Snow Stolnik

Tight junction modulation by chitosan nanoparticles: Comparison with chitosan solution

Present work investigates the potential of chitosan nanoparticles, formulated by the ionic gelation with tripolyphosphate (TPP), to open the cellular tight junctions and in doing so, improve the permeability of model macromolecules. A comparison is made with chitosan solution at equivalent concentrations. Initial work assessed cytotoxicity (through MTS and LDH assays) of chitosan nanoparticles and solutions on Calu-3 cells. Subsequently, a concentration of chitosan nanoparticles and solution exhibiting minimal toxicity was used to investigate the effect on TEER and macromolecular permeability across filter-cultured Calu-3 monolayer. Chitosan nanoparticles and solution were also tested for their effect on the distribution of the tight junction protein, zonnula occludens-1 (ZO-1). Chitosan nanoparticles produced a sharp and reversible decrease in TEER and increased the permeability of two FITC-dextrans (FDs), FD4 (MW 4 kDa) and FD10 (MW 10 kDa), with effects of a similar magnitude to chitosan solution. Chitosan nanoparticles produced changes in ZO-1 distribution similar to chitosan solution, indicating a tight junction effect. While there was no improvement in permeability with chitosan nanoparticles compared to solution, nanoparticles provide the potential for drug incorporation, and hence the possibility for providing controlled drug release and protection from enzymatic degradation.

Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells.

This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.

The colloidal properties of surfactant-free biodegradable nanospheres from poly(β-malic acid-co-benzyl malate)s and poly(lactic acid-co-glycolide)

Abstract The colloidal properties of surfactant-free nanospheres prepared from biodegradable polyesters, i.e. a range of benzyl esters of poly(β-malic acid) (PMLABeH) and poly(lactic acid-co-glycolide) (PLGA) have been investigated. The results reveal a significant difference in the colloidal behaviour of the nanosphere systems, depending on the composition of the copolymer used in their preparation. The nanospheres from the PLGA and fully benzylated PMLABe 100 polymer were relatively less stable to addition of electrolyte and coagulated around the pH which coincided with the dissociation constant of the carboxyl group. The coagulation was associated with a decrease in the zeta potential below a certain critical value needed for electrostatical stabilization. In comparison, nanospheres prepared from partly benzylated PMLABe 80 H 20 and PMLABe 90 H 10 copolymers did not coagulate at relatively high electrolyte concentrations and at low pH values of the suspending buffer. This may be interpreted in terms of an involvement of a steric component to nanosphere stabilization, whereby the steric component appears to be associated with the presence of an increased level of carboxyl groups on the nanosphere surface. The results suggest a different construction of nanospheres prepared from partly benzylated copolymers from those prepared from fully benzylated PMLABe 100 and PLGA copolymers.

Effect of PEGylation on the toxicity and permeability enhancement of chitosan

The aim of the present work is to investigate if conditions can be devised where PEGylation of chitosan would reduce its toxicity toward the nasal mucosa while maintaining its ability to open the cellular tight junctions and, consequently, produce an enhancement of macromolecular permeability. A series of mPEG-g-chitosan copolymers with varying levels of mPEG substitution, mPEG molecular weight, and chitosan molecular weight were synthesized by grafting carboxylic acid-terminated mPEGs (Mw 1.9 and 5.0 × 10(3) g mol(-1)) to chitosans (Mw 28.9 and 82.0 × 10(3) g mol(-1)) using a NHS/EDC coupling system. The synthesized mPEG-g-chitosans were fully characterized using a number of techniques, including FT-IR, (1)H NMR, and SEC-MALLS and their physicochemical properties were analyzed by TGA and DSC. Thereafter, the conjugates were tested for their cytotoxicity and tight junction modulating property in a relevant cell model, a mucus producing Calu-3 monolayer. mPEG-g-chitosan conjugates exhibited reduced toxicity toward cells, as compared to unmodified chitosan counterparts. Furthermore, the conjugates demonstrated a dramatic effect on cell monolayer transepithelial electrical resistance (TEER) and enhancement of permeability of model macromolecules. TEER and permeability-enhancing effects, as measurable indicators of tight junction modulation, were found to be pH-dependent and were notably more pronounced than those exhibited by unmodified chitosans. This work therefore demonstrates that conditions can be contrived where PEGylation improves the toxicity profile of chitosan, while preserving its effect on epithelial tight junctions in the nose.

Modular Construction of Multifunctional Bioresponsive Cell-Targeted Nanoparticles for Gene Delivery

Multifunctional and modular block copolymers prepared from biocompatible monomers and linked by a bioreducible disulfide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block copolymer architecture enabled well-defined nanoparticles to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folate-receptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to nonbioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tunable way.

The preparation of sub-200 nm biodegradable colloidal particles from poly(β-malic acid-co-benzyl malate) copolymers and their surface modification with Poloxamer and Poloxamine surfactants

Abstract In this paper a simple precipitation-solvent evaporation method for the preparation of stable spherical sub-200 nm colloids from novel biodegradable poly (β-malic acid-co-benzyl malate) (PMLABeH) copolymers is described. The particle size and zeta potential were found to be dependent on copolymer composition and concentration of polymer in the organic phase. A range of Poloxamer and Poloxamine polymers was used to modify the surface characteristics of the PMLABeH nanoparticles. The presence of the stabilizers led to a reduction in the surface potential and an increase in surface hydrophilicity of the PMLABeH nanoparticles. Differences observed in the adsorption behaviour of the Poloxamer and Poloxamine stabilizers on the PMLABeH colloids were attributed to an involvement of electrostatic interactions between the carboxyl groups on the surface of the nanoparticles and the tertiary nitrogens in the Poloxamine molecules. A significant difference in the coating thickness, surface hydrophobicity and zeta potential was seen between PMLABeH and polystyrene latexes coated with the Poloxamer and Poloxamine surfactants, suggesting a difference in the coating behaviour for these two pariculate systems.

