Gellan gum based physical hydrogels incorporating highly valuable endogen molecules and associating BMP-2 as bone formation platforms.

Physical hydrogels have been designed for a double purpose: as growth factor delivery systems and as scaffolds to support cell colonization and formation of new bone. Specifically, the polysaccharide gellan gum and the ubiquitous endogenous molecules chondroitin, albumin and spermidine have been used as exclusive components of these hydrogels. The mild ionotropic gelation technique was used to preserve the bioactivity of the selected growth factor, rhBMP-2. In vitro tests demonstrated the effective delivery of rhBMP-2 in its bioactive form. In vivo experiments performed in the muscle tissue of Wistar rats provided a proof of concept of the ability of the developed platforms to elicit new bone formation. Furthermore, this biological effect was better than that of a commercial formulation currently used for regenerative purposes, confirming the potential of these hydrogels as new and innovative growth factor delivery platforms and scaffolds for regenerative medicine applications.

Span poly-L-arginine nanoparticles are efficient non-viral vectors for PRPF31 gene delivery: An approach of gene therapy to treat retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common cause of inherited blindness in adults. Mutations in the PRPF31 gene produce autosomal dominant RP (adRP). To date there are no effective treatments for this disease. The purpose of this study was to design an efficient non-viral vector for human PRPF31 gene delivery as an approach to treat this form of adRP. Span based nanoparticles were developed to mediate gene transfer in the subretinal space of a mouse model of adRP carrying a point mutation (A216P) in the Prpf31 gene. Funduscopic examination, electroretinogram, optomotor test and optical coherence tomography were conducted to further in vivo evaluate the safety and efficacy of the nanosystems developed. Span-polyarginine (SP-PA) nanoparticles were able to efficiently transfect the GFP and PRPF31 plasmid in mice retinas. Statistically significant improvement in visual acuity and retinal thickness were found in Prpf31A216P/+ mice treated with the SP-PA-PRPF31 nanomedicine.

Nano and microcarriers to improve stem cell behaviour for neuroregenerative medicine strategies: Application to Huntington's disease.

The potential treatments for neurodegenerative disorders will be revolutionized by the transplantation of stem cells or neuronal progenitors derived from these cells. It is however crucial to better monitor their proliferation, improve their survival and differentiation and hence ameliorate their engraftment after transplantation. To direct stem cell fate, a delicate control of gene expression through RNA interference (RNAi) is emerging as a safe epigenetic approach. The development of novel biomaterials (nano and microcarriers) capable of delivering proteins, nucleic acids and cells, open the possibility to regulate cell fate while achieving neuroprotection and neurorepair and could be applied to Huntington's disease. This review first provides an overview of stem cell therapy for the neurodegenerative disorder Huntington's disease. Within that context, an integrative discussion follows of the control of stem cell behaviour by RNAi delivered by different nanocarriers in vitro prior to their transplantation. Finally, combined in vivo strategies using stem cells, biomaterials and epigenetic cell regulation are reported.

Comparison of different cationized proteins as biomaterials for nanoparticle-based ocular gene delivery.

Cationized polymers have been proposed as transfection agents for gene therapy. The present work aims to improve the understanding of the potential use of different cationized proteins (atelocollagen, albumin and gelatin) as nanoparticle components and to investigate the possibility of modulating the physicochemical properties of the resulting nanoparticle carriers by selecting specific protein characteristics in an attempt to improve current ocular gene-delivery approaches. The toxicity profiles, as well as internalization and transfection efficiency, of the developed nanoparticles can be modulated by modifying the molecular weight of the selected protein and the amine used for cationization. The most promising systems are nanoparticles based on intermediate molecular weight gelatin cationized with the endogenous amine spermine, which exhibit an adequate toxicological profile, as well as effective association and protection of pDNA or siRNA molecules, thereby resulting in higher transfection efficiency and gene silencing than the other studied formulations.

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.

Nanoparticles based on naturally-occurring biopolymers as versatile delivery platforms for delicate bioactive molecules: an application for ocular gene silencing.

