Polymer nanoparticle-based controlled pulmonary drug delivery.

The development of novel formulations for controlled pulmonary drug delivery purposes has gained remarkable interest in medicine. Although nanomedicine represents attractive concepts for the treatment of numerous systemic diseases, scant information is available on the controlled drug release characteristics of colloidal formulations following lung administration, which might be attributed to the lack of methods to follow their absorption and distribution behavior in the pulmonary environment.In this chapter, we describe the methods of preparation and characterization of drug-loaded polymeric nanoparticles prepared from biodegradable charge-modified branched polyesters, aerosolization of the nanosuspensions using a vibrating-mesh nebulizer, and evaluation of the pulmonary pharmacokinetics (i.e., absorption and distribution characteristics) of the nanoscale drug delivery vehicles following aerosol delivery to the airspace of an isolated lung model. The disclosed methodology may contribute to the design of advanced colloids for the treatment of respiratory disorders.

Cellular uptake mechanism and knockdown activity of siRNA-loaded biodegradable DEAPA-PVA-g-PLGA nanoparticles.

Efficient downregulation of gene expression depends on the uptake, intracellular distribution and efficient release of siRNA from their carrier. Therefore, the cellular uptake behavior and mechanism and intracellular localization of siRNA-loaded biodegradable nanoparticles were investigated. A biodegradable polymer, composed of poly(vinyl alcohol) (PVA) modified with diamine moieties and grafted with PLGA, abbreviated as DEAPA-PVA-g-PLGA, was used for the preparation of siRNA-loaded nanoparticles by solvent displacement. Particle sizes and morphology were determined by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The dependence of particle uptake into H1299-EGFP cells (lung cancer cells expressing green fluorescent protein) on both incubation time and temperature was studied by flow cytometry. Inhibition experiments focusing on clathrin- or caveolae-mediated uptake or uptake by macropinocytosis were performed. The intracellular localization was investigated by confocal laser scanning microscopy. The GFP knockdown efficiency was determined in vitro to establish the potential of the nanoparticles for the downregulation of gene expression. Nanoparticles with diameters of 120-180nm were successfully generated. In contrast to the uptake of standard PEI-polyplexes, which increased continuously over a period of 4h, nanoparticle uptake was complete within 2h. A decrease in particle uptake at 4°C (in comparison with 37°C) suggests an active uptake process. Inhibition experiments revealed the predominance of clathrin-mediated uptake for siRNA-loaded nanoparticles. The siRNA-loaded nanoparticles could be clearly located within cells, mainly in intracellular vesicles. Particle uptake could be increased by the addition of lung surfactant to the formulation. Bioactivity in terms of successful GFP knockdown in vitro was demonstrated and could be further optimized by the use of surfactant-modified particles. In conclusion, a high and rapid cellular uptake was shown for siRNA-loaded nanoparticles. Cell internalization is based on an energy-dependent and predominantly clathrin-mediated process. Particle localization in endosomes and lysosomes was demonstrated. Evidence for the efficient delivery of bioactive siRNA and specific GFP knockdown provides a solid basis for the application of DEAPA-PVA-g-PLGA-based particles for gene silencing in vivo.

Stabilization of aerosolizable nano-carriers by freeze-drying.

PURPOSE: This study investigates the feasibility of freeze-drying aerosolizable nano-carriers (NC) by the use of different lyoprotective agents (LPA) and the influence of the freeze-drying on the physicochemical properties of these nano-carriers and on their aerosolization. METHODS: Nano-carriers were prepared from fast-degrading polymers, DMAPA(24)-PVAL-g-PLGA(1:7.5) and DEAPA(26)-PVAL-g-PLGA(1:10), and freeze-dried using increasing concentrations of different LPA. The hydrodynamic diameter, zeta potential and morphology (atomic force microscopy) of NC were characterized before and after freeze-drying. The ability to aerosolize using a jet nebulizer and an electronic micro-pump nebulizer was also investigated. RESULTS: Freeze-drying with LPA led to a decreased zeta-potential of NC and changes in size about 20 nm without alteration in shape, whereas lyophilizates without LPA were found to aggregate. While freeze-drying was positively affected by increasing concentrations, it was not influenced by the type of LPA. The possibility for aerosolization was not influenced by any LPA. CONCLUSIONS: Freeze-drying with LPA is a suitable method to physically stabilize fast-degrading NC from aqueous suspensions without influencing the aerosolizability.

Branched polyesters based on poly[vinyl-3-(dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(D,L-lactide-co-glycolide): effects of polymer structure on in vitro degradation behaviour.

