The Involvement of Nano-Drug Delivery in Biosafety Issues

Nanotechnology encompasses a broad conjunct of techniques aimed to engineer, characterize and make use of structures of 1 (nanoplates), 2 (nanotubes) or 3 dimensions (nanoparticles) in the nanoscale, known as nano-objects. The upper limit of the nanoscale was fixed at 100 nm [1], but in the nanopharmaceutical field the nano-scale is accepted to rise up to 200-300 nm. Biosynthesized molecules (such as hormones, proteins, nucleic acids) and drugs, whose activity depends on a primary structure and not on new phenomena derived from its size in the nano-scale, do not fit into the definition of nano-object [2]. Also the lower limit of the nanoscale was fixed in 1 nm in order to exclude atoms [2]. Beyond these constraints, there is no restriction in chemical nature of nano-objects. Today, the global market of nanotechnological consumer product is gained by non biodegradable and mostly nondispersive nano-objects. This is underscored by the raise from 212 to 1317 products (nearly 521%) between 2006 and 2011 [3]. On the other hand, Nanomedicine is the emerging discipline that employs nanoobjects as tools to solve medical problems [4,5]. The volume market of Nanomedicine is expected to exceed $160 billion by 2015, according to a business report recently launched by the Global Industry Analysts Inc [6]. The main technological platform of Nanomedicine is nano-drug delivery, accounting for 78 % global sales and 58 % of patent filling worldwide [7,8] followed by development of nano-objects for in vitro/in vivo diagnosis [9] and tissue engineering [10]. The field is characterized by the advent of a different type of nano-objects, inherently dispersive or ‘free’.Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Toll like receptors agonists-based nanomedicines as veterinary immunotherapies

Many infections affecting animals enter across mucosa, needing of secretory immunity to reject the disease; protection against some pathogens must be early, fast, and specifically elicited, while from others must be wide enough to fight against variable serotypes. Today, however, most veterinary vac-cines are injectable (not recommended for chicken and small fishes), aimed to control clinical signs instead of eradicating the disease and poor inducers of secretory immunity. These drawbacks are com-pensated by vaccination with live attenuated agents, by using potentially toxic oily adjuvants and with indiscriminate use of antibiotics. In this review, the benefits of commercial and experimental nanomed-icines acting as immunostimulants and vaccine adjuvants made of toll-like receptor (TLR) agonistsloaded in nanoparticles (NP-TLR agonists), arepresented. Np-TLR agonists induce a magnified, site-limited triggering of innate immunity; also allow modifying the administration route from injectable to mucosal or nebulized, provide structural protection to TLR agonists and avoid their diffusion far from the administration site. Future implementation of immunotherapies based on Np-TLR agonists will be discussed as a function of scale production feasibility.Fil: Parra, Federico Leonel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Nanomedicine in Latin America

This special issue of Journal of Biomaterials and Tissue Engineering includes articles from Latin American researchers that work in an emerging discipline at the interface of biomaterials science, nanotechnology and therapeutics called Nanomedicine and that comprises the use of different mono, bi and three-dimensional nano-objects (e.g., nanoplates, nanoparticles, nanotubes, etc.) to address different medical problemsFil: Abraham, Gustavo Abel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Mar del Plata. Instituto de Investigación en Ciencia y Tecnología de Materiales (i); Argentina;Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina;Fil: Sosnik, Alejandro Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Nanomedicines, the essential is invisible… to the light microscope

