Marcos Garcia-Fuentes

Design of lipid nanoparticles for the oral delivery of hydrophilic macromolecules

Abstract Colloidal drug carriers prepared from solid triglycerides have been presented as a promising alternative to polymer nanoparticles. The present work is aimed at developing surface-modified lipid nanoparticles intended to encapsulate peptides within their structure and to study their physicochemical properties and in vitro stability in gastrointestinal fluids. The final goal is to explore their potential as oral delivery vehicles for macromolecules. The W/O/W multiple emulsion technique was originally applied and conveniently modified for the production of tripalmitin nanoparticles. This technique was selected because its makes the encapsulation of peptides feasible. Additionally, the surface of the particles could be modified through the incorporation of Poloxamer 188 or the lipid derivative PEG 2000-stearate into the formulation. This modification led to a reduction in the zeta potential values, varying from −34 mV for the non-coated particles to −20 mV for those prepared with PEG-stearate. Results of the stability of the nanoparticles in gastric and intestinal media indicate that the low pH of the gastric medium and the pancreatic enzymes in intestinal medium are responsible for the extensive aggregation and degradation of the non-coated lipid nanoparticles (80% degradation in 4 h). In contrast, PEG-stearate coated nanoparticles were more stable, as their polymer coating layer totally prevented aggregation in both media and significantly reduced pancreatin-induced degradation (40% approximately in 4 h). Preliminary studies showed that insulin, chosen as a model peptide, could be associated and released from PEG-stearate coated nanoparticles. Nevertheless, further work is required in order to optimize the release behavior of the entrapped peptide.

Chitosan-based drug nanocarriers: Where do we stand?

Chitosan-based nanocarriers have become one of the most intensively studied transmucosal nanometric drug delivery platforms. This is due to a number of factors, including their simple and mild preparation technique as well as their capacity to associate macromolecules and facilitate their transport across mucosal barriers. In this review, we first describe our contribution to the origin of chitosan nanocarriers in the mid 90s, and summarize the early work that has impacted the development of this delivery technology. Secondly, we present our perspective regarding the potential of chitosan nanocarriers for some relevant applications: (i) vaccination, (ii) transmucosal protein delivery and (iii) gene therapy. Finally, we offer our perspective on the plausible advances in this area in the near future.

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.

Optimization strategies for electrospun silk fibroin tissue engineering scaffolds

As a contribution to the functionality of scaffolds in tissue engineering, here we report on advanced scaffold design through introduction and evaluation of topographical, mechanical and chemical cues. For scaffolding, we used silk fibroin (SF), a well-established biomaterial. Biomimetic alignment of fibers was achieved as a function of the rotational speed of the cylindrical target during electrospinning of a SF solution blended with polyethylene oxide. Seeding fibrous SF scaffolds with human mesenchymal stem cells (hMSCs) demonstrated that fiber alignment could guide hMSC morphology and orientation demonstrating the impact of scaffold topography on the engineering of oriented tissues. Beyond currently established methodologies to measure bulk properties, we assessed the mechanical properties of the fibers by conducting extension at breakage experiments on the level of single fibers. Chemical modification of the scaffolds was tested using donor/acceptor fluorophore labeled fibronectin. Fluorescence resonance energy transfer imaging allowed to assess the conformation of fibronectin when adsorbed on the SF scaffolds, and demonstrated an intermediate extension level of its subunits. Biological assays based on hMSCs showed enhanced cellular adhesion and spreading as a result of fibronectin adsorbed on the scaffolds. Our studies demonstrate the versatility of SF as a biomaterial to engineer modified fibrous scaffolds and underscore the use of biofunctionally relevant analytical assays to optimize fibrous biomaterial scaffolds.

A comparative study of chitosan and chitosan/cyclodextrin nanoparticles as potential carriers for the oral delivery of small peptides ☆

The aim of this study was to characterize new nanoparticles (NPs) containing chitosan (CS), or CS/cyclodextrin (CDs), and evaluate their potential for the oral delivery of the peptide glutathione (GSH). More precisely, NP formulations composed of CS, CS/alpha-CD and CS/sulphobutyl ether-beta-cyclodextrin (SBE(7m)-beta-CD) were investigated for this application. CS/CD NPs showed particle sizes ranging from 200 to 500nm. GSH was loaded more efficiently in CS/SBE(7m)-beta-CD NPs by forming a complex between the tripeptide and the CD. X-ray Photoelectron Spectroscopy (XPS) analysis suggested that GSH is located in the core of CS/SBE(7m)-beta-CD NPs and that it is almost absent from the NP surface. Release studies performed in vitro at pH 1.2 and pH 6.8 showed that NP release properties can be modulated by selecting an appropriate CD. Transport studies performed in the frog intestine model confirmed that both CS and CS/CD nanoparticles could induce permeabilization of the intestinal epithelia. However, CS/SBE(7m)-beta-CD NPs provided absorption-enhancing properties in all segments of the duodenum, whereas CS NPs effect was restricted to the first segment of the duodenum. From the data obtained, we believe that CS/CD nanoparticles might represent an interesting technological platform for the oral administration of small peptides.

