"Off-the-shelf" microfluidic devices for the production of liposomes for drug delivery.

An "off-the-shelf" microfluidic chip approach, utilizing lowcost, commercially available components, for liposome production, is presented. Microfluidic devices with different geometries have been conveniently designed and assembled, allowing the production of narrowly dispersed unilamellar and very reproducible liposomes. The presented results indicate that off-the-shelf microfluidic devices can hold great promises for the efficient preparation of different lipid based colloidal systems for biomedical applications.

Effect of dynamic three-dimensional culture on osteogenic potential of human periodontal ligament-derived mesenchymal stem cells entrapped in alginate microbeads.

BACKGROUND AND OBJECTIVE: Bioreactors are devices that efficiently create an environment that enables cell cultures to grow in a three-dimensional (3D) context mimicking in vivo conditions. In this study, we investigate the effect of dynamic fluid flow on the osteogenic potential of human mesenchymal stem cells obtained from periodontal ligament and entrapped in alginate microbeads. MATERIAL AND METHODS: After proper immunophenotyping, cells were encapsulated in barium alginate, cultured in 3D static or 3D dynamic conditions represented by a bioreactor system. Calcein-AM/propidium iodide staining was used to assess cellular viability. Quantitative real-time polymerase chain reaction was used to analyze the expression of osteogenic markers (Runx2 and COL1). Alizarin Red S staining and the Fourier transform infrared spectroscopy were used to assess mineral matrix deposition. RESULTS: Optimal encapsulation procedure, in terms of polymer pumping rate, distance from droplet generator to the gelling bath and atomizing airflow was assessed. Cell viability was not affected by encapsulation in alginate microbeads. Bioreactor cell exposure was effective in anticipating osteogenic differentiation and improving mineral matrix deposition. CONCLUSION: For the first time human mesenchymal stem cells obtained from periodontal ligaments encapsulated in alginate microbeads were cultured in a bioreactor system. This combination could represent a promising strategy to create a cell-based smart system with enhanced osteogenic potential useful for many different dental applications.

Preparation and validation of low cost microfluidic chips using a shrinking approach.

The present paper describes the production of microfluidic chips using an approach based on shrinkable biocompatible polymers (i.e. agarose) for the production of size controlled microfluidic channels. In addition, all steps of chip production were carried out using an inexpensive approach that uses low cost chemicals and equipment. The produced chips were then validated by producing monodisperse polymeric microparticles for drug delivery and hydrogel microfibers for cell embedding.

Production of polymeric micelles by microfluidic technology for combined drug delivery: application to osteogenic differentiation of human periodontal ligament mesenchymal stem cells (hPDLSCs).

The current paper reports the production of polymeric micelles (PMs), based on pluronic block-copolymers, as drug carriers, precisely controlling the cellular delivery of drugs with various physico-chemical characteristics. PMs were produced with a microfluidic platform to exploit further control on the size characteristic of the PMs. PMs were designed for the co-delivery of dexamethasone (Dex) and ascorbyl-palmitate (AP) to in vitro cultured human periodontal ligament mesenchymal stem cells (hPDLSCs) for the combined induction of osteogenic differentiation. Mixtures of block-copolymers and drugs in organic, water miscible solvent, were conveniently converted in PMs within microfluidic channel leveraging the fast mixing at the microscale. Our results demonstrated that the drugs can be efficiently co-encapsulated in PMs and that different production parameters can be adjusted in order to modulate the PM characteristics. The comparative analysis of PM produced by microfluidic and conventional procedures confirmed that the use of microfluidics platforms allowed the production of PMs in a robust manner with improved controllability, reproducibility, smaller size and polydispersity. Finally, the analysis of the effect of PMs, containing Dex and AP, on the osteogenic differentiation of hPDLSCs is reported. The data demonstrated the effectiveness and safety of PM treatment on hPDLSC. In conclusion, this report indicates that microfluidic approach represents an innovative and useful method for PM controlled preparation, warrant further evaluation as general methodology for the production of colloidal systems for the simultaneous drug delivery.

Preparation and characterization of polysaccharidic microbeads by a microfluidic technique: application to the encapsulation of Sertoli cells.

Polysaccharides (e.g. alginate or agarose) represent a class of polymers commonly employed for the preparation of microparticles for cell entrapment and tissue engineering applications. The present work describes the production and characterization, by a microfluidic approach, of microbeads constituted of alginate and alginate/agarose blends, for the encapsulation of eukaryotic cells. The general production strategy is based on the formation of water-in-oil multiphase flow by a "Y" junction squeezing mechanism. The presented data demonstrate that the gelation step represents the crucial point for the production of morphologically excellent microbeads. In this respect, microfluidic methods appear to be an effective procedure for the production of microbeads intended for cell encapsulation, as proved by the high viability and maintenance of functional capability demonstrated by the encapsulated Sertoli cells.

Production and characterization of alginate microcapsules produced by a vibrational encapsulation device.

