Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic properties. This study provides an in vitro model for further investigation into the pathogenesis of Haemophilus species and the foundation for exploring whether H. haemolyticus can be used to prevent NTHi disease

Chitosan-coated mesoporous MIL-100(Fe) nanoparticles as improved bio-compatible oral nanocarriers

Nanometric biocompatible Metal-Organic Frameworks (nanoMOFs) are promising candidates for drug delivery. Up to now, most studies have targeted the intravenous route, related to pain and severe complications; whereas nanoMOFs for oral administration, a commonly used non-invasive and simpler route, remains however unexplored. We propose here the biofriendly preparation of a suitable oral nanocarrier based on the benchmarked biocompatible mesoporous iron(III) trimesate nanoparticles coated with the bioadhesive polysaccharide chitosan (CS). This method does not hamper the textural/ structural properties and the sorption/release abilities of the nanoMOFs upon surface engineering. The interaction between the CS and the nanoparticles has been characterized through a combination of high resolution soft X-ray absorption and computing simulation, while the positive impact of the coating on the colloidal and chemical stability under oral simulated conditions is here demonstrated. Finally, the intestinal barrier bypass capability and biocompatibility of CS-coated nanoMOF have been assessed in vitro, leading to an increased intestinal permeability with respect to the noncoated material, maintaining an optimal biocompatibility. In conclusion, the preservation of the interesting physicochemical features of the CS-coated nanoMOF and their adapted colloidal stability and progressive biodegradation, together with their improved intestinal barrier bypass, make these nanoparticles a promising oral nanocarrier

Enhancing Biopharmaceutical Attributes of Phospholipid Complex-loaded Nanostructured Lipidic Carriers of Mangiferin: Systematic Development, Characterization and Evaluation

Mangiferin (Mgf), largely expressed out from the leaves and stem bark of Mango, is a potent antioxidant. However, its in vivo activity gets tremendously reduced owing to poor aqueous solubility and inconsistent gastrointestinal absorption, high hepatic first-pass metabolism and high P-gp efflux. The current research work, therefore, was undertaken to overcome the biopharmaceutical hiccups by developing the Mgf-phospholipid complex (PLCs) loaded in nanostructured lipidic carriers (NLCs). The PLCs and NLCs were prepared using refluxing, solvent evaporation and hot emulsification technique, respectively with various molar ratios of Mgf and Phospholipon 90 G, i.e., 1:1; 1:2; and 1:3. The complex was evaluated for various physicochemical parameters like drug content (96.57%), aqueous solubility (25-fold improved) and oil-water partition coefficient (10-fold enhanced). Diverse studies on the prepared complex using FTIR, DSC, PXRD and SEM studies ratified the formation of PLCs at 1:1 ratio. The PLCs were further incorporated onto NLCs, which were systematically optimized employing a face centered cubic design (FCCD), while evaluating for particle size, zeta potential, encapsulation efficiency and in vitro drug release as the CQAs. Caco-2 cell line indicated insignificant cytotoxicity, and P-gp efflux, bi-directional permeability model and in situ perfusion studies specified enhanced intestinal permeation parameters. In vivo pharmacokinetic studies revealed notable increase in the values of Cmax (4.7-fold) and AUC (2.1-fold), respectively, from PLCs-loaded NLCs vis-à-vis Mgf solution. In a nutshell, the promising results observed from the present research work signified boosted biopharmaceutical potential of the optimized PLCs-loaded NLCs for potentially augmenting the therapeutic efficacy of Mgf

Cationic cyclodextrin/alginate chitosan nanoflowers as 5-fluorouracil drug delivery system

Cyclodextrins (CDs) have widely been used as component of drug delivery systems. However unmodified cyclodextrins are associated with cytotoxicity and poor water solubility thus limiting their use in pharmaceutical industry. The cationic-β-cyclodextrin (Cat-β-CD) polymer cores were synthesized using β-CD, epichlorohydrin and choline chloride via a one-step polycondensation process. The main aim of this study was to synthesize hierarchical nanoflowers composed of cationic-β-CD as polymeric core along with alginate and chitosan “petals” (Cat-β-CD/Alg-Chi nanoflowers) as carriers for oral delivery of 5-Fluorouracil (5-FU) via an ionic-gelation technique. The drug loading capacity, particle size, zeta potential and surface morphology of the synthesized nanoflowers were determined. The prepared nanoflowers were formed with an average size of 300 nm and a zeta potential of + 9.90 mV with good encapsulation efficiency of up to 77.3%. In vitro release of 5-FU from the loaded nanoflowers showed controlled and sustained release compared to the inclusion complex alone. Cat-β-CD/Alg-Chi nanoflowers were assessed against L929 cells and found to be effectively inhibiting the growth of L929 cells in a concentration dependent manner

Effect of Salt Forms of Chitosan on In Vitro Permeability Enhancement in Intestinal Epithelial Cells (Caco-2)