El poder de resolución del microscopio óptico es de unos 200 nm y si tenemos suerte, los nano-objetos más voluminosos apenas se distinguirán como puntos fluorescentes. ¿Podremos, mediante el control de la arquitectura de esos “puntos brillantes”, dirigir su acceso a ciertos sectores del cuerpo, donde ejecutarían exitosamente funciones terapéuticas? Por ahora estamos más lejos de lo que creemos en materializar ese ejercicio mental. Sin embargo, de todas las aplicaciones de la Nanotecnología al área de la salud, el campo terapéutico es el más importante. En este artículo nos adentraremos en los fenómenos que explican cómo funcionan los nano-objetos que transportan principios activos hasta los sitios blancos. A partir de allí será posible construir expectativas razonables sobre la irrupción de esta nueva tecnología en el corto futuro. Sus potencialidades y limitaciones son dependientes de nuestra capacidad de diseño racional, de desarrollo industrial y conocimiento de los fenómenos de nanotoxicidad, respectivamente.The resolution power of the optical microscope is nearly 200 nm and if we are lucky, the biggest nano-objects are saw as highly fluorescent dots. How the architecture of these shiny dots be controlled, to target them to specific body sites and successfully exert therapeutic actions? For the moment, we have been unable of meeting such issue. Nonetheless, drug delivery is by far the most important field of all the applications of Nanotechnology in health. In this review, we will address the phenomena underlying the action of nano-objects as drug delivery systems. On that knowledge, it will be possible to build reasonable expectancies to the irruption of these new medicines, in the near future. Potentialities and pitfalls of nanomedicines will depend on our skills for rational design, industrial development and insights gained on the new phenomena of nanotoxicity.Fil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Selective cytotoxicity of PAMAM G5 core-PAMAM G2.5 shell tecto-dendrimers on melanoma cells

Background: The controlled introduction of covalent linkages between dendrimer building blocks leads to polymers of higher architectural order known as tecto-dendrimers. Because of the few simple steps involved in their synthesis, tecto-dendrimers could expand the portfolio of structures beyond commercial dendrimers, due to the absence of synthetic drawbacks (large number of reaction steps, excessive monomer loading, and lengthy chromatographic separations) and structural constraints of high-generation dendrimers (reduction of good monodispersity and ideal dendritic construction due to de Gennes dense-packing phenomenon). However, the biomedical uses of tecto-dendrimers remain unexplored. In this work, after synthesizing saturated shell core-shell tecto-dendrimers using amine-terminated polyamidoamine (PAMAM) generation 5 (G5) as core and carboxyl-terminated PAMAM G2.5 as shell (G5G2.5 tecto-dendrimers), we surveyed for the first time the main features of their interaction with epithelial cells. Methods: Structural characterization of G5G2.5 was performed by polyacrylamide gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry, and microscopic techniques; their hydrodynamic size and Z-potential was also determined. Cellular uptake by human epidermal keratinocytes, colon adenocarcinoma, and epidermal melanoma (SK-Mel-28) cells was determined by flow cytometry. Cytotoxicity was determined by mitochondrial activity, lactate dehydrogenase release, glutathione depletion, and apoptosis/necrosis measurement. Results: The resultant 60%-67% saturated shell, 87,000-dalton G5G2.5 (mean molecular weight) interacted with cells in a significantly different fashion in comparison to their building blocks and to its closest counterpart, PAMAM G6.5. After being actively taken up by epithelial cells, G5G2.5 caused cytotoxicity only on SK-Mel-28 cells, including depletion of intracellular glutathione and fast necrosis that was manifested above 5 μM G5G2.5. It cannot be discounted that traces of LiCl within G5G2.5 were involved in such deleterious effects. Conclusion: These preliminary results suggest that at concentrations that do not damage healthy keratinocytes, G5G2.5 could display antimelanoma activity.Centro de Investigaciones Cardiovasculare

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

Background: Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined. Materials: 32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured. Results: The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA. Conclusion: The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the mediation of a direct brain delivery.Facultad de Ciencias MédicasCentro de Investigaciones Cardiovasculare

Increased brain radioactivity by intranasal 32P-labeled siRNA dendriplexes within in situ-forming mucoadhesive gels

