug mitoxantrone (MTX) to HeLa cancer cells by means of liposomes functionalized with the novel cell penetrating peptide gH625. This hydrophobic peptide enhances the delivery of doxorubicin to the cytoplasm of cancer cells, while the mechanism of this enhancement has not yet been understood. Here, in order to get a better insight into the role of gH625 on the mechanism of liposome-mediated drug delivery, we treated HeLa cells with liposomes functionalized with gH625 and loaded with MTX; liposome were characterized in terms of their physico-chemical properties and drug release kinetics. To quantify the MTX uptake and to study the subcellular drug distribution and interaction, we took advantage of the intrinsic fluorescence of MTX and of the fluorescence-based techniques like fluorescence-activated cell sorting (FACS) and confocal spectral imaging (CSI). gH625 liposomes showed an enhanced staining of the internalized drug is observed mainly in hydrophobic regions of the cytoplasm, where the increased presence of an oxidative metabolite of the drug is observed. MTX delivery with gH625-decorated nanoliposomes enhances the quantity of both the intracellular drug and of its oxidative metabolite and contributes to higher anticancer efficacy of the drug
Analysis of Bodily Fluids Using Vibrational Spectroscopy: a Direct Comparison of Raman Scattering and Infrared Absorption Techniques for the Case of Glucose in Blood Serum
Analysis of biomarkers present in the blood stream can potentially deliver crucial information on patient health and indicate the presence of numerous pathologies. The potential of vibrational spectroscopic analysis of human serum for diagnostic purposes has been widely investigated and, in recent times, infrared absorption spectroscopy, coupled with ultra-filtration and multivariate analysis techniques, has attracted increasing attention, both clinical and commercial. However, such methods commonly employ a drying step, which may hinder the clinical work flow and thus hamper their clinical deployment. As an alternative, this study explores the use of Raman spectroscopy, similarly coupled with ultra-filtration and multivariate analysis techniques, to quantitatively monitor diagnostically relevant changes of glucose in liquid serum samples, and compares the results with similar analysis protocols using infrared spectroscopy of dried samples. The analysis protocols to detect the imbalances in glucose using Raman spectroscopy are first demonstrated for aqueous solutions and spiked serum samples. As in the case of infrared absorption studies, centrifugal filtration is utilised to deplete abundant analytes and to reveal the spectral features of Low Molecular Weight Fraction analytes in order to improve spectral sensitivity and detection limits. Improved Root Mean Square Error of Cross Validation (RMSECV) was observed for Raman prediction models, whereas slightly higher R2 values were reported for infrared absorption prediction models. Summarising, it is demonstrated that the Raman analysis protocol can yield accuracies which are comparable with those reported using infrared absorption based measurements of dried serum, without the need for additional drying steps
Label-Free Quantification of Nanoencapsulated Piperonyl Esters in Cosmetic Hydrogels Using Raman Spectroscopy
Raman spectroscopy is a well-established technique for the molecular characterisation of samples and does not require extensive pre-analytical processing for complex cosmetic products. As an illustration of its potential, this study investigates the quantitative performance of Raman spectroscopy coupled with partial least squares regression (PLSR) for the analysis of Alginate nanoencapsulated Piperonyl Esters (ANC-PE) incorporated into a hydrogel. A total of 96 ANC-PE samples covering a 0.4% w/w–8.3% w/w PE concentration range have been prepared and analysed. Despite the complex formulation of the sample, the spectral features of the PE can be detected and used to quantify the concentrations. Using a leave-K-out cross-validation approach, samples were divided into a training set (n = 64) and a test set, samples that were previously unknown to the PLSR model (n = 32). The root mean square error of cross-validation (RMSECV) and prediction (RMSEP) was evaluated to be 0.142% (w/w PE) and 0.148% (w/w PE), respectively. The accuracy of the prediction model was further evaluated by the percent relative error calculated from the predicted concentration compared to the true value, yielding values of 3.58% for the training set and 3.67% for the test set. The outcome of the analysis demonstrated the analytical power of Raman to obtain label-free, non-destructive quantification of the active cosmetic ingredient, presently PE, in complex formulations, holding promise for future analytical quality control (AQC) applications in the cosmetics industry with rapid and consumable-free analysis
Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum
Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR—partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400–1500 cm−1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies
Monitoring Dermal Penetration and Permeation Kinetics of Topical Products; the Role of Raman Microspectroscopy
The study of human skin represents an important area of research and development in dermatology, toxicology, pharmacology and cosmetology, in order to assess the effects of exogenous agents, their interaction, their absorption mechanism, and/or their toxicity towards the different cutaneous structures. The processes can be parameterised by mathematical models of diffusion, of varying degrees of complexity, and are commonly measured by Franz cell diffusion, in vitro, and tape stripping, in vitro or in vivo, techniques which are recognised by regulatory bodies for commercialisation of dermally applied products. These techniques do not directly provide chemically specific measurement of the penetration and/or permeation of formulations in situ, however.
Raman microspectroscopy provides a non-destructive, non-invasive and chemically specific methodology for in vitro, and in vivo investigations, in-situ, and can provide a powerful alternative to the current gold standard methods approved by regulatory bodies.
This review provides an analysis of the current state of art of the field of monitoring dermal penetration and permeation kinetics of topical products, in vitro and in vivo, as well as the regulatory requirements of international guidelines governing them. It furthermore outlines developments in the analysis of skin using Raman microspectroscopy, towards the most recent demonstrations of quantitative monitoring of the penetration and permeation kinetics of topical products in situ, for in vitro and in vivo applications, before discussing the challenges and future perspectives of the field
Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells.
International audienceExpression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects
Bacterial cellulose hydrogel loaded with lipid nanoparticles for localized cancer treatment
The use of hybrid materials, where a matrix sustains nanoparticles controlling the release of the chemotherapeutic drug, could be beneficial for the treatment of primary tumors prior or after surgery. This localized chemotherapy would guarantee high drug concentrations at the tumor site while precluding systemic drug exposure minimizing undesirable side effects. We combined bacterial cellulose hydrogel (BC) and nanostructured lipid carriers (NLCs) including doxorubicin (Dox) as a drug model. NLCs loaded with cationic Dox (NLCs-H) or neutral Dox (NLCs-N) were fully characterized and their cell internalization and cytotoxic efficacy were evaluated in vitro against MDA-MB-231 cells. Thereafter, a fixed combination of NLCs-H and NLCs-N loaded into BC (BC-NLCs-NH) was assayed in vivo into an orthotopic breast cancer mouse model. NLCs-H showed low encapsulation efficiency (48%) and fast release of the drug while NLCs-N showed higher encapsulation (97%) and sustained drug release. Both NLCs internalized via endocytic pathway, while allowing a sustained release of the Dox, which in turn rendered IC50 values below of those of free Dox. Taking advantage of the differential drug release, a mixture of NLCs-N and NLCs-H was encapsulated into BC matrix (BC-NLCs-NH) and assayed in vivo, showing a significant reduction of tumor growth, metastasis incidence and local drug toxicities.Centro de Investigación y Desarrollo en Fermentaciones IndustrialesCentro de Química Inorgánic
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