Marco André Cardoso

The structure of a galactan sulfate from the red seaweed Bostrychia montagnei

The sulfated, methylated galactan isolated from the red seaweed Bostrychia montagnei, showed an unusually narrow structural dispersion. This agaran has the defining linear backbone of alternating 3-linked beta-D-galactopyranosyl units and 4-linked alpha-L-galactopyranosyl and 3,6-anhydrogalactopyranosyl residues. The D-units have C-6 methylation, C-6 single stubs of xylopyranosyl and minor to trace amounts of (possible) C-6 linked single stubs of galactopyranosyl. These units are mainly sulfated on C-4 with lesser sulfation at C-6 and minor at C-2. The L-residues are mainly methylated on C-2 of the 3,6-anhydrogalactopyranosyl and sulfated on C-3 of the L-galactopyranosyl; minor amounts of 2,3- and 3,6-disulfated and 2-O-methyl or 2-O-glycosyl 3-sulfated L-galactopyranosyl were also found.

A new HILIC-MS/MS method for the simultaneous analysis of carbidopa, levodopa, and its metabolites in human plasma.

Monitoring of the plasmatic levels of levodopa (LEV) and carbidopa (CAR) is necessary to adjust the dose of these drugs according to the individual needs of Parkinsons disease patients. To support drug therapeutic monitoring, a method using HILIC mode and LC-MS/MS was developed for the simultaneous determination of carbidopa, levodopa, and its metabolites (3-o-methyldopa (3-OMD) and dopamine (DOPA)) in human plasma. A triple quadrupole mass spectrometry was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. After straightforward sample preparation via protein precipitation, an Atlantis HILIC (150 × 2.1 mm, 3 μm, Waters, USA) column were used for separation under the isocratic condition of acetonitrile/water (79:21, v/v) containing 0.05% formic acid and 3 mmol/L ammonium formate and the total run time was 7 min. Deuterated LEV was used as internal standard for quantification. The developed method was validated in human plasma with a lower limit of quantitation of 75 ng/mL for LEV, 65 ng/mL for CAR and 3-OMD, and 20 ng/mL for DOPA. The calibration curve was linear within the concentration range of 75-800 ng/mL for LEV, 65-800 ng/mL for CAR and 3-OMD, and 20-400 ng/mL for DOPA (r>0.99). The assay was accurate and precise, with inter-assay and intra-assay accuracies within ±13.44% of nominal and inter-assay and intra-assay precision≤13.99%. All results were within the acceptance criteria of the US FDA and ANVISA guidelines for method validation. LEV, CAR, 3-OMD and DOPA were stable in the battery of stability studies, long-term, bench-top, autosampler, and freeze/thaw cycles. Samples from patients undergoing treatment were analyzed, and the results indicated that this new method is suitable for therapeutic drug monitoring in Parkinsons disease patients.

Simultaneous determination of levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma by an HPLC-MS/MS method.

BACKGROUND In this study, we developed and validated a HPLC-MS/MS method capable of simultaneously determining levodopa, carbidopa, entacapone, tolcapone, 3-O-methyldopa and dopamine in human plasma. RESULTS & METHODOLOGY: Chromatographic separation was achieved using a C8 column with a mobile phase consisting of a gradient of water and acetonitrile:methanol (90:10 v/v), both containing 0.1% formic acid. The developed method was selective, sensitive (LD<7.0 ng ml(-1)), linear (r>0.99), precise (RSD<11.3%), accurate (RE<11.8%) and free of residual and matrix effects. The developed method was successfully applied in plasma patients with Parkinsons disease using Stalevo®. CONCLUSION The new method can be used for the clinical monitoring of these substances and applied to adjustments in drug dosages.

