Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination

DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(d,l-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 μm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

Preparation and characterization of poly(lactic-co-glycolic acid) microparticles containing DNA molecules encoding a malaria vaccine candidate

Background A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of a malaria DNA vaccine is presented. A 40 kHz ultrasonic atomization device was used to create the microparticles from a feedstock containing 5 volumes of 0.5% w/v PLGA in acetone and 1 volume of condensed DNA which was fed at a flow rate of 18ml h-1. The plasmid DNA vectors encoding a malaria protein were condensed with a cationic polymer before atomization. Results High levels of gene expression in vitro were observed in COS-7 cells transfected with condensed DNA at a nitrogen to phosphate (N/P) ratio of 10. At this N/P ratio, the condensed DNA exhibited a monodispersed nanoparticle size (Z-average diameter of 60.8 nm) and a highly positive zeta potential of 38.8mV. The microparticle formulations of malaria DNA vaccine were quality assessed and it was shown that themicroparticles displayed high encapsulation efficiencies between 82-96% and a narrow size distribution in the range of 0.8-1.9 μm. In vitro release profile revealed that approximately 82% of the DNA was released within 30 days via a predominantly diffusion controlledmass transfer system. Conclusions This ultrasonic atomization technique showed excellent particle size reproducibility and displayed potential as an industrially viable approach for the formulation of controlled release particles.

Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization

An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-coglycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 μm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 μm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

Morfometria da cicatrização do cólon descendente de eqüinos submetidos a enterorrafias aposicional e invaginante

A pesquisa foi conduzida com o objetivo de comparar duas técnicas de enterorrafias no cólon descendente de eqüinos, uma aposicional e outra invaginante, utilizando-se de avaliações morfométricas. As intervenções cirúrgicas foram executadas em 15 eqüinos adultos sadios, pelo flanco esquerdo, sob anestesia geral inalatória. As enterotomias foram realizadas na tênia antimesentérica do cólon descendente e as enterorrafias foram feitas com fio de poliglactina 910 n° 0. Os eqüinos foram distribuídos aleatoriamente em cinco grupos de três animais cada e submetidos à eutanásia aos três, sete, 14, 21 e 35 dias de pós-operatório para colheita de segmentos intestinais dos locais das enterorrafias para análise morfométrica. Com o auxílio de analisador de imagens, foram contados os números de neutrófilos, células mononucleares e eosinófilos ao redor do fio de sutura. Na contagem de neutrófilos, houve diferença (P = 0,05) entre as enterorrafias, com maior número na aposicional. Quanto aos mononucleares, observou-se diferença (P = 0,01) na interação entre os dias de pós-operatório e as enterorrafias. Não foram observadas diferenças (P > 0,05) na contagem de eosinófilos. Concluiu-se que a enterorrafia invaginante causou reação inflamatória de menor intensidade que a aposicional, sendo a opção preferencial para uso neste órgão.

Aspectos morfométricos da cicatrização do cólon descendente de eqüinos submetidos a enterorrafias aposicionais com poliglactina 910 e com cianoacrilato

Foi realizado estudo morfométrico do cólon descendente de 15 eqüinos hígidos, nove fêmeas e seis machos, submetidos a enterorrafias aposicionais com fio de poliglactina 910 ou com cianoacrilato. Os animais foram distribuídos aleatoriamente em cinco grupos de três animais cada e sacrificados aos três, sete, 14, 35 e 70 dias de pós-operatório para colheita dos segmentos intestinais operados e posterior processamento histológico de rotina. A análise morfométrica foi feita em microscópio óptico adaptado a um sistema de análise de imagens, onde se quantificou o infiltrado inflamatório, mediante contagens de neutrófilos, eosinófilos, macrófagos, linfócitos e plasmócitos, não sendo observadas diferenças (P>0,05) entre as enterorrafias. Houve diferenças significativas entre os dias de pós-operatório para as contagens de neutrófilos, eosinófilos, macrófagos e linfócitos. Também se mensuraram as espessuras das regiões operadas (cicatriz) e das áreas contíguas não operadas (controle), registrando-se diferenças entre os dias de pós-operatório e as regiões (P<0,05), mas não entre as enterorrafias. O fio de poliglactina 910 e o cianoacrilato apresentam a mesma eficácia para serem usados em enterorrafias aposicionais do cólon descendente de eqüinos.

Análises histológica e morfométrica do uso de membrana biossintética de celulose em trocleoplastia experimental de cães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da concentração das fibras do tecido conjuntivo quando da associação de poliglactina 910 e silicone: estudo experimental em ratos

Neste estudo observamos a reação tecidual após implantação de próteses de silicone associadas à poliglactina 910. Telas de borracha de silicone com 1,5 x 1,5cm de diâmetro e 0,7mm de espessura, polifenestradas, foram implantadas no subcutâneo do dorso de ratos.Dois grupos foram observados, no primeiro apenas telas fenestradas eram implantadas, no segundo as fenestrações das telas eram preenchidas com fios de poliglactina 910. Os animais foram observados por 30, 60 e 90 dias quando eram sacrificados e as telas mais o tecido adjacente ressecados. Não se observou diferenças na quantidade de fibras colágenas entre os subgrupos de um mesmo grupo e ou quando comparados com os subgrupos do grupo em estudo. No grupo controle, onde se usou apenas as telas fenestradas de silicone houve uma diferença significante entre as fibras reticulares, em maior número nos grupos de 30 e 60 dias de observação, quando comparadas ao grupo de 90 dias. A poliglactina 910 associada ao silicone não alterou o número de fibras do tecido conjuntivo, contadas nas fenestrações das telas.

