Antibiotic-Loaded Amphiphilic Chitosan Nanoparticles Target Macrophages and Kill an Intracellular Pathogen.

In this work, levofloxacin (LVX), a third-generation fluoroquinolone antibiotic, is encapsulated within amphiphilic polymeric nanoparticles of a chitosan-g-poly(methyl methacrylate) produced by self-assembly and physically stabilized by ionotropic crosslinking with sodium tripolyphosphate. Non-crosslinked nanoparticles display a size of 29 nm and a zeta-potential of +36 mV, while the crosslinked counterparts display 45 nm and +24 mV, respectively. The cell compatibility, uptake, and intracellular trafficking are characterized in the murine alveolar macrophage cell line MH-S and the human bronchial epithelial cell line BEAS-2B in vitro. Internalization events are detected after 10 min and the uptake is inhibited by several endocytosis inhibitors, indicating the involvement of complex endocytic pathways. In addition, the nanoparticles are detected in the lysosomal compartment. Then, the antibacterial efficacy of LVX-loaded nanoformulations (50% w/w drug content) is assessed in MH-S and BEAS-2B cells infected with Staphylococcus aureus and the bacterial burden is decreased by 49% and 46%, respectively. In contrast, free LVX leads to a decrease of 8% and 5%, respectively, in the same infected cell lines. Finally, intravenous injection to a zebrafish larval model shows that the nanoparticles accumulate in macrophages and endothelium and demonstrate the promise of these amphiphilic nanoparticles to target intracellular infections.

Stimulating Extracellular Vesicles Production from Engineered Tissues by Mechanical Forces.

Extracellular vesicles (EVs) have emerged as a promising strategy to promote tissue regeneration. However, overcoming the low EV production yield remains a big challenge in translating EV-based therapies to the clinical practice. Current EV production relies heavily on 2D cell culture, which is not only less physiologically relevant to cells but also requires substantial medium and space. In this study, we engineered tissues seeded with stem cells from dental pulp or adipose tissues, or skeletal muscle cells, and significantly enhanced the EV production yield by applying mechanical stimuli, including flow and stretching, in bioreactors. Further mechanistic investigation revealed that this process was mediated by yes-associated protein (YAP) mechanosensitivity. EVs from mechanically stimulated dental pulp stem cells on 3D scaffolds displayed superior capability in inducing axonal sprouting than the 2D counterparts. Our results demonstrate the promise of this strategy to boost EV production and optimize their functional performance toward clinical translation.

Mixed Amphiphilic Polymeric Nanoparticles of Chitosan, Poly(vinyl alcohol) and Poly(methyl methacrylate) for Intranasal Drug Delivery: A Preliminary In Vivo Study.

Intranasal (i.n.) administration became an alternative strategy to bypass the blood-brain barrier and improve drug bioavailability in the brain. The main goal of this work was to preliminarily study the biodistribution of mixed amphiphilic mucoadhesive nanoparticles made of chitosan-g-poly(methyl methacrylate) and poly(vinyl alcohol)-g-poly(methyl methacrylate) and ionotropically crosslinked with sodium tripolyphosphate in the brain after intravenous (i.v.) and i.n. administration to Hsd:ICR mice. After i.v. administration, the highest nanoparticle accumulation was detected in the liver, among other peripheral organs. After i.n. administration of a 10-times smaller nanoparticle dose, the accumulation of the nanoparticles in off-target organs was much lower than after i.v. injection. In particular, the accumulation of the nanoparticles in the liver was 20 times lower than by i.v. When brains were analyzed separately, intravenously administered nanoparticles accumulated mainly in the "top" brain, reaching a maximum after 1 h. Conversely, in i.n. administration, nanoparticles were detected in the "bottom" brain and the head (maximum reached after 2 h) owing to their retention in the nasal mucosa and could serve as a reservoir from which the drug is released and transported to the brain over time. Overall, results indicate that i.n. nanoparticles reach similar brain bioavailability, though with a 10-fold smaller dose, and accumulate in off-target organs to a more limited extent and only after redistribution through the systemic circulation. At the same time, both administration routes seem to lead to differential accumulation in brain regions, and thus, they could be beneficial in the treatment of different medical conditions.

Oral Pharmacokinetics of a Chitosan-Based Nano- Drug Delivery System of Interferon Alpha.