Purpose: To investigate the effect of chitosan (CS) salt forms and pH condition on the transepithelial electrical resistance (TEER) of Caco-2 cell monolayer for enhanced permeability. Methods: Solutions (2 %w/v) of four different salt forms of CS-aspartate (CS-A), CS-ethylene diamine tetraacetate (CS-EDTA), CS-hydroxybenzotriazole (CS-HOBt) and CS-thiamine pyrophosphate (CS-TPP) - were prepared and tested on TEER using fluorescein isothiocyanate dextran 4,400 (FD-4) as the permeant across Caco-2 cell monolayer in both pH 6.2 and 7.4 (physiological pH) environment. Results: The results show that CS-salt solutions, at pH of 6.2, increased cell permeability in a dose-dependent manner and caused relatively reversible effects only at low doses of 0.001 - 0.010 %w/v. At CS-salt solution concentration of 0.01 %w/v, accumulation of FD-4 in the acceptor compartment was in the rank order: CS-EDTA > CS-TPP > CS-A > CS-HOBt. All CS-salt solutions significantly (p < 0.05) increased the transport of FD-4. On the other hand, at pH 7.4, only CS-EDTA at a concentration of 0.5 %w/v enhanced the transport of FD-4. CS-EDTA was also the most toxic CS salt. Conclusion: The salt forms of CS are capable of enhancing the transport of FD-4 across Caco-2 cell monolayer, with CS-EDTA the most promising of them

Oral Methylated N-Aryl Chitosan Derivatives for Inducing Immune Responses to Ovalbumin

Purpose: To investigate different structures of modified chitosan containing different chain lengths and aromatic moieties for vaccine delivery capacity. Methods: The characteristics of the modified chitosan, namely, methylated N-(4-N,Ndimethylaminobenzyl) chitosan (TM-Bz-CS), methylated N-(4-N,N-dimethylaminocinnamyl) chitosan (TM-CM-CS) and methylated N-(4-pyridinylmethyl) chitosan (TM-Py-CS), with Eqiva degree (equivalent degree) were studied by in vitro absorption enhancement on the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers as well as by in vivo adjuvant activity against ovalbumin (OVA), a model antigen, via oral administration to BALB/c mice. Results: At the same concentration and pH (0.1 mg/ml, pH 7.4), TM65CM50CS exhibited the highest in vitro enhancing paracellular permeability and also the highest in vivo adjuvant activity following oral administration to mice. OVA-specific serum immunoglobulin G (IgG) antibody levels of mice that received OVA in TM65CM50CS were significantly (p < 0.05) higher than those that received OVA in TM65CS, TM56Bz42CS and TM53Py40CS. On the other hand, TM65CS and TM56Bz42CS exhibited in vitro enhancing paracellular permeability but showed no immune responses, while TM53Py40CS failed to enhance paracellular permeability and did not elicit immune responses as well. Conclusion: This study demonstrates that addition of hydrophobic moiety (dimethylaminocinnamyl) to CS backbone can increase both its absorption enhancing property and adjuvant activity. The chemical structure and the positive charge location play an important role for binding affinity, absorption enhancement and immune responses

Oral Methylated N-Aryl Chitosan Derivatives for Inducing Immune Responses to Ovalbumin

Purpose: To investigate different structures of modified chitosan containing different chain lengths and aromatic moieties for vaccine delivery capacity. Methods: The characteristics of the modified chitosan, namely, methylated N-(4-N,Ndimethylaminobenzyl) chitosan (TM-Bz-CS), methylated N-(4-N,N-dimethylaminocinnamyl) chitosan (TM-CM-CS) and methylated N-(4-pyridinylmethyl) chitosan (TM-Py-CS), with Eqiva degree (equivalent degree) were studied by in vitro absorption enhancement on the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers as well as by in vivo adjuvant activity against ovalbumin (OVA), a model antigen, via oral administration to BALB/c mice. Results: At the same concentration and pH (0.1 mg/ml, pH 7.4), TM65CM50CS exhibited the highest in vitro enhancing paracellular permeability and also the highest in vivo adjuvant activity following oral administration to mice. OVA-specific serum immunoglobulin G (IgG) antibody levels of mice that received OVA in TM65CM50CS were significantly (p < 0.05) higher than those that received OVA in TM65CS, TM56Bz42CS and TM53Py40CS. On the other hand, TM65CS and TM56Bz42CS exhibited in vitro enhancing paracellular permeability but showed no immune responses, while TM53Py40CS failed to enhance paracellular permeability and did not elicit immune responses as well. Conclusion: This study demonstrates that addition of hydrophobic moiety (dimethylaminocinnamyl) to CS backbone can increase both its absorption enhancing property and adjuvant activity. The chemical structure and the positive charge location play an important role for binding affinity, absorption enhancement and immune responses