Acellular Human Amniotic Membrane Scaffold Loaded with Nanoparticles Containing 15d-PGJ2: A New System Local Anti-Inflammatory Treatment of Eye Diseases

The pathogenesis of chronic inflammatory eye diseases is multifactorial and includes factors as tissue injuries, metabolic disorder and autoimmune diseases. The 15-deoxy-Δ12, 14-PG J2 is known for its anti-inflammatory, antioxidant and immunmodulatory properties. In vivo adhesions between cells and the extracellular matrix play a crucial role in cell differentiation, proliferation, and migration as well as tissue remodeling. Here, we present a simple method to incorporate 15d-PGJ2 nanoparticles in acellular human amniotic membrane (HAM) scaffold, as potential local anti-inflammatory delivery system. After completely removing the cells on the amniotic membrane with a sodium dodecyl sulphate and mechanical approach, we seeded Vero cells incorporate 15d-PGJ2 nanoparticles on it. The morphology of the Vero cells and nanoparticles was observed by scanning electron microscopy (SEM). The cells cultivated observed by scanning electron microscopy (SEM) presented the incorporation of the nanoparticles smooth surface and spherical shape. Our results indicate that the HAM may be an ideal candidate as a nanoparticule-matrix adhesion substrate to study a new system for local anti-inflammatory therapy.

DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE DETERMINATION OF CHLORHEXIDINE AND P-CHLOROANILINE IN VARIOUS PHARMACEUTICAL FORMULATIONS

□ A simple, rapid and sensitive isocratic reversed-phase (RP) high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of chlorhexidine (CHX) and p-chloroaniline (CAL) in various pharmaceutical formulations. Compound separation was achieved in less than 10 min with an XBridge C18 column that was maintained at 40°C and a mobile phase consisting of 32:68 (v/v) of acetonitrile and a pH 3.0 phosphate buffer solution (a 0.05 M monobasic sodium phosphate solution containing 0.2% of triethylamine). Analyses were performed at a flow rate of 2 mL min−1 and at a detection wavelength of 239 nm. The method was shown to be selective, linear, accurate, and precise in intra-day and inter-day analyses. The robustness of the method was shown by slightly changing the flow rate, column oven temperature, and proportion of acetonitrile in the mobile phase. The method was found, however, to be very sensitive to the pH of the mobile phase buffer. The method was successfully validated following the guidelines of the International Conference on Harmonization (ICH). This validation proved that the method was suitable for the determination of CHX and CAL in toothpaste, mouthwash, wound cleanser, and skin and hand disinfectants.

15d-PGJ2-loaded in nanoparticles associated with decellurazied human amniotic membrane scaffold: A potential anti-inflammatory delivery system