Interferon alpha (IFNα) is a protein drug used to treat viral infections and cancer diseases. Due to its poor stability in the gastrointestinal tract, only parenteral administration ensures bioavailability, which is associated with severe side effects. We hypothesized that the nanoencapsulation of IFNα within nanoparticles of the mucoadhesive polysaccharide chitosan would improve the oral bioavailability of this drug. In this work, we produced IFNα-loaded chitosan nanoparticles by the ionotropic gelation method. Their hydrodynamic diameter, polydispersity index and concentration were characterized by dynamic light scattering and nanoparticle tracking analysis. After confirming their good cell compatibility in Caco-2 and WISH cells, the permeability of unmodified and poly(ethylene glycol) (PEG)-modified (PEGylated) nanoparticles was measured in monoculture (Caco-2) and co-culture (Caco-2/HT29-MTX) cell monolayers. Results indicated that the nanoparticles cross the intestinal epithelium mainly by the paracellular route. Finally, the study of the oral pharmacokinetics of nanoencapsulated IFNα in BalbC mice revealed two maxima and area-under-the-curve of 56.9 pg*h/mL.

Mixed Mucoadhesive Amphiphilic Polymeric Nanoparticles Cross a Model of Nasal Septum Epithelium in Vitro.

Intranasal administration of nano-drug-delivery systems emerged as an appealing strategy to surpass the blood-brain barrier and thus increase drug bioavailability in the central nervous system. However, a systematic study of the effect of the structural properties of the nanoparticles on the nose-to-brain transport is missing. In this work, we synthesized and characterized mixed amphiphilic polymeric nanoparticles combining two mucoadhesive graft copolymers, namely, chitosan- g-poly(methyl methacrylate) and poly(vinyl alcohol)- g-poly(methyl methacrylate), for the first time. Chitosan enables the physical stabilization of the nanoparticles by ionotropic cross-linking with tripolyphosphate and confers mucoadhesiveness, while poly(vinyl alcohol) is also mucoadhesive and, owing to its nonionic nature, it improves nanoparticle compatibility in nasal epithelial cells by reducing the surface charge of the nanoparticles. After a thorough characterization of the mixed nanoparticles by dynamic light scattering and nanoparticle tracking analysis, we investigated the cell uptake by fluorescence light and confocal microscopy and imaging flow cytometry. Mixed nanoparticles were readily internalized at 37 °C, while the uptake was inhibited almost completely at 4 °C, indicating the involvement of energy-dependent mechanisms. Finally, we assessed the nanoparticle permeability across liquid-liquid and air-liquid monolayers of a nasal septum epithelial cell line and studied the effect of nanoparticle concentration and temperature on the apparent permeability. Overall, our findings demonstrate that these novel amphiphilic nanoparticles cross this in vitro model of intranasal epithelium mainly by a passive (paracellular) pathway involving the opening of epithelial tight junctions.

Chitosan-graft-poly(methyl methacrylate) amphiphilic nanoparticles: Self-association and physicochemical characterization.

In this work, we synthesized and characterized the self-assembly behavior of a chitosan-poly(methyl methacrylate) graft copolymer and the properties of the formed nanoparticles by static and dynamic light scattering, small-angle neutron scattering, and transmission electron microscopy. Overall, our results indicate that the hydrophobization of the chitosan side-chain with PMMA leads to a complex array of small unimolecular and/or small-aggregation number "building blocks" that further self-assemble into larger amphiphilic nanoparticles.

Chitosan ascorbate hydrogel improves water uptake capacity and cell adhesion of electrospun poly(epsilon-caprolactone) membranes.

The most important prerequisites for wound coverage matrices are biocompatibility, adequate porosity, degradability and exudate uptake capacity. A moderate hydrophilicity and exudate uptake capacity can often favour cell adhesion and wound healing potential, however, most of the synthetic polymers like polycaprolactone (PCL) are hydrophobic. Hydrogels based on natural polymers can improve the hydrophilicity and exudate uptake capacity of synthetic dressings and improve healing. In this work, we report the development of chitosan ascorbate-infiltrated electrospun PCL membranes. Our study demonstrated that chitosan ascorbate infiltration improves the hydrophilicity as well as water uptake capacity of the membranes and highly favoured the adhesion of human umbilical vein endothelial cells and human mesenchymal stem cells on the membranes.

Permeability of Novel Chitosan-g-Poly(Methyl Methacrylate) Amphiphilic Nanoparticles in a Model of Small Intestine In Vitro.