Silk fibroin/hyaluronan scaffolds for human mesenchymal stem cell culture in tissue engineering.

The design of new bioactive scaffolds mimicking the physiologic environment present during tissue formation is an important frontier in biomaterials research. Herein, we evaluated scaffolds prepared from blends of two biopolymers: silk fibroin and hyaluronan. Our rationale was that such blends would allow the combination of silk fibroins superior mechanical properties with the biological characteristics of hyaluronan. We prepared scaffolds with porous microstructures by freeze-drying aqueous solutions of silk fibroin and hyaluronan and subsequent incubation in methanol to induce water insolubility of silk fibroin. Hyaluronan acted as an efficient porogenic excipient for the silk fibroin scaffolding process, allowing the formation of microporous structures within the scaffolds under mild processing conditions. Mesenchymal stem cells were seeded on silk fibroin/hyaluronan scaffolds and cultured for three weeks. Histology of the constructs after cell culture showed enhanced cellular ingrowth into silk fibroin/hyaluronan scaffolds as compared to plain silk fibroin scaffolds. In the presence of tissue-inductive stimuli, in vitro stem cell culture on silk fibroin/hyaluronan scaffolds resulted in more efficient tissue formation when measured by glycosaminoglycan and type-I and type-III collagen gene expression, as compared to plain silk fibroin scaffolds. In conclusion, our data encourages further exploration of silk fibroin/hyaluronan scaffolds as biomimetic platform for mesenchymal stem cells in tissue engineering.

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.

Protein delivery based on uncoated and chitosan-coated mesoporous silicon microparticles

Mesoporous silicon is a biocompatible, biodegradable material that is receiving increased attention for pharmaceutical applications due to its extensive specific surface. This feature enables to load a variety of drugs in mesoporous silicon devices by simple adsorption-based procedures. In this work, we have addressed the fabrication and characterization of two new mesoporous silicon devices prepared by electrochemistry and intended for protein delivery, namely: (i) mesoporous silicon microparticles and (ii) chitosan-coated mesoporous silicon microparticles. Both carriers were investigated for their capacity to load a therapeutic protein (insulin) and a model antigen (bovine serum albumin) by adsorption. Our results show that mesoporous silicon microparticles prepared by electrochemical methods present moderate affinity for insulin and high affinity for albumin. However, mesoporous silicon presents an extensive capacity to load both proteins, leading to systems were protein could represent the major mass fraction of the formulation. The possibility to form a chitosan coating on the microparticles surface was confirmed both qualitatively by atomic force microscopy and quantitatively by a colorimetric method. Mesoporous silicon microparticles with mean pore size of 35 nm released the loaded insulin quickly, but not instantaneously. This profile could be slowed to a certain extent by the chitosan coating modification. With their high protein loading, their capacity to provide a controlled release of insulin over a period of 60-90 min, and the potential mucoadhesive effect of the chitosan coating, these composite devices comprise several features that render them interesting candidates as transmucosal protein delivery systems.

Hyaluronic acid/Chitosan nanoparticles as delivery vehicles for VEGF and PDGF-BB.

The development of a vascular network in tissue-engineered constructs is a fundamental bottleneck of bioregenerative medicine, particularly when the size of the implant exceeds a certain limit given by diffusion lengths and/or if the host tissue shows a very active metabolism. One of the approaches to achieve the vascularization of tissue constructs is generating a sustained release of proangiogenic factors from the ischemic site. This work describes the formation and characterization of hyaluronic acid-chitosan (HA/CS) nanoparticles for the delivery of two pro-angiogenic growth factors: vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF-BB). These nanoparticles were prepared by an ionic gelification technique, and different formulations were developed by encapsulating the growth factors in association with two stabilizing agents: bovine serum albumin or heparin sodium salt. These carriers were characterized with regard to their physicochemical properties, their stability in biological media, and their cytotoxicity in the C3a hepatoma cell line. The results show that nanoparticles around 200 nm can be prepared by this method. HA/CS nanoparticles were stable when incubated in EMEM cell culture medium or in water at 37°C for 24 h. Cell culture tests confirmed that HA/CS nanoparticles are not cytotoxic within the concentration range used for growth factor delivery. Moreover, HA/CS nanoparticles were able to entrap efficiently both growth factors, reaching association values of 94% and 54% for VEGF and PDGF, respectively. In vitro release studies confirm that PDGF-BB is released from HA/CS nanoparticles in a sustained manner over ∼ 1 week. On the other hand, VEGF is completely released within the first 24 h.