The optimization, through a Design of Experiments (DoE) approach, of a microencapsulation procedure for isolated neonatal porcine islets (NPI) is described. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An alginate/polyornithine encapsulation procedure, developed and validated in our laboratory for almost a decade, was used to embody pancreatic islets. We analyzed different experimental parameters including frequency of vibration, amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. We produced calcium-alginate gel microbeads with excellent morphological characteristics as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology, viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

Peptide-based cationic molecules for the production of positive charged liposomes and micelles.

This paper describes the synthesis and the physico-chemical characterization of cationic peptides (CPs) for possible application as non-viral gene delivery systems. Particularly, the production of cationic liposomes and micelle solutions was considered. Liposomes were prepared by REV-phase and extrusion presenting an average diameter reflecting the pore size of the membrane used for the extrusion. After DNA complexation the mean diameter of complexes decreased by increasing the number of positive charges. The non-complexed liposome preparations showed a net positive zeta potential comprised between 17.8-30 mV. After adding Defibrotide (DFT) to liposomes (at a 1:4 +/- molar ratio) the zeta potential fell down to a net negative value indicating the formation of the ionic complex. Concerning micelles, before complexation it was not possible to measure their size by PCS. However, after DFT complexation the size of complexes highly increased. In addition, as previously seen for liposomes, before complexation, the five CPs solutions showed a positive zeta potential ranging from 10-17.8 mV, while after addition of DFT the zeta potential fell to negative values. Concerning toxicity studies, in general CP-liposomes displayed a lower toxicity towards K562 cells as compared to the corresponding CP-solution. Taking into account these results, the studied CPs could be efficiently used to obtain both cationic liposomes and micelles. Moreover they are able to complex DNA with different interaction strength, depending on the type of peptide-based cationic molecule used.

Liposomes and micellar dispersions for delivery of benzoheterocyclic derivatives of distamycin A.

In this article we describe the production and characterization of specialized delivery systems for some distamycin derivatives (DD), namely liposomes and micellar dispersions. All the formulations were designed to increase the solubility of DD in an aqueous environment and to reduce the possible toxicity problems related to the administration of these drugs. For instance, liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters, then characterized in terms of dimensions, morphology, and encapsulation efficacy. The analysis of their in vitro antiproliferative activity on cultured human and mouse leukemic cells demonstrated that liposomes and micellar dispersions containing DD exert quite different effects. These effects were compared with those shown by the free drug depending on type of drug and also cell line used.

Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells.

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Peptide nucleic acids (PNA)-DNA chimeras targeting transcription factors as a tool to modify gene expression.

Peptide nucleic acids (PNAs)-DNA chimeras have been recently described as DNA mimics constituted of a part of PNA and of a part of DNA. We have demonstrated that double stranded molecules based on PNA-DNA chimeras bind to transcription factors in a sequence-dependent manner. Accordingly, these molecules can be used for transcription factor decoy (TFD) pharmacotherapy. Effects of double stranded PNA-DNA chimeras targeting NF-kappaB and Sp1 were determined on in vitro cultured human cells and were found to be comparable to those observed using double-stranded DNA decoys. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments for non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

Optimized parameters for microencapsulation of pancreatic islet cells: an in vitro study clueing on islet graft immunoprotection in type 1 diabetes mellitus.

Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical barrier to prevent islet graft (TX)-directed immune destruction. However, extent of the achieved immunoprotection will continue to be variable and unpredictable until the role of the individual mechanisms involved with TX-related inflammatory cell and immune reactivity are clarified. Macrophages (M) are believed to play a pivotal role in controlling the host/TX interaction and its consequences. We then have studied the effects of isolated rat M and their secretory products on allogeneic islets enveloped in variably sized and configured microcapsules, within in vitro mixed islet-M cocultures. In particular, we aimed to determine the sequence of immune or not immune specific cascade of early events that derive from such on interaction. One of the specific aims was to assess whether the membrane's physical intactness and conversely its even minimal rupture, along with the microcapsules' size (i.e., large vs. small) would significantly impact M reactivity and, thereby, the encapsulated islet viability and function. Special care was taken to evaluate extent of the elicited reactivity by meticulously monitoring cytokine, N2 derivative, and other proinflammatory protein curve profiles during the early M activation process. The study has preliminarily shown that, for equally formulated microcapsules, the capsular size and membrane's morphologic thoroughness are key to prevent M reactivity and possibly avoid the intracapsular islet cell damage. While elucidation of pathways involved with the encapsulated islet TX-directed host's responsiveness actually is in progress, it has clearly emerged that microcapsules should comply with well-defined physical properties and formulation specifications in order to obviate the primum movens of the inflammatory reaction process.

Liposomes containing distamycins: preparation, characterization and antiproliferative activity.

This article describes the production and characterization of two liposome formulations containing antitumor drugs, namely distamycin A (Dist) and a new alkyl derivative of distamycin A (C16-Dist). Egg-PC/cholesterol liposomes (4:1 mol/mol) were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. The encapsulation efficiency was found to be almost complete for C16-Dist (99.8%), while native distamycin A showed a lower yield (19.0%). The in vitro antiproliferative activity of the distamycins-containing liposomes determined on human leukaemic K562 cells, was 11-fold and 8-fold higher for native and alkyl derivative distamycin A, respectively, compared with that of the corresponding free drugs. Liposomal formulations show an increase in the activity and specificity of distamycins in experimental antitumor therapy.

