Noemi Csaba

Ionically crosslinked chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery

Ionically crosslinked nanoparticles based on high and low molecular weight chitosans (CS) were formulated with plasmid DNA or dsDNA oligomers using the ionic gelation technique with pentasodium tripolyphospate (TPP) as crosslinking agent. The resulting CS/TPP nanoparticles were investigated with regard to their physical-chemical properties, in vitro transfection efficiency, toxicity, cellular uptake, and in vivo gene expression following intratracheal administration to mice. The effects of co-formulating the nanoparticles with a model protein, BSA, were also studied. CS/TPP nanoparticles showed high encapsulation efficiencies both for plasmid DNA and dsDNA oligomers (20-mers), independent of CS molecular weight. TEM images revealed a spherical shape of the CS/TPP nanoparticles in contrast to the heterogeneous and irregular morphology displayed by conventional chitosan polyplexes. The nanoparticles showed high physical stability and no DNA release could be detected in diverse release media, nor even after incubation with heparin. Low molecular weight (LMW) CS/TPP nanoparticles gave high gene expression levels in HEK 293 cells already 2 days after transfection, reaching a plateau of sustained and high gene expression between 4 and 10 days. The inclusion of BSA into the nanostructures did not alter the inherent transfection efficiency of the nanoparticles. Confocal studies suggest endocytotic cellular uptake of the nanoparticles and a subsequent release into the cytoplasm within 14 h. LMW CS/TPP nanoparticles mediated a strong beta-galactosidase expression in vivo after intratracheal administration. The results of this study forward ionically crosslinked CS/TPP nanoparticles as a biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.

Nanoparticles for nasal vaccination.

The great interest in mucosal vaccine delivery arises from the fact that mucosal surfaces represent the major site of entry for many pathogens. Among other mucosal sites, nasal delivery is especially attractive for immunization, as the nasal epithelium is characterized by relatively high permeability, low enzymatic activity and by the presence of an important number of immunocompetent cells. In addition to these advantageous characteristics, the nasal route could offer simplified and more cost-effective protocols for vaccination with improved patient compliance. The use of nanocarriers provides a suitable way for the nasal delivery of antigenic molecules. Besides improved protection and facilitated transport of the antigen, nanoparticulate delivery systems could also provide more effective antigen recognition by immune cells. These represent key factors in the optimal processing and presentation of the antigen, and therefore in the subsequent development of a suitable immune response. In this sense, the design of optimized vaccine nanocarriers offers a promising way for nasal mucosal vaccination.

The performance of nanocarriers for transmucosal drug delivery.

Most of the newly designed drug molecules are characterised by low solubility in aqueous medium, low permeability through biological membranes and/or an insufficient stability in the biological environment. Fundamental studies have provided proof-of-concept of the potential of particulate nanocarriers for overcoming these unsuitable properties. For example, it is known that polymeric nanosystems may enhance transmucosal transport of drugs with poor penetration capacities while preserving their biological activity. Moreover, in recent years it has become clear that through an appropriate selection of the nanosystem components it is possible to enhance its affinity for the mucosa and, hence, the residence time of the drug in contact with the absorptive epithelium. These properties, combined with a suitably tailored release profile can markedly increase the efficacy of pharmaceuticals. Overall, the properties that have been identified as critical for the performance of these delivery systems are particle size, surface charge and surface chemical composition. These properties are known to affect the physical and chemical stability of the nanoparticles in the biological environment as well as their ability to interact (unspecific bioadhesion, receptor-mediated interaction and so on) and, eventually, overcome biological barriers. The present article aims to critically review the latest advances in this area and to provide some insights into these complex issues. Thus, herein the most widely investigated transmucosal drug delivery nanosystems and their most promising applications are reported.

Nanoparticles as protein and gene carriers to mucosal surfaces

One of the most exciting and challenging applications of nanotechnology in medicine is the development of nanocarriers for the intraepithelial delivery of biomacromolecules through mucosal surfaces. These biomacromolecules represent an increasingly important segment of the therapeutic arsenal; however, their potential is still limited by their instability and inability to cross biological barriers. Nanoparticle carriers have emerged as one of the most promising technologies to overcome this limitation, owing mainly to their demonstrated capacity to interact with biological barriers. In this review, we summarize the current advances made on nanoparticles designed for transmucosal delivery. Supported by the examples of a variety of therapeutic macromolecules - peptides and proteins, gene medicines and vaccines - we review the lessons learned from the past and we offer a future perspective for this field.

Vaccine delivery carriers: insights and future perspectives.

Vaccination is undoubtedly the most effective health intervention for disease prevention and eradication. Nevertheless, currently there is still a need for improving immunization coverage worldwide. A promising strategy to achieve this goal nowadays relies on the use of delivery carriers capable of inducing an effective immune response and providing improved stability, safety and cost effectiveness. This article focuses on analyzing the critical aspects in the design of these carriers, and reviewing the state of the art of currently marketed formulations and those in advanced clinical development. These vaccine delivery carriers include emulsions, liposomes and polymeric particulate carriers. Finally, particular attention is given to the evolution in the design of polymeric nanocarriers, which have been receiving increasing attention and hold promise to generate novel platforms for needle-free administration and single-dose vaccination.