Ana Paula Perez1, Cecilia Mundiña-Weilenmann2, Eder Lilia Romero1, Maria Jose Morilla11Programa de Nanomedicinas, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Buenos Aires, 2Centro de Investigaciones Cardiovasculares, CCT-La Plata, CONICET, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, ArgentinaBackground: Molecules taken up by olfactory and trigeminal nerve neurons directly access the brain by the nose-to-brain pathway. In situ-forming mucoadhesive gels would increase the residence time of intranasal material, favoring the nose-to-brain delivery. In this first approach, brain radioactivity after intranasal administration of 32P-small interference RNA (siRNA) complexed with poly(amidoamine) G7 dendrimers (siRNA dendriplexes) within in situ-forming mucoadhesive gels, was determined.Materials: 32P-siRNA dendriplexes were incorporated into in situ-forming mucoadhesive gels prepared by blending thermosensitive poloxamer (23% w/w) with mucoadhesive chitosan (1% w/w, PxChi) or carbopol (0.25% w/w, PxBCP). Rheological properties, radiolabel release profile, and local toxicity in rat nasal mucosa were determined. The best-suited formulation was intranasally administered to rats, and blood absorption and brain distribution of radioactivity were measured.Results: The gelation temperature of both formulations was 23°C. The PxChi liquid showed non-Newtonian pseudoplastic behavior of high consistency and difficult manipulation, and the gel retained 100% of radiolabel after 150 minutes. The PxCBP liquid showed a Newtonian behavior of low viscosity and easy manipulation, while in the gel phase showed apparent viscosity similar to that of the mucus but higher than that of aqueous solution. The gel released 35% of radiolabel and the released material showed silencing activity in vitro. Three intranasal doses of dendriplexes in PxCBP gel did not damage the rat nasal mucosa. A combination of 32P-siRNA complexation with dendrimers, incorporation of the dendriplexes into PxCBP gel, and administration of two intranasal doses was necessary to achieve higher brain radioactivity than that achieved by intravenous dendriplexes or intranasal naked siRNA.Conclusion: The increased radioactivity within the olfactory bulb suggested that the combination above mentioned favored the mediation of a direct brain delivery.Keywords: nucleic acids, gel, dendrimers, mucosa, olfactory bul

Uptake and intracellular traffic of siRNA dendriplexes in glioblastoma cells and macrophages

Gene silencing using small interfering RNA (siRNA) is a promising new approach for glioblastoma. The endocytic uptake and delivery of siRNA to intracellular compartments could be enhanced by complexation with polyamidoamine dendrimers.In the present work, the uptake mechanisms and intracellular traffic of siRNA/generation 7 dendrimer complexes (siRNA dendriplexes) were screened in T98G glioblastoma and J774 macrophages. Methods: The effect of a set of chemical inhibitors of endocytosis on the uptake and silencing capacity of dendriplexes was determined by flow cytometry. Colocalization of fluorescent dendriplexes with endocytic markers and occurrence of intracellular dissociation were assessed by confocal laser scanning microscopy. Results: Uptake of siRNA dendriplexes by T98G cells was reduced by methyl-beta-cyclodextrin, genistein, and cytochalasine D, silencing activity was reduced by genistein; dendriplexes colocalized with cholera toxin subunit B. Therefore, caveolin-dependent endocytosis was involved both in the uptake and silencing activity of siRNA dendriplexes. On the other hand, uptake of siRNA dendriplexes by J774 cells was reduced by methyl-beta-cyclodextrin, genistein, chlorpromazine, chloroquine, cytochalasine D, and nocodazole, the silencing activity was not affected by chlorpromazine, genistein or chloroquine, and dendriplexes colocalized with transferrin and cholera toxin subunit B. Thus, both clathrin-dependent and caveolin-dependent endocytosis mediated the uptake and silencing activity of the siRNA dendriplexes. SiRNA dendriplexes were internalized at higher rates by T98G but induced lower silencing than in J774 cells. SiRNA dendriplexes showed relatively slow dissociation kinetics, and their escape towards the cytosol was not mediated by acidification independently of the uptake pathway. Conclusion: The extent of cellular uptake of siRNA dendriplexes was inversely related to their silencing activity. The higher silencing activity of siRNA dendriplexes in J774 cells could be ascribed to the contribution of clathrin-dependent and caveolin-dependent endocytosis vs only caveolin-dependent endocytosis in T98G cells.Fil: Perez, Ana Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Cosaka, María Luz. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Romero, Eder Lilia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morilla, María José. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Reparation of an inflamed air-liquid interface cultured a549 cells with nebulized nanocurcumin