Chronic inflammatory diseases are disorders multifactorial growing on elderly and diabetic populations. 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) acts in protective role against oxidative stress and inflammation both in vivo and in vitro. Here, we present a simple method to incorporate 15d-PGJ2 nanoparticles in acellular human amniotic membrane scaffold, a potential local anti-inflammatory delivery system. After completely removing the cells on the amniotic membrane with a sodium dodecyl sulphate and mechanical approach, we seeded Vero cells incorporated with 15d-PGJ2 nanoparticles. The morphology of the Vero cells and nanoparticles was observed by scanning electron microscopy (SEM) and phase contrast microscopy. The cells cultivated presented the incorporation of the nanoparticles, smooth surface and spherical shape. Within this study it was shown that amniotic membrane can be used to incorporate cells and anti-inflammatory loaded nanoparticles for prevention and treatment of various diseases. Introduction The incidence of people with chronic inflammatory diseases has been increasing over the last three decades, threatening human health. A number of factors are recognized as causes of the pathogenesis as for example the autoimmune diseases, metabolic disorders and chronic respiratory disease [1]. The 15-Deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) is a natural ligand of peroxisome proliferator activated receptor γ(PPARγ). This molecule has anti-inflammatory property and is involved in a variety of physiological and pathological processes, including rheumatoid arthritis, myocardial infarction, neural damage, and tumorigenesis [2]. The nanomaterials could provide a revolution in technology that will soon impact the diseases treatment methods through new nanoparticles delivery systems [3]. The development of new strategies for regenerative medicine is one of the most active research in the areas of nanotechnology[4]. Current advances in biotechnology uses acellular scaffolds to regenerate tissues, this is an approach highly beneficial in terms of biocompatibility and biofunctionality [5]. The decellularized human amniotic membrane is the commonest sources for scaffolds used in tissue regeneration because of it good biocompatibility and biodegradability [6]. In this study, we aimed to incorporate 15-deoxy-Δ12,14-PG J2 nanoparticles in acellular human amniotic membrane scaffold as a potential local anti-inflammatory delivery system. Methods Preparation of acellular human amniotic scaffold The study was approved by the Hospital Pequeno Príncipe Ethical Committee for the usage of biological material for research purposes approved under article number 0948-11. All materials were used in compliance with ethical guidelines by the Brazilian National Health Council. Fresh human amniotic membrane (HAM) was obtained after caesarian deliveries. Maternal donors provided informed consent and were serologically negative for HIV, hepatitis B, hepatitis C, and syphilis. Briefly, blood clots were immediately cleaned off the placenta after surgery with phosphate buffered saline (PBS) solution containing 100 U/mL penicillin and 100 mg/mL streptomycin. The acellular human amniotic scaffold (AHAS) was prepared as Correspondence to: Julio Cesar Francisco, Cell Therapy and Biotechnology in Regenerative Medicine Research Group, Pelé Pequeno Príncipe Institute, Paraná, Brazil, E-mail: julio.apfr@gmail.com Received: March 10, 2016; Accepted: April 05, 2016; Published: April 11, 2016 Francisco JC (2016) 15d-PGJ2-loaded in nanoparticles associated with decellurazied human amniotic membrane scaffold: A potential anti-inflammatory delivery system Volume 2(2): 111-113 Front Nanosci Nanotech, 2016 doi: 10.15761/FNN.1000118 described by Riau et al., 2010 [7]. Part of the human HAM was then deprived of amniotic epithelial cells to obtain AHAS by sodium dodecyl sulphate in PBS, and incubated with shaking rate of 100 rpm at 37°C for 24 h. Finally, the prepared AHAS was rewashed 3 times with PBS. Nanoencapsulation of 15d-PGJ2 in poly(D,L-lactide-co-glycolide) (PLGA) nanocapsules (15d-PGJ2-NC). The 15d-PGJ2-NC were prepared by the nanoprecipitation method, as described by Fessi et al., 1989, and supplied by Dr. Napimoga from Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center [8,9]. Culture and seeding of VERO cells with 15d-PGJ2-NC Vero cells (ATCC® CCL-81TM) were cultured for 7 days in DMEM (Dulbecco’s modified Eagle medium, Invitrogen) supplemented with 10% FBS (fetal bovine serum, Invitrogen) and 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C with 5% CO2. The culture medium was changed every 2 days. The Vero cells were seeded on plastic plate wells over AHAS and with 15d-PGJ2-NC in concentration of 10 μg/ mL for 24h in order to cells incorporate the nanoparticles, after the incubation time the plate was washed with PBS. Phase contrast images were obtained with Axio Vert.A1 (Zeiss). Scanning electron microscopy (SEM) The morphology and structure of the cells with 15d-PGJ2-NC cultivated in AHAS were examined in a JEOL 1200EX II microscope (Jeol ltda, Akishima) operating at 80 kV. In order to perform the SEM analysis, material was fixed on top coverslip, dried, mounted on a stub for SEM, fixed in 2.5% (v/v) glutaraldehyde (Sigma-Aldrich) in PBS and post-fixed with 1% (v/v) and 0.1 M sodium cacodylate trihydrate (Sigma-Aldrich). Results SEM morphology Nanoparticles characteristics of 15d-PGJ2 in the Vero cells cultivated on acellular amniotic membrane were revealed by SEM analysis. Figure 1 shows the vero cells cultivated on AHAS (a), Vero cells incubated with 15d-PGJ2 10 μg/mL (b). Vero cell cultivated on AHAS and incubated with 15d-PGJ2 10 μg/mL at 6.500x (c) and 13.000x (d). The presence of 15d-PGJ2 in the surface the amniotic membrane can be noticed. At 15d-PGJ2 concentration (10 μg/mL), a more uniform covering of the cell is observed. Discussion In recent decades, the nanotechnology has progressed rapidly. One of the most important factors in regenerative medicine is the preparation of a scaffold. Acellular amniotic membrane human scaffolds have recently become the focus of interest mainly due to the possible beneficial and applications in regenerative medicine [10]. So far, various decellularization methods have been suggested to develop extracellular matrix derived scaffolds. Tissue engineering using acellular scaffods has introduced a new field of repair in the treatment of wounds tissues or diseases [11]. HAM is an appropriate substitute for reconstruction of various organs and tissues due to its availability, low cost and low risk of viral disease transmission and immunologic rejection [12]. The overall aim of this study was to develop a type of surface covered by nanoparticles and cells with tissue engineering applications in the form of a 3D scaffold. The use of chemical approach for decellularization shown in SEM completely removal of cells while leaving the matrix intact, unlike in other studies[13]. An intact extracellular matrix is essential for recellularization, because the extra cellular matrix contains components necessary for cell attachment, proliferation, and ultimately tissue remodeling [14]. In this study, we demonstrated the recellularization efficiency with the cultivation of VERO cells in AHAS provide an adequate in vitro microenvironment for proliferation and incorporation 15d-PGJ2 nanoparticles in acellular scaffold. Conflict of interest None declared. Conclusion 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of the potent ligand of peroxisome proliferator-activated receptor γ (PPARγ), suppresses proliferation and induces intracellular apoptosis in different events in excessive inflammation. This study showed that AHAS could be used as a biomaterial and be applied as a scaffold for regenerative medicine and nanoparticle delivery.