Branched polyesters of the general structure poly[vinyl-3-(dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(D,L-lactide-co-glycolide) have shown potential for nano- and micro-scale drug delivery systems. Here the in vitro degradation behaviour with a special emphasis on elucidating structure-property relationships is reported. Effects of type and degree of amine substitution as well as PLGA side chain length were considered. In a first set of experiment, the weight loss of solvent cast films of defined size from 19 polymers was measured as a function of incubation in phosphate buffer (pH 7.4) at 37 degrees C over a time of 21 days. A second study was initiated focusing on three selected polymers in a similar set up, but with additional observation of pH influences (pH 2 and pH 9) and determination of water uptake (swelling) and molecular weights during degradation. Scanning electron micrographs have been recorded at selected time points to characterize film specimens morphologically after degradation. Our investigations revealed the potential to influence the degradation of this polymer class by the degree of amine substitution, higher degrees leading to faster erosion. The erosion rate could further be influenced by the type of amine functionality, DEAPA-modified polyesters degrading as fast as or slightly faster than DMAPA-modified polyesters and these degrading faster than DEAEA-PVA-g-PLGA. As a third option the degradation rate could be modified by the PLGA side chain length, shorter side chains leading to faster erosion. As compared to linear PLGA, remarkably shorter degradation times could be achieved by grafting short PLGA side chains onto amine-modified PVA backbones. Erosion times from less than 5 days to more than 4 weeks could be realized by selecting the type of amine functionality, the degree of amine substitution and the PLGA side chain length at the time of synthesis. In addition, the pathway of hydrolytic degradation can be tuned to be either mainly bulk or surface erosion.

On the design of in situ forming biodegradable parenteral depot systems based on insulin loaded dialkylaminoalkyl-amine-poly(vinyl alcohol)-g-poly(lactide-co-glycolide) nanoparticles.

The feasibility to generate in situ forming parenteral depot systems from insulin loaded dialkylaminoalkyl-amine-poly(vinyl alcohol)-g-poly(lactide-co-glycolide) nanoparticles, was investigated. Biodegradable nanoparticles formed polymeric semi-solid depots upon injection into isotonic phosphate buffered saline (PBS) with no additional initiators. Nanoparticles (NP) prepared from the different amine-modified polyesters displayed a pronounced positive zeta-potential of >25 mV. Diethylaminopropyl-amine-poly(vinyl alcohol)-g-poly(lactide-co-glycolide) (DEAPA(68)-PVAL-g-PLGA(1:20)), diethylaminoethyl-amine-poly(vinyl alcohol)-g-poly(lactide-co-glycolide) (DEAEA(33)-PVAL-g-PLGA(1:20)), and dimethylaminopropyl-amine-poly(vinyl alcohol)-g-poly(lactide-co-glycolide) (DMAPA(33)-PVAL-g-PLGA(1:20)), formed in situ depots by an ion-mediated aggregation with subsequent fusion of nanoparticles, related to a decreased glass transition temperature in the presence of PBS. Moreover, two factors, namely, polymer and insulin-nanocomplex concentration, were evaluated using a response surface design with respect to nanoparticles formation and insulin loading. Nanoparticles and implants were investigated by atomic force microscopy (AFM). The in vitro release from implants loaded with 2% insulin was carried out in a flow trough cell and quantified by high performance liquid chromatography (HPLC). The release showed a triphasic profile with an initial burst, pore diffusion and diffusion from the swollen matrix over more than two weeks. Insulin distribution in the implants during the release was followed by confocal laser scanning microscopy (CLSM). These findings combined with the protection of the model peptide against competitive macromolecules and the possibility to get dry powders by lyophilization make these nanoparticles-based depots suitable candidates for the design of controlled release devices for bioactive macromolecules.

Thermosensitive polymers: synthesis, characterization, and delivery of proteins.

Three triblock copolymers based on the poly(lactide) or poly(lactide-co-glycolide) and poly(ethylene glycol) or poly(ethylene oxide) blocks were synthesized and characterized. The weight average molecular weight and number average molecular weight were determined by gel permeation chromatography and proton nuclear magnetic resonance spectroscopy, respectively. Fourier transform infrared spectroscopy was used to determine the completion of synthesis of polymers. Thermoreversible sol-gel transition temperature and concentration were determined by an inverted tube method. Two formulations each of three synthesized polymers containing 5% (w/v) of lysozyme or bromelain but differing in polymer concentrations (20-30%, w/v) were prepared and studied for in vitro release of the incorporated protein. In vitro biocompatibility of the delivery systems was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Biological activities of lysozyme and bromelain were determined by enzyme activity assays. Critical gelling concentrations were found in the range of 20-30% (w/v). In vitro biocompatibility study showed that all the formulations were biocompatible. Increasing the polymer concentration led to a decrease in burst release and extended the in vitro release of proteins. Furthermore, biological activities of lysozyme and bromelain in released samples were found to be significantly (p<0.05) greater in comparison to the control. Thus, the above thermosensitive polymers were able to deliver proteins in biologically active forms at a controlled rate for 2-8 weeks.