Nanoparticles based on naturally-occurring biopolymers, most of them endogenous macromolecules, were designed as a versatile generation of delivery platforms for delicate bioactive molecules. The design of these nanosystems was specifically based on our recent finding about the ability of endogenous polyamine spermine (SPM) to interact with anionic biopolymers (ABs) generating ionically cross-linked nanosystems. The initial first generation of these delivery platforms, based on glycosaminoglycans and other polysaccharides, showed a very high association capacity for some delicate bioactive proteins such as growth factors, but a limited capacity to associate negatively charged molecules, such as pDNA and siRNA. However, the versatility of these nanosystems in terms of composition allowed us to customise the association of active ingredients and their physicochemical characteristics. Concretely, we prepared and incorporated gelatine cationized with spermine (CGsp) to their composition. The resulting modified formulations were characterised by a nanometric size (150-340 nm) and offer the possibility to modulate their zeta potential (from -35 to 28 mV), providing an efficient association of nucleic acids. The biological evaluation of these optimised nanosystems revealed that they are able to be internalised in vivo into corneal and conjunctival tissues and also to provide a significant siRNA gene silencing effect.

Current strategies for DNA therapy based on lipid nanocarriers.

INTRODUCTION: Lipid based nanocarriers represent one of the most widely used strategies for the delivery of gene molecules. This review focuses on current strategies for the use of these nanocarriers that could open new horizons in DNA therapy and offer an opportunity to support the transition from resource-based approaches towards knowledge-based strategies. AREAS COVERED: The present review highlights the most promising approaches focusing on the development of safe, stable, and effective lipid-based carriers capable of delivering DNA to the proper target sites and cells. In addition, we intend to provide some insights in to future strategies that should be considered in order to break down barriers in the transformation of DNA basic-science breakthroughs into clinical applications. EXPERT OPINION: On the basis of the significant advances in the design of lipid nanocarriers our impression is that they are, with respect to other systems, in a 'pole' position in the DNA therapy development race.

Spermidine cross-linked hydrogels as a controlled release biomimetic approach for cloxacillin.

The intrinsic ability of albumin to bind active substances in the physiological fluids has been explored to endow hydrogels with improved capability to regulate drug release. To develop such biomimetic-functional hydrogels, it is critical that albumin conformation is not altered and that the protein remains retained inside the hydrogel keeping its conformational freedom, i.e., it should be not chemically cross-linked. Thus, the hydrogels were prepared with various proportions of albumin by physical cross-linking of anionic polysaccharides (gellan gum and chondroitin sulfate) with the cationic endogen polyamine spermidine under mild conditions in order to prevent albumin denaturation. Texture and swelling properties of hydrogels with various compositions were recorded, and the effect of the preparation variables was evaluated applying neurofuzzy logic tools for hydrogels prepared with and without albumin and associating the antibiotic cloxacillin. Developed hydrogel systems were extensively analyzed by means of nuclear magnetic resonance (NMR) to determine weak-to-medium and strong binding modes and the equilibrium constants of the albumin-cloxacillin association. NMR techniques were also employed to demonstrate the successful modulation of the cloxacillin release from the albumin-containing hydrogels. In vitro microbiological tests carried out with Staphylococcus aureus and Staphylococcus epidermidis confirmed the interest of the albumin-containing hydrogels as efficient platforms for cloxacillin release in its bioactive form.

Progress in the characterization of bio-functionalized nanoparticles using NMR methods and their applications as MRI contrast agents.

Significant progress has been made over the last three decades in the field of NMR, a technique which has proven to have a variety of applications in many scientific disciplines, including nanotechnology. Herein we describe how NMR enables the characterization of nanosystems at different stages of their formation and modification (raw materials, bare or functionalized nanosystems), even making it possible to study in vivo nanoparticle interactions, thereby importantly contributing to nanoparticle design and subsequent optimization. Furthermore, the unique characteristics of nanosystems can open up new prospects for site-targeted, more specific contrast agents, contributing to the development of certain nuclear magnetic resonance applications such as MRI.

Application of NMR spectroscopy in the development of a biomimetic approach for hydrophobic drug association with physical hydrogels.