Co-cultivated cells integration into bacterial cellulose scaffold as a new device for tissue regeneration

Regenerative medicine has now emerged as one of the most promising treatments for the patients suffering from damaged tissue. Tissue engineering has currently been based on the technology using bacterial celluloses (BC) scaffolds as alternatives for extracellular matrix. Several types of the matrix were projected successfully to cultivate different types of cells, such as, for example, Chitosan [poly-(β-1/4)-2-amino-2-deoxy-D-glucopyranose], alginate, gelatin or collagen scaffolds. Nonetheless, insufficient cell proliferation into scaffolds and owing to inflammatory reaction due to scaffold biodegradation remain problems to be solved. We propose seeding cell to construct functional devices from products obtained from bacterial cellulose origin. SEM images revealed of cells attached at the BC. The BC configured an excellent device which offers enormous potential for tissue regeneration and cells cultivation. Introduction New devices composed of natural or synthetic materials are developing as therapeutic options specially designed to be applied in special clinical conditions, aiming to replace or regenerate damaged using tissue engineering[1]. Cellular therapy is a new clinical approach for the repair of damaged tissue. Its potential use in reconstructive surgery cardiac surgery is becoming a reality. A number of biomaterials used as scaffolds have been developed and investigated, in vitro and in vivo, for potential use in tissue engineering. Bacterial cellulose (BC) is considered to be an important potential source of scaffolding material owing to its biocompatibility [2]. BC has been used in the food industry for applications such as lowcalorie desserts, salads, and fabricated food, in the paper manufacturing industry to enhance paper strength, in acoustic diaphragms for audio speakers, and in the pharmaceutical industry as a filtration membrane, wound dressing and artificial skin [3,4] Currently, there is a great enthusiasm toward attracting desired stem cells to the surface of bacterial cellulose devices as part of tissueengineering applications especially in the field of regenerative medicine. Medical devices are used for a variety of different applications and are intended generally to attract endothelial progenitor cells (EPCs) to the surface of the membrane. The EPCs drawn to the surface would increase the potential of endothelialization, which consequently brings about smaller thrombotic events triggered by the coarse surface of a non-autologous [5] Thus, in this article, we present the membrane of bacterial cellulose as a new device for the adhesion and proliferation of skeletal muscle and mesenchymal stem cells. Methods The study was approved by the Institutional Animal Ethics Committee Pontifical Catholic for usage of biological material for research purposes approved under article number 555. Bacterial Cellulose The bacterial cellulose was obtained commercially from the Membracel biotechnological products, Brazil. Cell isolation, proliferation assay, and cell seeding Skeletal myoblasts were isolated after biopsy of the skeletal muscle of the lower limb, according to the technique of Delaporte [6]. Mesenchymal cells were isolated through bone marrow aspiration of the poster superior iliac crest of Wistar rats 14. After cell centrifugation and separation according to density using the Ficoll-Paque PLUS solution (Amersham Biosciences), the mesenchymal cells adhered to the surface of the plate, while those of hematopoietic origin did not. After centrifugation of the bone marrow cells, the mononuclear cells were distributed in flasks, and, after 48 hours, they were washed with PBS, and only the stromal cells remained adhered, containing the mesenchymal cells. The assays were performed in 25-cm2 flasks, and Correspondence to: Julio Cesar Francisco, Experimental Laboratory School of Health Sciences of Pontifical Catholic University of Paraná (PUCPR), Curitiba, PR, Brazil, E-mail: julio.apfr@gmail.com