Aluminum and zinc complexes based on an amino-bis(pyrazolyl) ligand: synthesis, structures, and use in MMA and lactide polymerization.

The coordination chemistry of bis[2-(3,5-dimethyl-1-pyrazolyl)ethyl]amine (1, LH) with aluminum- and zinc-alkyls has been studied. Reaction of 1 with AlR3 affords the adducts [LH] x AlR3 (R = Me, 2; Et, 3), which undergo alkane elimination upon heating to yield the amido complexes [L]AlR2 (R = Me, 4; Et, 5). Reaction of LiO(iPrO)C=CMe2 with 2 proceeds via N-H deprotonation to give Li[L]AlMe3 (6), while the former enolate adds to 4 to generate [Me2C=C(OiPr)OLi] x [L]AlMe2 (7). Similarly, the 1:1 reaction of ZnEt2 with 1 gives [LH] x ZnEt2 (9), which is transformed into [L]ZnEt (10) upon heating. When an excess of ZnEt2 was used in the latter reaction, the bimetallic complex [L]ZnEt x ZnEt2 (11) was isolated beside 10. Performing the same reaction in the presence of O2 traces yielded selectively the dinuclear ethyl-ethoxide complex [L]Zn2Et2(mu-OEt) (12), which was alternatively prepared from the reaction of 10 and ZnEt(OEt). Zinc chloride complexes [LH] x ZnRCl (R = Et, 13; p-CH3C6H4CH2, 14) and [L]ZnCl (15) were prepared in high yields following similar strategies. Ethyl abstraction from 10 with B(C6F5)3 yields [L]Zn+EtB(C6F5)3- (16). All complexes have been characterized by multinuclear nuclear magnetic resonance (NMR), elemental analysis, and single-crystal X-ray diffraction studies for four-coordinate Al complexes 2, 4, and 6 and Zn complexes 9-12 and 14. Aluminate species 6 and 7 initiate the polymerization of methyl methacrylate, and the monomer conversions are improved in the presence of neutral complexes 2 or 4, respectively; however, these methyl methacrylate (MMA) polymerizations are uncontrolled. Polymerization of rac-lactide takes place at 20 degrees C in the presence of zinc ethoxide complex 12 to yield atactic polymers with controlled molecular masses and relatively narrow polydispersities.

In vitro evaluation of a poly(lactide-co-glycolide)-collagen composite scaffold for bone regeneration.

Numerous materials have been proposed for bone tissue regeneration. However, none has been shown to be entirely satisfactory. In this study we fabricated a hybrid composite scaffold composed of poly(D,L-lactide-co-glycolide) (PLGA) and a naturally derived collagen matrix derived from porcine bladder submucosa matrix (BSM), and evaluated the biological activities and physical properties of the scaffold for use in bone tissue regeneration. The BSM-PLGA composite scaffolds are able to promote cellular interactions and possess uniformly interconnected pores with adequate structural integrity. The composite scaffolds were tested with both human embryonic stem (hES) cells and bovine osteoblasts (bOB). Cells seeded on the composite scaffolds readily attached, infiltrated and proliferated, as confirmed by cell viability and mitochondrial metabolic activity. Use of the composite scaffolding system with cells may enhance the formation of bone tissue for therapeutic regeneration.

Surfactant-free, biodegradable nanoparticles for aerosol therapy based on the branched polyesters, DEAPA-PVAL-g-PLGA.