The anti-inflammatory, antifibrotic and antimicrobial activities of curcumin (CUR) are missed because of its low solubility in aqueous media, low bioavailability, and structural lability upon oral intake. Soft nanoparticles such as nanoliposomes are not efficient as CUR carriers, since crystalline CUR is expelled from them to physiological media. Nanostructures to efficiently trap and increase the aqueous solubility of CUR are needed to improve both oral or nebulized delivery of CUR. Here we showed that SRA1 targeted nanoarchaeosomes (nATC) [1:0.4 w:w:0.04] archaeolipids, tween 80 and CUR, 155 ± 16 nm sized of −20.7 ± 3.3 z potential, retained 0.22 mg CUR ± 0.09 per 12.9 mg lipids ± 4.0 (~600 µM CUR) in front to dilution, storage, and nebulization. Raman and fluorescence spectra and SAXS patterns were compatible with a mixture of enol and keto CUR tautomers trapped within the depths of nATC bilayer. Between 20 and 5 µg CUR/mL, nATC was endocytosed by THP1 and A549 liquid–liquid monolayers without noticeable cytotoxicity. Five micrograms of CUR/mL nATC nebulized on an inflamed air–liquid interface of A549 cells increased TEER, normalized the permeation of LY, and decreased il6, tnfα, and il8 levels. Overall, these results suggest the modified pharmacodynamics of CUR in nATC is useful for epithelia repair upon inflammatory damage, deserving further deeper exploration, particularly related to its targeting ability.Fil: Altube, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; ArgentinaFil: Caimi, Lilen Ivonne. Fundación Instituto Leloir; ArgentinaFil: Huck Iriart, Cristián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; ArgentinaFil: Morilla, María José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; ArgentinaFil: Romero, Eder Lilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Diseño de Estrategias de Targeting de Drogas; Argentin

Selective cytotoxicity of PAMAM G5 core-PAMAM G2.5 shell tecto-dendrimers on melanoma cells

Background: The controlled introduction of covalent linkages between dendrimer building blocks leads to polymers of higher architectural order known as tecto-dendrimers. Because of the few simple steps involved in their synthesis, tecto-dendrimers could expand the portfolio of structures beyond commercial dendrimers, due to the absence of synthetic drawbacks (large number of reaction steps, excessive monomer loading, and lengthy chromatographic separations) and structural constraints of high-generation dendrimers (reduction of good monodispersity and ideal dendritic construction due to de Gennes dense-packing phenomenon). However, the biomedical uses of tecto-dendrimers remain unexplored. In this work, after synthesizing saturated shell core-shell tecto-dendrimers using amine-terminated polyamidoamine (PAMAM) generation 5 (G5) as core and carboxyl-terminated PAMAM G2.5 as shell (G5G2.5 tecto-dendrimers), we surveyed for the first time the main features of their interaction with epithelial cells. Methods: Structural characterization of G5G2.5 was performed by polyacrylamide gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry, and microscopic techniques; their hydrodynamic size and Z-potential was also determined. Cellular uptake by human epidermal keratinocytes, colon adenocarcinoma, and epidermal melanoma (SK-Mel-28) cells was determined by flow cytometry. Cytotoxicity was determined by mitochondrial activity, lactate dehydrogenase release, glutathione depletion, and apoptosis/necrosis measurement. Results: The resultant 60%-67% saturated shell, 87,000-dalton G5G2.5 (mean molecular weight) interacted with cells in a significantly different fashion in comparison to their building blocks and to its closest counterpart, PAMAM G6.5. After being actively taken up by epithelial cells, G5G2.5 caused cytotoxicity only on SK-Mel-28 cells, including depletion of intracellular glutathione and fast necrosis that was manifested above 5 μM G5G2.5. It cannot be discounted that traces of LiCl within G5G2.5 were involved in such deleterious effects. Conclusion: These preliminary results suggest that at concentrations that do not damage healthy keratinocytes, G5G2.5 could display antimelanoma activity.Centro de Investigaciones Cardiovasculare