13 research outputs found
Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells
Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosanâhsa-miRNA-145 (CSâmiRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CSâmiRNA nanocomplexes were produced with a range of (+/â) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200ânm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CSâmiRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CSâmiRNA complexes offer a promising non-viral platform for breast cancer gene therapy
Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells
Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500âÎŒM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability
In Vitro and Sensory Evaluation of Capsaicin-Loaded Nanoformulations
Capsaicin has known health beneficial and therapeutic properties. It is also able to enhance the permeability of drugs across epithelial tissues. Unfortunately, due to its pungency the oral administration of capsaicin is limited. To this end, we assessed the effect of nanoencapsulation of capsaicin, under the hypothesis that this would reduce its pungency. Core-shell nanocapsules with an oily core and stabilized with phospholipids were used. This system was used with or without chitosan coating. In this work, we investigated the in vitro release behavior of capsaicin-loaded formulations in different physiological media (including simulated saliva fluid). We also evaluated the influence of encapsulation of capsaicin on the cell viability of buccal cells (TR146). To study the changes in pungency after encapsulation we carried out a sensory analysis with a trained panel of 24 students. The in vitro release study showed that the systems discharged capsaicin slowly in a monotonic manner and that the chitosan coating had an effect on the release profile. The cytotoxic response of TR146 cells to capsaicin at a concentration of 500 ÎŒM, which was evident for the free compound, was reduced following its encapsulation. The sensory study revealed that a chitosan coating results in a lower threshold of perception of the formulation. The nanoencapsulation of capsaicin resulted in attenuation of the sensation of pungency significantly. However, the presence of a chitosan shell around the nanoformulations did not mask the pungency, when compared with uncoated systems
Nanoencapsulated capsaicin changes migration behavior and morphology of madin darby canine kidney cell monolayers
We have developed a drug delivery nanosystem based on chitosan and capsaicin. Both substances have a wide range of biological activities. We investigated the nanosystemâs influence on migration and morphology of Madin Darby canine kidney (MDCK-C7) epithelial cells in comparison to the capsaicin-free nanoformulation, free capsaicin, and control cells. For minimally-invasive quantification of cell migration, we applied label-free digital holographic microscopy (DHM) and single-cell tracking. Moreover, quantitative DHM phase images were used as novel stain-free assay to quantify the temporal course of global cellular morphology changes in confluent cell layers. Cytoskeleton alterations and tight junction protein redistributions were complementary analyzed by fluorescence microscopy. Calcium influx measurements were conducted to characterize the influence of the nanoformulations and capsaicin on ion channel activities. We found that both, capsaicin-loaded and unloaded chitosan nanocapsules, and also free capsaicin, have a significant impact on directed cell migration and cellular motility. Increase of velocity and directionality of cell migration correlates with changes in the cell layer surface roughness, tight junction integrity and cytoskeleton alterations. Calcium influx into cells occurred only after nanoformulation treatment but not upon addition of free capsaicin. Our results pave the way for further studies on the biological significance of these findings and potential biomedical applications, e.g. as drug and gene carriers
Low-Molecular-Weight Dextran Sulfate Nanocapsules Inhibit the Adhesion of Helicobacter pylori to Gastric Cells
The Gram-negative bacterium Helicobacter pylori is the most common bacterial pathogen in humans, infecting 24â79% of the population at any time. Standard eradication protocols involve multi-target therapy including combinations of antibiotics, which has promoted the emergence of resistant strains. To address this challenge, we prepared antibiotic-free colloidal nanoparticles designed to interfere with the adhesion mechanisms of H. pylori and thus prevent both the onset and recurrence of infection. Our colloidal particles comprised a nanocapsule (NC) formulation based on an oil-core nanoemulsion co-stabilized with lysozyme and lecithin, coated with negatively charged low-molecular-weight (DexS40-NC) or high-molecular-weight (DexS500-NC) dextran sulfate, or positively charged chitosan (CSHMC+30-NC). The oil core of all NC formulations was also loaded with curcumin, a model lipophilic phytochemical substance with well-documented anti-inflammatory and anti-tumor activities. Our proof-of-principle experiments showed that the DexS40-NC formulation inhibited the adhesion of H. pylori to AGS stomach cells in a dose-dependent manner. DexS40-NC achieved more potent inhibition than DexS500-NC or uncoated control nanoemulsions, whereas the effect of CSHMC+30-NC was not clear-cut given the ability of this formulation to aggregate bacteria. DexS40-NC, unlike DexS500-NC, showed no cytotoxic effects against AGS, Caco-2, or MDCK cell lines. DexS40-NC is, therefore, a promising candidate for further development as an alternative or complementary therapy against H. pylori infections
A national program for detection of alpha 1-antitrypsin deficiency in Italy. Gruppo I.D.A.
alpha 1-antitrypsin (AAT) deficiency is an inherited condition characterized by low serum levels of AAT and an increased risk of developing pulmonary emphysema. The disease occurs mainly in Caucasians, but Southern Europe, including Italy, is considered a low prevalence area. We developed a national program in Italy in order to improve our knowledge of the epidemiology of AAT deficiency and to establish a registry of the AAT-deficient individuals. The program had two phases: the first lasted 36 months, during which blood from coupons mailed by respiratory physicians from throughout the country, was isoelectrofocused by the Central Laboratory in Rome. The second phase started in February 1996, and the Registry was established. Up to August 1998, 151 subjects with AAT deficiency have been identified and 64 have been enrolled in the Registry. We believe that such a program plays a crucial role in identifying AAT deficiency in a country such as Italy, with low prevalence and low awareness of this rare condition
Chitosan-based nanocapsules: physical characterization, stability in biological media and capsaicin encapsulation
International audienc
Structure of Chitosan Determines Its Interactions with Mucin
Synthetic
and natural mucoadhesive biomaterials in optimized galenical
formulations are potentially useful for the transmucosal delivery
of active ingredients to improve their localized and prolonged effects.
Chitosans (CS) have potent mucoadhesive characteristics, but the exact
mechanisms underpinning such interactions at the molecular level and
the role of the specific structural properties of CS remain elusive.
In the present study we used a combination of microviscosimetry, zeta
potential analysis, isothermal titration calorimetry (ITC) and fluorescence
quenching to confirm that the soluble fraction of porcine stomach
mucin interacts with CS in water or 0.1 M NaCl (at <i>c</i> < <i>c</i>*; relative viscosity, η<sub>rel</sub>, ⌠2.0 at pH 4.5 and 37 °C) via a heterotypic stoichiometric
process significantly influenced by the degree of CS acetylation (DA).
We propose that CSâmucin interactions are driven predominantly
by electrostatic binding, supported by other forces (e.g., hydrogen
bonds and hydrophobic association) and that the DA influences the
overall conformation of CS and thus the nature of the resulting complexes.
Although the conditions used in this model system are simpler than
the typical in vivo environment, the resulting knowledge will enable
the rational design of CS-based nanostructured materials for specific
transmucosal drug delivery (e.g., for Helicobacter
pylori stomach therapy)
Raster-scanning optoacoustic mesoscopy for gastrointestinal imaging at high resolution.
In vivo optical imaging modalities are mostly limited to cell cultures, superficial tissues, and intravital imaging since they lack either resolution or penetration depth.1 In contrast, optoacoustic (OA) imagingâcombining features of optical and ultrasound imagingâhas been used to visualize hemoglobin in depths of approximately 3 cm in patients with Crohnâs disease.2,3 Realizing an even higher resolution, raster-scanning OA mesoscopy (RSOM) provides intrinsic optical tissue contrast down to 10-20 ÎŒm resolution at still high penetration depths of several millimeters