PURPOSE: This study describes the development of surfactant-free, biodegradable nanoparticle systems with varying physicochemical properties and their suitability for pulmonary application via nebulization. METHODS: Nanoparticle suspensions were formulated from the branched polyester, diethylaminopropyl amine-poly(vinyl alcohol)-grafted-poly(lactide-co-glycolide) (DEAPA-PVAL-g-PLGA) alone, as well as with increasing amounts of carboxymethyl cellulose (CMC). Particle size, zeta potential, turbidity, and morphology (atomic force microscopy) were characterized. Three formulations were chosen for further study: Cationic nanoparticles without CMC, cationic nanoparticles with CMC, and anionic nanoparticles with an excess of CMC. Nanoparticle degradation was characterized, as well as stability during nebulization. Nanoparticle-cell interactions were investigated and quantified using confocal laser scanning microscopy and fluorescence spectrometry. RESULTS: Nanoparticles ranged in size from 70-250 nm and displayed zeta potentials of +58.9 to -46.6 mV. Anionic nanoparticles showed the highest stability during nebulization. The degradation rate of each nanoparticle formulation decreased with increasing amounts of CMC. Cell association was highest among cationic nanoparticles (57% and 30%, respectively), although these were not internalized. Despite a lower rate of cell association (3%), anionic nanoparticles were internalized by A549 cells. CONCLUSIONS: Surfactant-free nanoparticles from DEAPA-PVAL-g-PLGA are versatile drug delivery systems; however, only the anionic formulations investigated were proven suitable for aerosol therapy.

Loading of tetanus toxoid to biodegradable nanoparticles from branched poly(sulfobutyl-polyvinyl alcohol)-g-(lactide-co-glycolide) nanoparticles by protein adsorption: a mechanistic study.

PURPOSE: Mucosal delivery of vaccine-loaded nanoparticles (NP) is an attractive proposition from an immunologic perspective. Although numerous NP preparation methods are known, sufficient antigen loading of NP remains a challenge. The aim of this study was to evaluate adsorptive loading of NP with a negatively charged surface structure using tetanus toxoid (TT) as a model vaccine. METHODS: Blank NP, consisting of poly(sulfobutyl-polyvinyl alcohol)-g-(lactide-co-glycolide), as well as poly(lactide-co-glycolide) NP were prepared by a solvent displacement technique. The use of polymers with different degrees of substitution resulted in NP with different negative surfaces charges. Adsorption of TT to NP was performed varying to NP surface properties, protein equilibrium concentration, and loading conditions. RESULTS: The protein adsorption was controlled by NP surface properties, and maximum TT adsorption occurred at highly negatively charged NP surfaces. Results from isothermal titration calorimetry and zeta-potential measurement suggest an adsorption process governed by electrostatic interactions. The adsorption followed the Langmuir isotherm in the concentration ranges studied. TT withstood this gentle loading procedure in a nonaggregated, enzyme-linked immunoabsorbant assay-active form. CONCLUSION: The results demonstrate that negatively charged NP consisting of poly(sulfobutyl-polyvinyl alcohol)-g-(lactide-co-glycolide) are suitable for adsorptive loading with TT and may have potential for mucosal vaccination.

Sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide)s facilitate the preparation of small negatively charged biodegradable nanospheres.

The manufacturing conditions for small nanoparticles (NP) in the range of 100-500 nm are difficult to control. Novel biodegradable, brush-like branched polyesters with a negatively charged hydrophilic backbone, poly(2-sulfobutyl-vinyl alcohol)-g-poly(lactide-co-glycolide), facilitate their preparation by a modified solvent displacement procedure. Furthermore, the structure and the surface properties of the colloidal systems are investigated. NP were characterized by photon correlation spectroscopy (PCS), zeta-potential measurement (ZPM), particle charge detection (PCD), nuclear magnetic resonance spectroscopy (NMR) and transmission electron microscopy (TEM). Varying the manufacturing conditions NP with mean diameters of about 100 up to 500 nm and, depending on polymer composition, negatively charged surfaces were obtained. The NP visualized by TEM showed smooth surfaces. Furthermore, surface characterization and NMR studies suggested a core/corona structure of the particles. This study demonstrates that a simple solvent displacement technique can be used for the reproducible preparation of discrete NP with defined negatively charged surfaces and narrow size distributions. These NP may have potential for peroral or parenteral protein delivery systems.

Uso do adesivo biológico de fibrina para reinserçäo de músculos retos superiores em coelhos: II - estudo histopatológico / Fibrin biological adhesive for reinsertion of superior rectus muscles: an histopathological study in rabbits

A procura de novos métodos de adesäo tecidual tem sido uma constante nas diversas áreas da cirurgia. Recentemente, atençäo especial tem sido dada a um tipo de adesivo biológico composto por fibrinogênio e trombina, que quando comparado a outros adesivos como o cianoacrilato, apresenta a vantagem de ser totalmente absorvível e é considerado o melhor entre os adesivos teciduais. O presente estudo foi realizado em coelhos e teve como objetivo avaliar, através de exame histopatológico, a reaçäo tecidual produzida pelo adesivo biológico de fibrina na junçäo músculo escleral e compará-la com a reaçäo produzida pela poliglactina 910. A amostra constituída por 24 animais foi separada de acordo com o período