Physicochemical characterization of FRET-labelled chitosan nanocapsules and model degradation studies

Sub-micron o/w emulsions coated with chitosan have been used for drug delivery, quorum sensing inhibition, and vaccine development. To study interactions with biological systems, nanocapsules have been fluorescently labelled in previous works, but it is often difficult to distinguish the released label from intact nanocapsules. In this study, we present advanced-labelling strategies based on Förster Resonance Energy Transfer (FRET) measurements for chitosan-coated nanocapsules and investigate their dissolution and degradation. We used FRET measurements of nanocapsules loaded with equimolar concentrations of two fluorescent dyes in their oily core and correlated them with dynamic light scattering (DLS) count rate measurement and absorbance measurements during their disintegration by dissolution. Using count rate measurements, we also investigated the enzymatic degradation of nanocapsules using pancreatin and how protein corona formation influences their degradation. Of note, nanocapsules dissolved in ethanol, while FRET decreased simultaneously with count rate, and absorbance was caused by nanocapsule turbidity, indicating increased distance between dye molecules after their release. Nanocapsules were degradable by pancreatin in a dose-dependent manner, and showed a delayed enzymatic degradation after protein corona formation. We present here novel labelling strategies for nanocapsules that allow us to judge their status and an in vitro method to study nanocapsule degradation and the influence of surface characteristics

Co-assembly of chitosan and phospholipids into hybrid hydrogels

Novel hybrid hydrogels were formed by adding chitosan (Ch) to phospholipids (P) self-assembled particles in lactic acid. The effect of the phospholipid concentration on the hydrogel properties was investigated and was observed to affect the rate of hydrogel formation and viscoelastic properties. A lower concentration of phospholipids (0.5% wt/v) in the mixture, facilitates faster network formation as observed by Dynamic Light Scattering, with lower elastic modulus than the hydrogels formed with higher phospholipid content. The nano-porous structure of Ch/P hydrogels, with a diameter of 260±20 nm, as observed by cryo-scanning electron microscopy, facilitated the penetration of water and swelling. Cell studies revealed suitable biocompatibility of the Ch/P hydrogels that can be used within life sciences applications

Differences of the tumour cell glycocalyx affect binding of capsaicin-loaded chitosan nanocapsules

The glycocalyx regulates the interaction of mammalian cells with extracellular molecules, such as cytokines. However, it is unknown to which extend the glycocalyx of distinct cancer cells control the binding and uptake of nanoparticles. In the present study, exome sequencing data of cancer patients and analysis of distinct melanoma and bladder cancer cell lines suggested differences in cancer cell-exposed glycocalyx components such as heparan sulphate. Our data indicate that glycocalyx differences affected the binding of cationic chitosan nanocapsules (Chi-NCs). The pronounced glycocalyx of bladder cancer cells enhanced the internalisation of nanoencapsulated capsaicin. Consequently, capsaicin induced apoptosis in the cancer cells, but not in the less glycosylated benign urothelial cells. Moreover, we measured counterion condensation on highly negatively charged heparan sulphate chains. Counterion condensation triggered a cooperative binding of Chi-NCs, characterised by a weak binding rate at low Chi-NC doses and a strongly increased binding rate at high Chi-NC concentrations. Our results indicate that the glycocalyx of tumour cells controls the binding and biological activity of nanoparticles. This has to be considered for the design of tumour cell directed nanocarriers to improve the delivery of cytotoxic drugs. Differential nanoparticle binding may also be useful to discriminate tumour cells from healthy cells

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 nanosystem’s 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

Cytotoxicity of silica nanoparticles through exocytosis of von Willebrand factor and necrotic cell death in primary human endothelial cells.

