Begoña Seijo

Chitosan-based nanostructures: a delivery platform for ocular therapeutics.

Nanoscience and nanotechnology has caused important breakthroughs in different therapeutic areas. In particular, the application of nanotechnology in ophthalmology has led to the development of novel strategies for the treatment of ocular disorders. Indeed, the association of an active molecule to a nanocarrier allows the molecule to intimately interact with specific ocular structures, to overcome ocular barriers and to prolong its residence in the target tissue. Over the last decade, our group has designed and developed a delivery platform based on the polysaccharide chitosan, which suits the requirements of the topical ocular route. These nanosystems have been specifically adapted for the delivery of hydrophilic and lipophilic drugs and also polynucleotides onto the eye surface. The results collected up until now suggest the potential of this delivery platform and the subsequent need of a full preclinical evaluation in order to satisfy the specific regulatory demands of this mode of administration.

Bioadhesive hyaluronan–chitosan nanoparticles can transport genes across the ocular mucosa and transfect ocular tissue

Gene transfer is considered to be a promising alternative for the treatment of several chronic diseases that affect the ocular surface. The goal of the present work was to investigate the efficacy and mechanism of action of a novel DNA nanocarrier made of hyaluronan (HA) and chitosan (CS), specifically designed for topical ophthalmic gene therapy. With this goal in mind, we first evaluated the transfection efficiency of the plasmid DNA-loaded nanoparticles in a human corneal epithelium cell model. Second, we investigated the bioadhesion and internalization of the nanoparticles in the rabbit ocular epithelia by confocal laser scanning microscopy. Third, we determined the in vivo efficacy of these nanocarriers in terms of their ability to transfect ocular tissues. The results showed that HA–CS nanoparticles and, in particular, those made of low molecular weight CS (10–12 kDa), led to high levels of expression of secreted alkaline phosphatase in the human corneal epithelium model. In addition, we observed that, following topical administration to rabbits, these nanoparticles entered the corneal and conjunctival epithelial cells and, then, become assimilated by the cells. More importantly, these nanoparticles provided an efficient delivery of the associated plasmid DNA inside the cells, reaching significant transfection levels. Therefore, we conclude that these nanoparticles may represent a new strategy toward the gene therapy of several ocular diseases.

Microencapsulated chitosan nanoparticles for pulmonary protein delivery: In vivo evaluation of insulin-loaded formulations

This work presents a new dry powder system consisting of microencapsulated protein-loaded chitosan nanoparticles (CS NPs). The developed system was evaluated in vivo in rats in order to investigate its potential to transport insulin (INS), a model protein, to the deep lung, where it is absorbed into systemic circulation. The INS-loaded CS NPs were prepared by ionotropic gelation and characterized for morphology, size, zeta potential, association efficiency and loading capacity. Afterwards, the NPs were co-spray dried with mannitol resulting in a dry powder with adequate aerodynamic properties for deposition in deep lungs. The assessment of the plasmatic glucose levels following intratracheal administration to rats revealed that the microencapsulated INS-loaded CS NPs induced a more pronounced and prolonged hypoglycemic effect compared to the controls. Accordingly, the developed system constitutes a promising alternative to systemically deliver therapeutic macromolecules to the lungs, but it can also be used to provide a local effect.

Microspheres containing lipid/chitosan nanoparticles complexes for pulmonary delivery of therapeutic proteins

Chitosan/tripolyphosphate nanoparticles have already been demonstrated to promote peptide absorption through several mucosal surfaces. We have recently developed a new drug delivery system consisting of complexes formed between preformed chitosan/tripolyphosphate nanoparticles and phospholipids, named as lipid/chitosan nanoparticles (L/CS-NP) complexes. The aim of this work was to microencapsulate these protein-loaded L/CS-NP complexes by spray-drying, using mannitol as excipient to produce microspheres with adequate properties for pulmonary delivery. Results show that the obtained microspheres are spherical and present appropriate aerodynamic characteristics for lung delivery (aerodynamic diameters around 2-3 microm and low apparent tap density of 0.4-0.5 g/cm3). The physicochemical properties of the L/CS-NP complexes are affected by the phospholipids composition. Phospholipids provide a controlled release of the encapsulated protein (insulin), which was successfully associated to the system (68%). The complexes can be easily recovered from the mannitol microspheres upon incubation in aqueous medium, maintaining their morphology and physicochemical characteristics. Therefore, this work demonstrates that protein-loaded L/CS-NP complexes can be efficiently microencapsulated, resulting in microspheres with adequate properties to provide a deep inhalation pattern. Furthermore, they are expected to release their payload (the complexes and, consequently, the encapsulated macromolecule) after contacting with the lung aqueous environment.

Novel Hyaluronan-Based Nanocarriers for Transmucosal Delivery of Macromolecules

The goal of this work was to design a new nanocarrier composed of the glycosaminoglycan hyaluronan and the polysaccharide chitosan, intended for the transmucosal delivery of macromolecules. The nanoparticles were characterized for their size and superficial charge. The incorporation of hyaluronan was verified by agarose gel electrophoresis and Fourier transform infrared (FT-IR) spectroscopy. The ability of the nanosystems to encapsulate macromolecules was studied taking the hydrophilic protein bovine serum albumin (BSA) and the hydrophobic polypeptide Cyclosporine A (CyA) as models. Results showed that the experimental conditions could be conveniently adjusted in order to modulate the physicochemical properties of the carriers and their composition. Moreover, the nanoparticles provided high association efficiencies of the selected macromolecules.