Rigid internal fixation with titanium versus bioresorbable miniplates in the repair of mandibular fractures in rabbits

The purpose of this study was to compare by qualitative histology the efficacy of rigid internal fixation with titanium system and the Lacto Sorb® system in mandibular fractures in rabbits. Thirty male adult rabbits Oryctolagus cuniculus were used. Unilateral mandibular osteotomies were performed between the canine and first premolar. The animals were divided into two groups: for Group I - rigid internal fixation was performed with titanium system 1.5 mm (Synthes, Oberdorf, Switzerland), with two screws of 6 mm (bicortical) on each side of the osteotomy. For Group II-rigid internal fixation was performed with PLLA/PGA system 1.5 mm (Lacto Sorb®, WLorenz, Jacksonville, FL, USA). The histological analysis evaluated the presence of inflammatory reaction, degree of bone healing and degree of resorption of the Lacto Sorb® screws. The results of both fixation systems were similar, only with a small difference after 15 and 30 days. In Group I a faster bony healing was noted. But after 60 days, bony healing was similar in both groups. It is concluded that both PLLA/PGA and titanium plates and screws provide sufficient strength to permit mandibular bone healing. The resorption process of PLLA/PGA osteosynthesis material did not cause acute or chronic inflammatory reaction or foreign body reaction during the studied period. © 2004 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Comparative study of the healing process when using Vicryl®, Vicryl Rapid®, Vicryl Plus®, and Monocryl® sutures in the rat dermal tissue

Introduction: Various types of sutures are available in the market with different constitutions. However, there is a lack of research to assess and quantify the behavior of these materials. Resources and materials: This study comes benchmark wires polyglactin 910 (Vicryl ®), irradiated polyglactin 910 (Vicryl Rapid ®), polyglactin 910 treated with triclosan (Vicryl Plus ®), and poliglecaprone 25 (Monocryl ®). For this, we used 40 rats that were divided into two groups, underwent two skin incisions longitudinal 2-cm long. In Group A, simple interrupted sutures using irradiated polyglactin 910 on the right and left side of polyglactin 910, and in group B, polyglactin 910 with triclosan on the right and the left poliglecaprone 25 were made. At 2, 7, 14, and 28 days after surgery, the ten animals were killed per period, and the samples were processed for histomorphologic and histometric analysis. Conclusions: The results demonstrated that the wire poliglecaprone 25 showed better biological response, with less inflammatory infiltrates and rapid organization of connective tissue. © 2012 Springer-Verlag Berlin Heidelberg.

Surgical Treatment of Severe Frontal Bone Fracture

Craniofacial trauma can lead to several complications. The combined fractures of anterior and posterior walls of the frontal bone are almost always followed by lesions in nasofrontal orifices and disruption of nasofrontal ostia or ducts, a significant factor for the development of early and late complications after sinus fractures. This article reports a case of trauma patient, who underwent neurological evaluation and at first showed good general condition. Computed tomography noted fracture of the anterior and posterior walls of the frontal sinus and small foci of pneumocephalus in the cerebral cortex. The patient was monitored periodically and 9 days after trauma showed increased areas of pneumocephalus in prefrontal cortex, cerebrospinal fluid draining, and large dura mater lesion, with signs of necrosis and inflammation (meningitis). The necrotic tissues were removed, and dura mater was repaired through the approximation with resorbable wire polyglactin 910 5-0, oxidized cellulose application, and bonding with human fibrin sealant (fibrinogen, thrombin, and calcium chloride). Sinusectomy, frontal sinus, and nasofrontal duct obliteration with pedicled pericranium flap were performed. Tomographically, a reanatomization was noted in frontal region, and a 12-month follow-up showed no complication. The use of fibrin glue to repair dura mater lacerations, as well as the pedicle pericranium flap for frontal sinus and nasofrontal duct obliteration, is an efficient method for treating fractures of the frontal bone.

Effects of fibre reinforcement on the mechanical properties of brushite cement

In this study the effect of structure and amount of polyglactin fibre incorporation into a brushite forming calcium phosphate cement system and the effect of mechanical compaction on the fibre modified system were investigated. In comparison the effect of resorbable polycaprolactone surface coating of cement specimens was investigated. The results showed that, apart from the mechanical properties of the reinforcing material, the structure of the incorporated fibres, regular or random, is crucial for the resulting flexural strength and modulus of elasticity. Fibre reinforcement could also be combined with mechanical compaction of the cement/fibre composite paste leading to a possible 7-fold increase in flexural strength or an almost 5-fold increase in modulus of elasticity. Reinforcement of the tensile surface of cement grafts may ultimately improve strength where required, especially in conjunction with bone fixation devices. © 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.