Ana Grenha

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.

Biocompatibility of chitosan carriers with application in drug delivery.

Chitosan is one of the most used polysaccharides in the design of drug delivery strategies for administration of either biomacromolecules or low molecular weight drugs. For these purposes, it is frequently used as matrix forming material in both nano and micron-sized particles. In addition to its interesting physicochemical and biopharmaceutical properties, which include high mucoadhesion and a great capacity to produce drug delivery systems, ensuring the biocompatibility of the drug delivery vehicles is a highly relevant issue. Nevertheless, this subject is not addressed as frequently as desired and even though the application of chitosan carriers has been widely explored, the demonstration of systems biocompatibility is still in its infancy. In this review, addressing the biocompatibility of chitosan carriers with application in drug delivery is discussed and the methods used in vitro and in vivo, exploring the effect of different variables, are described. We further provide a discussion on the pros and cons of used methodologies, as well as on the difficulties arising from the absence of standardization of procedures.

Chitosan/carrageenan nanoparticles: Effect of cross-linking with tripolyphosphate and charge ratios

Chitosan/carrageenan/tripolyphosphate nanoparticles were prepared by polyelectrolyte complexation/ionic gelation, the latter compound acting as cross-linker. The incorporation of the three components in the nanoparticle matrix was assessed by analytical techniques (FTIR, XPS and TOF-SIMS). Using chitosan/carrageenan nanoparticles as control, the effect of the cross-linker in the particles properties was studied. A decrease in size (from 450-500 nm to 150-300 nm) and in zeta potential (from +75 - +85 mV to +50 - +60 mV), and an increase in production yield (from 15-20% to 25-35%), and in stability (from one week to up to 9 months) were observed. Also, a correlation between positive to negative charge ratios in the formulations and the above characteristics was established. The small size and high positive surface charge make the developed chitosan/carrageenan/tripolyphosphate nanoparticles potential tools for an application in mucosal delivery of macromolecules.

Chitosan nanoparticles: a survey of preparation methods

The application of macromolecules in therapy is frequently hindered by stability and/or permeation issues. These limitations have been addressed by the pharmaceutical industry through the development of suitable noninjectable drug carriers. In this context, nanoparticles have emerged as one of the most exciting tools due to the increased surface-to-volume ratio, which provides an intimate interaction with epithelial surfaces. Nanoparticles further enable the encapsulated molecules to retain their biological activity, from the production steps to the final release. Chitosan has reached a prominent position as carrier-forming material, as diverse methods can be applied to produce nanoparticles using that excipient. These involve either hydrophilic or lipophilic environments that generally result in mild conditions or aggressive and time-consuming processes, respectively. In this review, a detailed description of methods used to produce chitosan nanocarriers is provided, accompanied by illustrative schemes of the procedures. The emphasis is on the variables reported to affect the final properties of the vehicles.

Sulfated Seaweed Polysaccharides as Multifunctional Materials in Drug Delivery Applications

In the last decades, the discovery of metabolites from marine resources showing biological activity has increased significantly. Among marine resources, seaweed is a valuable source of structurally diverse bioactive compounds. The cell walls of marine algae are rich in sulfated polysaccharides, including carrageenan in red algae, ulvan in green algae and fucoidan in brown algae. Sulfated polysaccharides have been increasingly studied over the years in the pharmaceutical field, given their potential usefulness in applications such as the design of drug delivery systems. The purpose of this review is to discuss potential applications of these polymers in drug delivery systems, with a focus on carrageenan, ulvan and fucoidan. General information regarding structure, extraction process and physicochemical properties is presented, along with a brief reference to reported biological activities. For each material, specific applications under the scope of drug delivery are described, addressing in privileged manner particulate carriers, as well as hydrogels and beads. A final section approaches the application of sulfated polysaccharides in targeted drug delivery, focusing with particular interest the capacity for macrophage targeting.

Locust bean gum: Exploring its potential for biopharmaceutical applications.

Polysaccharides have been finding, in the last decades, very interesting and useful applications in the biomedical and, specifically, in the biopharmaceutical field. Locust bean gum is a polysaccharide belonging to the group of galactomannans, being extracted from the seeds of the carob tree (Ceratonia siliqua). This polymer displays a number of appealing characteristics for biopharmaceutical applications, among which its high gelling capacity should be highlighted. In this review, we describe critical aspects of locust bean gum, contributing for its role in biopharmaceutical applications. Physicochemical properties, as well as strong and effective synergies with other biomaterials are described. The potential for in vivo biodegradation is explored and the specific biopharmaceutical applications are discussed.

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.

MUCOSAL DELIVERY OF LIPOSOME― CHITOSAN NANOPARTICLE COMPLEXES

Designing adequate drug carriers has long been a major challenge for those working in drug delivery. Since drug delivery strategies have evolved for mucosal delivery as the outstanding alternative to parenteral administration, many new drug delivery systems have been developed which evidence promising properties to address specific issues. Colloidal carriers, such as nanoparticles and liposomes, have been referred to as the most valuable approaches, but still have some limitations that can become more inconvenient as a function of the specific characteristics of administration routes. To overcome these limitations, we developed a new drug delivery system that results from the combination of chitosan nanoparticles and liposomes, in an approach of combining their advantages, while avoiding their individual limitations. These lipid/chitosan nanoparticle complexes are, thus, expected to protect the encapsulated drug from harsh environmental conditions, while concomitantly providing its controlled release. To prepare these assemblies, two different strategies have been applied: one focusing on the simple hydration of a previously formed dry lipid film with a suspension of chitosan nanoparticles, and the other relying on the lyophilization of both basic structures (nanoparticles and liposomes) with a subsequent step of hydration with water. The developed systems are able to provide a controlled release of the encapsulated model peptide, insulin, evidencing release profiles that are dependent on their lipid composition. Moreover, satisfactory in vivo results have been obtained, confirming the potential of these newly developed drug delivery systems as drug carriers through distinct mucosal routes.

Hybrid nanosystems based on natural polymers as protein carriers for respiratory delivery: Stability and toxicological evaluation.

Chitosan/carrageenan/tripolyphosphate nanoparticles were previously presented as holding potential for an application in transmucosal delivery of macromolecules, with tripolyphosphate demonstrating to contribute for both size reduction and stabilisation of the nanoparticles. This work was aimed at evaluating the capacity of the nanoparticles as protein carriers for pulmonary and nasal transmucosal delivery, further assessing their biocompatibility pattern regarding that application. Nanoparticles demonstrated stability in presence of lysozyme, while freeze-drying was shown to preserve their characteristics when glucose or sucrose were used as cryoprotectants. Bovine serum albumin was associated to the nanoparticles, which were successfully microencapsulated by spray-drying to meet the aerodynamic requirements inherent to pulmonary delivery. Finally, a satisfactory biocompatibility profile was demonstrated upon exposure of two respiratory cell lines (Calu-3 and A549 cells) to the carriers. A negligible effect on cell viability along with no alterations on transepithelial electrical resistance and no induction of inflammatory response were observed.