Nanoparticle transport in epithelial cells: pathway switching through bioconjugation

The understanding and control of nanoparticle transport into and through cellular compartments is central to biomedical applications of nanotechnology. Here, it is shown that the transport pathway of 50 nm polystyrene nanoparticles decorated with vitamin B12 in epithelial cells is different compared to both soluble B12 ligand and unmodified nanoparticles, and this is not attributable to B12 recognition alone. Importantly, the study indicates that vitamin B12 -conjugated nanoparticles circumnavigate the lysosomal compartment, the destination of soluble vitamin B12 ligand. Whereas cellular trafficking of soluble B12 is confirmed to occur via the clathrin-mediated pathway, transport of B12 -conjugated nanoparticles appears to predominantly take place by a route that is perturbed by caveolae-specific inhibitors. This data suggests that, following its conjugation to nanoparticles, in addition to dramatically increasing the cellular uptake of nanoparticles, the normal cell trafficking of B12 is switched to an alternative pathway, omitting the lysosomal stage: a result with important implications for oral delivery of nanoparticulate diagnostics and therapeutics.

Barrier characteristics of epithelial cultures modelling the airway and intestinal mucosa: a comparison.

The barrier characteristics of polarized layers of Calu-3 and Caco-2 cell lines, as commonly used in vitro models of intestinal and airway mucosa, respectively, were investigated by assessing the translocation of model macromolecules and nanoparticles. The barrier capacity of the cell layers towards the movement of macromolecules and nanoparticulates differed considerably between the cell lines. Permeability studies revealed the existence of a notably larger solute molecular weight limit for paracellular diffusion in Caco-2 monolayers compared to Calu-3 cells. Removal of mucus in Calu-3 cells resulted in cell layers exhibiting a larger macromolecular permeability, in addition to improved nanoparticle translocation. Microscopic examination of the tight junctions, as cellular features that play a major role in preventing transepithelial movement of macromolecules, revealed that the appearance of cell-cell boundaries was notably different in the two cell lines, which could explain the differences in macromolecular permeability. The data overall showed that epithelial layers of airway Calu-3 and intestinal Caco-2 cell cultures in vitro exhibit a different level of restrictiveness and this is due to the cell morphology and the presence of mucus.

Uptake and transport of B12-conjugated nanoparticles in airway epithelium

Non-invasive delivery of biotherapeutics, as an attractive alternative to injections, could potentially be achieved through the mucosal surfaces, utilizing nanoscale therapeutic carriers. However, nanoparticles do not readily cross the mucosal barriers, with the epithelium presenting a major barrier to their translocation. The transcytotic pathway of vitamin B12 has previously been shown to ‘ferry’ B12-decorated nanoparticles across intestinal epithelial (Caco-2) cells. However, such studies have not been reported for the airway epithelium. Furthermore, the presence in the airways of the cell machinery responsible for transepithelial trafficking of B12 is not widely reported. Using a combination of molecular biology and immunostaining techniques, our work demonstrates that the bronchial cell line, Calu-3, expresses the B12-intrinsic factor receptor, the transcobalamin II receptor and the transcobalamin II carrier protein. Importantly, the work showed that sub-200 nm model nanoparticles chemically conjugated to B12 were internalised and transported across the Calu-3 cell layers, with B12 conjugation not only enhancing cell uptake and transepithelial transport, but also influencing intracellular trafficking. Our work therefore demonstrates that the B12 endocytotic apparatus is not only present in this airway model, but also transports ligand-conjugated nanoparticles across polarised epithelial cells, indicating potential for B12-mediated delivery of nanoscale carriers of biotherapeutics across the airways.

Epithelial toxicity of alkylglycoside surfactants

Alkylglycoside surfactants have been proposed as drug delivery excipients with the potential to enhance mucosal drug absorption of therapeutic macromolecules. Previous work reported their drug absorption-promoting potential by demonstrating that several compounds within this class of surfactants improve mucosal absorption of peptides, proteins and other macromolecules. However, detailed investigation of their toxicity has not been conducted. Using Calu-3 epithelial cell layers as a model of the airway mucosa, and liposomes as models of cell membranes, this work investigates the cytotoxicity of dodecylmaltoside, tridecylmaltoside and tetradecylmaltoside, as representative alkylglycosides. A combination of different toxicity assays and other tests indicating cell membrane disruption were used to assess cytotoxicity. The alkylglycosides tested induced a dramatic reduction in cell viability, cell membrane and liposome-disruptive effects, as well as abrogation of transepithelial electrical resistance that did not recover completely. Importantly, these phenomena were noted at concentrations markedly lower than those typically used in the literature studies demonstrating the absorption-enhancing properties of alkylglycosides. This work therefore demonstrates that alkylglycosides exhibit significant toxicity towards airway epithelial cells, most likely resulting from a membrane-damaging effect, highlighting a need for further evaluation of their safety as absorption-enhancing excipients.