The clinical application of sparingly soluble drugs is hampered by the wide range of problems associated to their delivery. Herein we present a new physical hydrogel as a delivery system for these drugs. The strategy behind the design of this delivery system involved the incorporation of the protein albumin into the hydrogel with the aim of exploiting its intrinsic capacity to bind small hydrophobic molecules. Prednisolone and ketoconazole were used as model drug molecules. A combination of the saturation transfer difference (STD) spectra and a novel double titration assay followed by NMR was applied to study all of the possible binding modes between albumin and each drug. Finally, the ability of the hydrogel system to release the two model drugs was corroborated. The results of the release studies were in agreement with the drug binding capacities derived from the NMR studies, thus confirming that the potential of the NMR approach as a predictive technique could be useful in evaluating the designs of new drug delivery systems.

Spermidine-cross-linked hydrogels as novel potential platforms for pharmaceutical applications.

Endogen polyamines are known to be molecules of high biological value. Herein, a new generation of physical hydrogels was developed through the mild ionotropic gelantion technique, using the endogen polyamine spermidine as a physical cross-linker. The main negatively charged polymer of the hydrogel is the natural polysaccharide gellan gum. Optionally, interesting endogen molecules, such as chondroitin sulfate and albumin, can be included as part of the formulation. These new hydrogels were characterized and the influence of the different components on their final properties was carefully analyzed, ultimately demonstrating the possibility to modulate these properties as well as the system's versatility in terms of composition. On the contrary, in vitro cell studies showed the absence of cytotoxicity of these hydrogels. Finally, the in vitro-release profiles obtained for different model molecules evidenced the potential of these systems as novel drug delivery platforms.

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.

Study of the stability of proteoliposomes as vehicles for vaccines against Neisseria meningitidis based on recombinant porin complexes.

Although effective against epidemic serogroup B Neisseria meningitidis strains, vaccines based on outer membrane vesicles continue to present important limitations, and great efforts are currently being focused in the development of a variety of new vaccine candidates and in the reformulation of currently existing ones. In this work, three N. meningitidis proteins, the PorA and PorB porins and the RmpM protein, were cloned, purified and incorporated into liposomes to build defined systems. The ability of proteoliposomes to allow the refolding porin complexes, and their stability during storage at 4°C and after lyophilization in presence of two cryoprotection agents, glucose and trehalose, were evaluated. This approach allowed to mimic the porin complexes present in natural OMVs, reducing the content of hypervariable protein PorA. During storage at 4°C, our systems showed some changes in the morphology and aggregation after three months, while after lyophilization the systems maintained their properties during the whole nine months of storage checked, with glucose allowing the best preservation of the antigenic properties of the proteins in the proteoliposomes.

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.

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.

Chemically modified gelatin as biomaterial in the design of new nanomedicines.

The synthesis of new polymers has led to dramatic enhancements in the medical field. In particular, new chemical entities provided new prospects in tissue engineering, cellular therapy and drug delivery. However, significant efforts still need to be taken in consideration in order to achieve diverse clinical applications in these fields, which is challenging because of the lack of suitable materials with desired microstructure, permeability, degradation rates, products, and suitable mechanical properties. For these reasons some chemical strategies are focused in back to the nature approaches or, in other words, in improving the properties of natural polymers by chemical modifications. We report that by using a simple chemical modification technique we can obtain new biomaterials, specifically suitable for biomedical applications. Concretely, we describe the chemical modification of gelatin and the suitable characteristics of the modified protein to develop new nanomedicines. This protein was selected because of its enormous potential in biomedicine, which is currently limited due to the difficulty of its use without toxic chemical crosslinkers. The modification of the protein was based on the transformation of the carboxylic group into amido groups after their reaction with polyamines, leading to a positively charged biopolymer. To cationize the gelatin two polyamines where used: ethylenediamine and spermine, the latter being one of the endogenous polyamines which has a very positive influence over cells. This modification was monitored by physico-chemical techniques such as NMR, spectrophotometry and potentiometry. With the most promising modified gelatins we were able to develop nanoparticles using the ionotropic gelation technique. In order to determine the ability of these new nanosystems to associate bioactive molecules we selected a model plasmid DNA. The developed nanosystems were characterized corroborating their ability to associate the genetic material. In conclusion, we were able to obtain a semi-synthetic biomaterial with tunable physico-chemical properties, which can be used to develop new nanosystems with the ability to associate genetic material. We therefore propose that the gelatin, with its chemical modification, provide a unique biomaterial for the development of new nanomedicines.

Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells.

PURPOSE: Nanoparticles are a promising alternative for ocular drug delivery, and our group has proposed that they are especially suited for ocular mucosal disorders. The goal of the present study was to determine which internalization pathway is used by cornea-derived and conjunctiva-derived cell lines to take up hyaluronic acid (HA)-chitosan oligomer (CSO)-based nanoparticles (HA-CSO NPs). We also determined if plasmids loaded onto the NPs reached the cell nucleus. METHODS: HA-CSO NPs were made of fluoresceinamine labeled HA and CSO by ionotropic gelation and were conjugated with a model plasmid DNA for secreted alkaline phosphatase. Human epithelial cell lines derived from the conjunctiva and the cornea were exposed to HA-CSO NPs for 1 h and the uptake was investigated in living cells by fluorescence microscopy. The influence of temperature and metabolic inhibition, the effect of blocking hyaluronan receptors, and the inhibition of main endocytic pathways were studied by fluorometry. Additionally, the metabolic pathways implicated in the degradation of HA-CSO NPs were evaluated by lysosome identification. RESULTS: There was intracellular localization of plasmid-loaded HACSO NPs in both corneal and conjunctival cells. The intracellular presence of NPs diminished with time. HA-CSO NP uptake was significantly reduced by inhibition of active transport at 4 °C and by sodium azide. Uptake was also inhibited by blocking hyaluronan receptors with anti-CD44 Hermes-1 antibody, by excess HA, and by filipin, an inhibitor of caveolin-dependent endocytosis. HA-CSO NPs had no effect on cell viability. The transfection efficiency of the model plasmid was significantly higher in NP treated cells than in controls. CONCLUSIONS: HA-CSO NPs were internalized by two different ocular surface cell lines by an active transport mechanism. The uptake was mediated by hyaluronan receptors through a caveolin-dependent endocytic pathway, yielding remarkable transfection efficiency. Most of HA-CSO NPs were metabolized within 48 h. This uptake did not compromise cell viability. These findings further support the potential use of HA-CSO NPs to deliver genetic material to the ocular surface.

Ocular tolerance to a topical formulation of hyaluronic acid and chitosan-based nanoparticles.

PURPOSE: Hyaluronic acid-chitosan nanoparticles (HA-CS NPs) have the potential to serve as a reliable drug delivery system to topically treat ocular surface disorders. We evaluated the in vivo uptake by ocular structures, the acute tolerance, and possible alterations of tear film physiology in rabbits. METHODS: Fluorescent HA-CS NPs (fl-HA-CS NPs) were prepared by ionotropic gelation using fluoresceinamine-labeled hyaluronic acid and resuspended in buffer. fl-HA-CS NPs (30 microL, 0.5 mg/mL) and fluoresceinamine-HA conjugate (30 microL) were instilled into rabbit eyes every 30 minutes for 6 hours. In vivo uptake and acute tolerance were characterized 24 hours after the first instillation and compared with preinstillation measurements. Clinical signs, including tear production, lacrimal drainage system patency and reflux, and ocular surface pathology, were evaluated. RESULTS: The rabbits showed no signs of ocular discomfort or irritation after exposure to HA-CS NPs. No macroscopic alteration in ocular surface structures was observed. fl-HA-CS NPs were present inside conjunctival and corneal epithelial cells, although the distribution within the cells was different. The fl-HA-CS NPs had no significant effects on tissue morphology and functionality, tear production, or drainage. CONCLUSION: Taken together, these data demonstrate that HA-CS NPs are a safe drug carrier for ocular surface application.

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.

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.