Nanoparticle-induced endothelial cell (EC) dysfunction, due to the induction of inflammation and/or the activation of the coagulation system, is associated with pulmonary and ischemic cardiovascular diseases. Although it is contigent on several mechanisms, involving formation of reactive oxygen species and inflammatory cytokines such as interleukin (IL)-6 and 8, the involvement of the coagulation system is not well understood. The results of toxicity assays using the tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) release showed that silica NP-induced cytotoxicity depends on the size and the dose of applied NP. Moreover, propidium iodide (PI) stainings and caspase 3/7 assays identified increased necrosis in ECs. Exposing human umbilical vein endothelial cells (HUVECs) to SiO(2) NP with diameters of 304 nm and 310 nm led to significant increase of Weibel-Palade body (WPB) exocytosis, associated with the release of von Willebrand factor (VWF) and the formation of ultralarge fibers (ULVWF). High resolution microscopy techniques revealed that internalization and perinuclear localization of perylene-labeled NP with a size of 310 nm affect not only viability, but also cell migration and proliferation. In conclusion, our data indicate that NP-induced activation and dysfunction of ECs is reflected by release of VWF and necrotic cell death

Bladder cancer-derived interleukin-1 converts the vascular endothelium into a pro-inflammatory and pro-coagulatory surface

Background!#!Bladder cancer cells orchestrate tumour progression by pro-inflammatory cytokines. Cytokines modulate the local tumour microenvironment and increase the susceptibility of tumour distant tissues for metastasis. Here, we investigated the impact of human bladder cancer cell derived factors on the ability to modulate and activate human vascular endothelial cells.!##!Methods!#!The pro-inflammatory and pro-coagulatory potential of four different bladder cancer cell lines was accessed by qRT-PCR arrays and ELISA. Modulation and activation of endothelial cells was studied in microfluidic devices. Clinical relevance of our findings was confirmed by immune histology in tissue samples of bladder cancer patients and public transcriptome data.!##!Results!#!The unbalanced ratio between interleukin (IL)-1 and IL-1 receptor antagonist (IL-1ra) in the secretome of bladder cancer cells converted the quiescent vascular endothelium into a pro-adhesive, pro-inflammatory, and pro-coagulatory surface. Microfluidic experiments showed that tumour cell induced endothelial cell activation promoted leukocyte recruitment and platelet adhesion. Human bladder cancer tissue analysis confirmed that loss of IL-1ra and elevated IL-1 expression was associated with enhanced cancer progression.!##!Conclusions!#!Our data indicate that IL-1 and IL-1ra were dysregulated in bladder cancer and could facilitate tumour dissemination through endothelial cell activation. Targeting the IL-1/IL-1ra axis might attenuate tumour-mediated inflammation and metastasis formation

CD33-related siglecs in inflammatory responses

Ca2+ ions act as second messengers to transduce extracellular stimuli (e.g. those from hormones, neurotransmitters and growth factors) into a variety of intracellular activities which include cell division, contraction and cell death. Ca2+ is able to control and coordinate so many cellular functions because extracellular stimuli each evoke complex, spatially- and temporally-organized changes in cytoplasmic Ca2+ concentration ([Ca2+]c). Of particular significance, is the ability of cells to localize cytoplasmic Ca2+ signals to certain regions of the cell. It is this localization which enables the control of specific cellular activities to proceed from distinct regions. In these regions (microdomains) the Ca2+ concentration may significantly exceed the bulk average [Ca2+]c value. For example, depolarization of the plasma membrane activates a voltage-dependent Ca2+ current to evoke a rise in [Ca2+] in the subplasma membrane space and bulk cytoplasm. The rise in the bulk cytoplasm ([Ca2+]c) is approximately uniform throughout the cell - a feature that would usefully promote even contraction of the muscle cell. However, simultaneously, the Ca2+ rise in subplasma membrane space ([Ca2+]PM) has a wide range of amplitudes and time courses. The [Ca2+]PM variations reflect an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma and enables local activation of specific effectors. In contrast to depolarisation-evoked Ca2+ rises, Ca2+ release from the internal store is not uniform throughout the bulk cytoplasm but begins at one site and progresses from there as a travelling spatial gradient of Ca2+ (a Ca2+ wave). Ca2+ waves are modulated in small regions of the cell by the local control of IP3R and RyR. Local activity of mitochondria specifically regulates the activity of IP3R to promote or inhibit wave progression. IP3R or RyR may also be controlled by endogenous proteins associated with the receptors, such as the 12 kDa FK506 binding protein (FKBP12), which may locally regulate release directly or indirectly via inhibition of the phosphatase calcineurin or via the kinase mammalian target of rapamycin (mTOR). Together, these processes and controls contribute to the generation of the complex Ca2+ signalling patterns that are required to transduce extracellular stimuli to physiological responses