Chitosan nanoparticles for drug delivery to the eye

The purpose of this review is to provide the reader with an overview of the advances made in ocular delivery of bioactive molecules by means of chitosan-based nanosystems, and their potential relevance in clinical use. The studies described clearly emphasise that chitosan-based nanostructures are versatile systems that can be tailor-made according to required compositions, surface characteristics and particle size. Such parameters, which are known to influence their in vivo performance, can be modulated by adjusting the formulation conditions of the nanotechnologies responsible for their formation, by incorporating additional materials in the preparation steps, and/or by using synthetically modified chitosan. Moreover, this review illustrates how the advances achieved in the understanding of the interaction of nanosystems with the ocular structures should result in the coming years, logically, into challenging innovations in ocular nanomedicines with significant impact on clinical practice.

Hybrid Nanoparticle Design Based on Cationized Gelatin and the Polyanions Dextran Sulfate and Chondroitin Sulfate for Ocular Gene Therapy

We describe the development of hybrid nanoparticles composed of cationized gelatin and the polyanions CS and DS for gene therapy in the ocular surface. The physicochemical properties of the nanoparticles that impact their bioperformance, such as average size and zeta potential, can be conveniently modulated by changing the ratio of polymers and the crosslinker. These systems associate plasmid DNA and are able to protect it from DNase I degradation. We corroborate that the introduction of CS or DS in the formulation decreases the in vitro toxicity of the nanoparticles to human corneal cells without compromising the transfection efficiency. These nanoparticles are potential candidates for the development of safer and more effective nanomedicines for ocular therapy.

Colon-specific delivery of budesonide from microencapsulated cellulosic cores: evaluation of the efficacy against colonic inflammation in rats

Budesonide (BDS) is a potent corticosteroid that has important implications in the pharmaco‐therapy of inflammatory bowel disease, especially in the treatment of ulcerative colitis and Crohns disease. BDS is available on the market in the form of enteric‐coated preparations. However these products, similar to other available site‐specific dosage forms, are not sufficiently selective to treat colonic inflammatory bowel disease. The objective of this study was to evaluate the efficacy of a new microparticulate system containing BDS, to treat experimentally induced colitis in rats. This microparticulate system consisted of BDS‐containing hydrophobic cores, microencapsulated within an enteric polymer, which solubilizes at above pH 7, thus combining pH‐sensitive and controlled‐release properties. Colonic injury and inflammation were assessed by measuring colon/bodyweight ratio, myeloperoxidase (MPO) activity, and by scoring macroscopic and histological damage in colitic rats. Rats were treated orally with BDS, included in the developed system, once a day for 4 days after the induction of inflammation. A BDS suspension and BDS‐containing enteric microparticles were included as control formulations in the experimental design. The administration of the new BDS delivery system significantly reduced the colon/bodyweight ratio compared with the administration of control formulations. Similarly, MPO activity and macroscopic and histological damage of the inflamed colonic segments decreased significantly when the BDS formulation was administered, compared with the results obtained after oral administration of the drug suspension. There were no significant differences, however, when the new treatment was compared with the control formulation consisting of simple enteric microparticles.

Pullulan-based nanoparticles as carriers for transmucosal protein delivery

Polymeric nanoparticles have revealed very effective in transmucosal delivery of proteins. Polysaccharides are among the most used materials for the production of these carriers, owing to their structural flexibility and propensity to evidence biocompatibility and biodegradability. In parallel, there is a preference for the use of mild methods for their production, in order to prevent protein degradation, ensure lower costs and easier procedures that enable scaling up. In this work we propose the production of pullulan-based nanoparticles by a mild method of polyelectrolyte complexation. As pullulan is a neutral polysaccharide, sulfated and aminated derivatives of the polymer were synthesized to provide pullulan with a charge. These derivatives were then complexed with chitosan and carrageenan, respectively, to produce the nanocarriers. Positively charged nanoparticles of 180-270 nm were obtained, evidencing ability to associate bovine serum albumin, which was selected as model protein. In PBS pH 7.4, pullulan-based nanoparticles were found to have a burst release of 30% of the protein, which maintained up to 24h. Nanoparticle size and zeta potential were preserved upon freeze-drying in the presence of appropriate cryoprotectants. A factorial design was approached to assess the cytotoxicity of raw materials and nanoparticles by the metabolic test MTT. Nanoparticles demonstrated to not cause overt toxicity in a respiratory cell model (Calu-3). Pullulan has, thus, demonstrated to hold potential for the production of nanoparticles with an application in protein delivery.

Expression of MUC5AC in ocular surface epithelial cells using cationized gelatin nanoparticles.

Decreased production of the mucin MUC5AC in the eye is related to several pathological conditions, including dry eye syndrome. A specific strategy for increasing the ocular levels of MUC5AC is not yet available. Using a plasmid specially designed to encode human MUC5AC, we evaluated the ability of hybrid cationized gelatin nanoparticles (NPs) containing polyanions (chondroitin sulfate or dextran sulfate) to transfect ocular epithelial cells. NPs were developed using the ionic gelation technique and characterized by a small size (<200 nm), positive zeta potential (+20/+30 mV), and high plasmid association efficiency (>95%). MUC5AC mRNA and protein were detected in conjunctival cells after in vitro transfection of the NPs. The in vivo administration of the NPs resulted in significantly higher MUC5AC expression in the conjunctiva compared to untreated control and naked plasmid. These results provide a proof-of-concept that these NPs are effective vehicles for gene therapy and candidates for restoring the MUC5AC concentration in the ocular surface.