Aminated polysaccharide microspheres as DNA delivery systems.

This article describes the production and characterization of cationic microparticles based on pullulan and starch for the delivery of nucleic acids. The microparticles were prepared by chemically cross-linkinking of a polymer solution dispersed in organic phase, followed by amination with N, N-diethyl-2-chloroethyl amine hydrochloride, or N-glycidyl-N,N-dimethyl-N-methylammonium chloride. The association of desoxyribonucleotide (DNA) with positively charged microparticles was determined. The association capacity and the affinity of microspheres for DNA were investigated as a function of type of polysaccharide, content and basicity of the amino groups. It was found that the both types of carriers synthetized display a high affinity for defibrotide due to the high porosity of polysaccharide microspheres (PMs). The in vitro release kinetics from microspheres showed an initial fast release of DNA (30 min) followed by slower release rate over 14 days. DNA release was influenced by the ionic strength of the receiving fluid. In addition, DNA release was slightly more rapid from pullulan than from starch complexes. DNA stability studies were performed by agarose gel, indicating no degradation even after 14 days. All the produced cationic microspheres were able to quantitatively load DNA. The release of DNA from PMs was strongly affected by the ionic strength of the receiving fluid. Finally, agarose gel electrophoresis of DNA released from microspheres indicated that no DNA degradation occurs even after 14 days of release from PMs.

Delivery systems for DNA-binding drugs as gene expression modulators.

Despite the large number of publications describing the synthesis and physicocharacterization of the binding between drugs and DNA, relatively few examine drug delivery systems (DDSs) for these molecules. The aim to find DDSs for DNA-binding drugs (DBDs) was prompted mainly to reduce the toxicity and/or enhance the tumor specificity of systemically administered drugs. With this in mind, we have reviewed the biological effects of some DBDs that are currently used as antitumor drugs and describe a brief selection of DDSs currently in clinical trials or on the market.

Acrylic microspheres for oral controlled release of the biguanide buformin.

Spherical microparticles based on methacrylic acid-methyl methacrylate copolymer have been developed. The method chosen for the preparation of such microparticles was suspension radical copolymerization of acrylic comonomers in the presence of the ethyleneglycol dimethacrylate as crosslinking agent. The microparticles obtained were characterised by inverse size exclusion chromatography, scanning electron microscopy, swelling degree and exchange capacity. The porous volume of the microspheres ranged from 0.086 ml/g for the microparticles produced by a methacrylic acid/methyl methacrylate ratio of 1/3 and a 10% degree of crosslinking, to 8.57 ml/g for the microparticles produced by a methacrylic acid/methyl methacrylate ratio of 3/1 and 2% degree of crosslinking (in 0.1 N NaCl in phosphate buffer pH 7.4). Also the pore diameter of the swollen microparticles ranged from a few to 120 A. Buformin tosylate - a classical hypoglycaemic drug - was included in the polymer network of the microparticles during the polymerization process. Due to the water solubility of the drug and its low solubility in the organic phase, the entrapment yield did not exceed 15%. However the amount of encapsulated drug as well as the drug released from the microparticles, was dependent on the methacrylic acid/methyl methacrylate ratio, the degree of crosslinking and solvent/comonomers ratio.

Pectin-based microspheres: a preformulatory study.

This paper reports on (a) the production of pectin microspheres and (b) the influence of different experimental parameters and ionic crosslinking on morphological and dimensional characteristics of pectin microspheres. Morphological and dimensional characteristics of pectin were analyzed as a function of the type of pectin, the dispersing phase, the stirring speed, and the emulsifying agent. Crosslinking by calcium chloride and the encapsulation of antibiotics (i.e., metronidazol and tetracycline) gave particles morphologically similar to empty particles but with slower swelling kinetic.

Improved function of rat islets upon co-microencapsulation with Sertoli's cells in alginate/poly-L-ornithine.

The purpose of this study was to assess whether Sertoli's cells would improve functional performance of homologous pancreatic islets within microcapsules. Purified rat Sertoli's cells were co-enveloped with islets in microcapsules that had been fabricated with alginic acid and poly-L-ornithine. Confocal laser microscopy was used to determine any mitogenic effects of Sertoli's cells on islets beta-cells. Insulin secretion from islets, with or without Sertoli's cells, was examined, and grafts of Sertoli's cells with islets in microcapsules into diabetic mice were carried out. Co-incubation of Sertoli's cells with islets resulted in a significant increase in the islet beta-cell mitotic rate, which was coupled with significantly higher insulin release under glucose stimulation, as compared to controls. Grafts of co-microencapsulated Sertoli's cells with islets resulted in prolongation of the achieved normoglycemia in the animals receiving Sertoli's cells with islets as compared to controls that received islets only. Sertoli's cells do promote mitogenic activities upon in vitro co-incubation with islets, whose in vitro functional and in vivo post-transplant consequences were evident. Sertoli's cells could, therefore, be co-microencapsulated with islets for transplantation in diabetic recipients.