Concomitant delivery of a CTL-restricted peptide antigen and CpG ODN by PLGA microparticles induces cellular immune response

Poly(lactide-co-glycolide) (PLGA) microparticles (MP) possess immunological adjuvant properties. Yet, exploitation of their full potential has just begun. The purpose of this study was to explore opportunities arising from surface modifications, and attachment and entrapment of combinations of antigen and a Toll-like receptor (TLR) ligand. The cytotoxic T lymphocyte (CTL)-restricted OVA ovalbumin peptide SIINFEKL was microencapsulated into bare, chitosan-coated, and protamine-coated PLGA MP using a microextrusion-assisted solvent extraction process. A TLR-ligand (CpG ODN) was either covalently coupled or physically adsorbed onto the MP surface. The peptide encapsulation efficiency decreased from 71% for uncoated particles to 62% and 45% upon coating with chitosan and protamine, respectively. CpG adsorption efficiency decreased from 93% for protamine-coated particles to 19% and 8% for chitosan and bare particles. Release of the adsorbed CpG was slow and incomplete (23% within 7 days) with the protamine coating, intermediate (>90% within 3 days) with the chitosan coating, and immediate (100% within 3 h) without coating. Interestingly, only the uncoated PLGA MP with adsorbed CpG mediated a prominent CTL response in mice at 6 days after immunization, as determined from IFN-γ release from antigen-specific CD8+ cells; failure of the other MP formulations was ascribed to the low release of antigen and CpG within the first week after immunization. The study illustrates novel opportunities for PLGA MP vaccines by combining antigens and immunostimulatory ligands.

Surface coating of PLGA microparticles with protamine enhances their immunological performance through facilitated phagocytosis.

Surface modifications of poly(lactide-co-glycolide) microparticles with different polycationic electrolytes have mainly been studied for conjugation to antigens and/or adjuvants. However, the in vivo immunological effects of using surface charged particles have not been address yet. In this study, microparticles were coated or not with protamine, a cationic and arginine-rich electrolyte that confers microparticles with a positively surface charge. We then evaluated the potential of protamine-coatings to assist the induction of immune responses in mice. Interestingly, enhanced antibodies and T-cell responses were observed in mice treated with the coated particles. In vitro studies suggested that the improved immunological performance was mediated by an increased uptake. Indeed, protamine-coated particles that carried a plasmid were even internalised into non-phagocytic cells and to cause their transfection. These results open the way for further research into a novel technology that combines the use protamine for facilitated cell penetration of that and biodegradable microparticles for prolonged antigen or drug release.

Design and characterisation of new nanoparticulate polymer blends for drug delivery

The aim of the present work was the design of novel nanoparticle compositions based on poly(lactic acid/glycolic acid) (PLGA): poloxamer and PLGA: poloxamine blend matrices. For this purpose, we have applied a modified solvent diffusion technique that allows the preparation of the nanoparticles without the use of high energy sources. Nanoparticles have been prepared with different PLGA: poloxamer and PLGA: poloxamine ratios using PEO-derivatives with different molecular weights (M w) and hydrophilia–lipophilia balance (HLB) values. Our results show that the physicochemical characteristics of the nanoparticles, such as size and zeta potential, are influenced by the type of PEO-derivative associated to the PLGA matrix. The 1H-NMR analysis of the different nanoparticle compositions showed that the extent of incorporation of the PEO-derivative depends strongly on its HLB and also on the nanoparticles preparation conditions. The capacity of these nanoparticles as drug delivery devices was evaluated using bovine insulin as a model drug. The insulin-encapsulation efficiency was shown to be dependent on the composition of the nanoparticles, those containing hydrophilic PEO-derivatives being the most effective in entrapping the drug molecules. The formation of the blend system displayed positive effects on the release characteristics of the nanoparticles. Nanoparticles exhibited a reduced initial burst and a nearly linear, constant release rate over a time period of two weeks.

Nanotherapies for the treatment of ocular diseases

The topical route is the most frequent and preferred way to deliver drugs to the eye. Unfortunately, the very low ocular drug bioavailability (less than 5%) associated with this modality of administration, makes the efficient treatment of several ocular diseases a significant challenge. In the last decades, it has been shown that specific nanocarriers can interact with the ocular mucosa, thereby increasing the retention time of the associated drug onto the eye, as well as its permeability across the corneal and conjunctival epithelium. In this review, we comparatively analyze the mechanism of action and specific potential of the most studied nano-drug delivery carriers. In addition, we present the success achieved until now using a number of nanotherapies for the treatment of the most prevalent ocular pathologies, such as infections, inflammation, dry eye, glaucoma, and retinopathies.

Polypeptides and polyaminoacids in drug delivery

Introduction: Advances achieved over the last few years in drug delivery have provided novel and versatile possibilities for the treatment of various diseases. Among the biomaterials applied in this field, it is worth highlighting the increasing importance of polyaminoacids and polypeptides. The appealing properties of these polymers are very promising for the design of novel compositions in a variety of drug delivery applications. Areas covered: This review provides an overview on the general characteristics of polyaminoacids and polypeptides and briefly discusses different synthetic pathways for their production. This is followed by a detailed description of different drug delivery applications of these polymers, emphasizing those examples that already reached advanced preclinical development or have entered clinical trials. Expert opinion: Polyaminoacids and polypeptides are gaining much attention in drug delivery due to their exceptional properties. Their application as polymers for drug delivery purposes has been sped up by the significant achievements related to their synthesis. Certainly, cancer therapy has benefited the most from these advances, although other fields such as vaccine delivery and alternative administration routes are also being successfully explored. The design of new entities based on polyaminoacids and polypeptides and the improved insight gained in drug delivery guarantee exciting findings in the near future.