Engineering of drug nanocarriers combining fine-tuned mucoadhesive/mucopenetrating properties is currently being investigated to ensure more efficient mucosal drug delivery. Aiming to improve the transmucosal delivery of hydrophobic drugs, we designed a novel nanogel produced by the self-assembly of amphiphilic chitosan graft copolymers ionotropically crosslinked with sodium tripolyphosphate. In this work, we synthesized, for the first time, chitosan-g-poly(methyl methacrylate) nanoparticles thiolated by the conjugation of N-acetyl cysteine. First, we confirmed that both non-crosslinked and crosslinked nanoparticles in the 0.05⁻0.1% w/v concentration range display very good cell compatibility in two cell lines that are relevant to oral delivery, Caco-2 cells that mimic the intestinal epithelium and HT29-MTX cells that are a model of mucin-producing goblet cells. Then, we evaluated the effect of crosslinking, nanoparticle concentration, and thiolation on the permeability in vitro utilizing monolayers of (i) Caco-2 and (ii) Caco-2:HT29-MTX cells (9:1 cell number ratio). Results confirmed that the ability of the nanoparticles to cross Caco-2 monolayer was affected by the crosslinking. In addition, thiolated nanoparticles interact more strongly with mucin, resulting in a decrease of the apparent permeability coefficient (Papp) compared to the pristine nanoparticles. Moreover, for all the nanoparticles, higher concentration resulted in lower Papp, suggesting that the transport pathways can undergo saturation.

Protoporphyrin IX-modified chitosan-g-oligo(NiPAAm) polymeric micelles: from physical stabilization to permeability characterization in vitro.

Two main hurdles persist towards the more extensive bench-to-bed side translation of non-parenteral polymeric micelles. The first pertains to their thermodynamically-driven disassembly under uncontrolled dilution conditions in the biological milieu and upon interaction with biomacromolecules (e.g., proteins). The second is related to the relatively poor understanding of the pathways by which polymeric micelles improve the bioavailability of the payload by mucosal routes (e.g., intestinal). In this work, a chitosan-g-oligo(N-isopropylacrylamide) (CS-g-oligo(NiPAAm)) copolymer was modified with non-cytotoxic amounts of protoporphyrin IX (PP), a planar molecule of amphiphilic character that undergoes self-aggregation in water by forming π-π stacked supramolecular structures, to induce micellization under disfavored conditions and to serve as a fluorescent tracer for the measurement of the micelle permeability across a model of the intestinal epithelium in vitro. Findings indicated that the conjugation of PP amounts as low as 2% w/w induced the formation of micelles at temperatures below the lower critical solution temperature of oligo(NiPAAm) (30-32 °C). Moreover, permeability studies conducted at both 4 °C and 37 °C strongly suggested that despite the relatively large size of the micelles (200-300 nm), they cross the epithelial monolayer mainly by a paracellular pathway due to the opening of tight junctions. Complementary uptake studies by flow cytometry indicated that no endocytosis, though due to passive or facilitated diffusion, some internalization takes place.

Mucoadhesive nanogels by ionotropic crosslinking of chitosan-g-oligo(NiPAam) polymeric micelles as novel drug nanocarriers.

AIM: To investigate a novel kind of mucoadhesive nanogel based on the supramolecular aggregation of chitosan-g-oligo(N-isopropylacrylamide) copolymers. MATERIALS & METHODS: Copolymers were synthesized by the graft-free radical polymerization of N-isopropylacrylamide on chitosan. The aggregation was studied by dynamic light scattering and nanoparticle tracking analysis (NTA), the nanostructure by transmission electron microscopy(TEM)/cryo-TEM, the mucoadhesiveness in vitro with mucin and the cytocompatibility in Caco2 cells. RESULTS: Copolymers (36-74% w/w N-isopropylacrylamide content) showed critical micellar concentration between 2.0 and 40.0 × 10(-3)% w/v and micelles were nanometric and positively charged. Physical stabilization was achieved with ionotropic crosslinking. TEM/cryo-TEM revealed multimicellar aggregates with good mucoadhesion and cytocompatibility properties. Micellar systems (1-10% w/v) increased the solubility of efavirenz up to 1249-fold. CONCLUSION: Results support the potential of these nano-drug delivery systems for improved mucosal administration of hydrophobic drugs.