S456 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-M.81]

Fluorinated molecules and molecular devices as MRI tools

B. La Ferla 1, C. Zona 1,∗, D. Aurilia 1, C. Musicanti 2, G. Riccio 2, G.Vanhoutte 3

1 Department of Biotechnology and Biosciences, University of Milano -Bicocca, Italy2 Nanovector srl, Italy3 University of Antwerp, Bio-Imaging Lab, Antwerp, BelgiumKeywords: MRI; Fluorine; Nanoparticles; Alzheimer’s disease

Among the many contrast agents used in MRI, 19F is gaininggreat importance due to its high natural abundance and absencein biological tissue so the endogenous 19F background noise sig-nal is low. At the same time it was shown that 19F substitutioneffects drug efficacy changing the lipophilicity and electronic char-acter of the drug which effects delivery. The presence of 19F inorganic molecules may improve efficacy and decrease toxicity dueto enhanced solubility and reduced hydrophilicity. However, theintensities of 19F NMR signals are dramatically reduced in thebrain and thus have seen very few applications for the imagingpathologies within this organ. In a project devoted to the gen-eration of nanoparticles (NPs) for the diagnosis and therapy ofAlzheimer’s disease (AD) we designed fluorinated beta amyloid(Abeta) plaques/oligomers/fibrils ligands and NPs with the aim ofdetecting these � amyloid elements directly in the brain tissuein vivo. For this purpose we designed and synthesised curcuminederivatives, identified in a previous work as good (Abeta) ligands,with improved solubility, and carrying form 3 to 6 magneticallyequivalent fluorine atoms. We also synthesised monomers with atrifluoro acetamido group for the generation of fluorinated solid-lipid nanoparticles (SLN), and generated NPs with a good fluorineloading (1.3 mg/ml). Both ligands and NP will be subjected to pre-liminary in vitro evaluation.

The research leading to these results has received fundingfrom the European Community’s Seventh Framework Programme(FP7/2007-2013) under grant agreement n◦ 212043.

doi:10.1016/j.jbiotec.2010.09.669

[P-M.82]

Electron-Beam Radiation as An Effective Measure for EnhancingViral Safety of Allo Bone Transplants

Jung Eun Bae 1,∗, Eun Kyo Jeong 1, Jae Il Lee 1, Seong Hyun Jeon 2,Ji-Hwa Chae 2, In Seop Kim 1

1 Department of Biological Sciences, Hannam University, Daejon 305-811, Republic of Korea2 Hans Daedeok R&D Center, Hans Biomed Corp., Daejeon 305-811,Republic of KoreaKeywords: E-beam radiation; Demineralized bone matrix; Virusinactivation

Human demineralized bone matrix (DBM) has been clinicallyused as it is osteoinductive. Safety of biological materials suchas DBM is often questioned because materials from human ori-gin might be contaminated with infectious pathogen. The abilityto remove and/or inactivate known and potential viral contami-nants during the manufacturing process of DBM has become animportant parameter in assessing the safety of the products. Vali-dation of the process for viral removal and/or inactivation can playan essential and important role in establishing the safety of DBM.In order to increase the safety of DBM, E-beam radiation processhas been optimized, because it destroys the reproductive cells ofmicroorganisms by destroying their DNA chains. The aim of presentstudy was to evaluate the efficacy of 15 kGy E-beam radiation ininactivating viruses during the manufacture of DBM. Viral clear-ance validation method to evaluate efficacy of E-beam radiationwas developed and then the effect of E-beam radiation in inactivat-ing viruses was kinetically determined. A variety of experimentalmodel viruses for human pathogenic viruses, including the humanimmunodeficiency virus (HIV), hepatitis A virus (HAV), bovine her-pes virus (BHV), bovine viral diarrhea virus (BVDV), and porcineparvovirus (PPV) were selected for this study. All the envelopedviruses such as HIV, BHV, and BVDV were completely inactivatedto undetectable levels with 5 kGy radiation. Non-enveloped virusessuch as HAV and PPV were gradually inactivated with increasingdoses of E-beam radiation. The log reduction factors achieved were≥4.00 for HIV, ≥5.45 for BHV, ≥5.77 for BVDV, 4.92 for HAV, and5.73 for PPV. These results indicate that 15 kGy E-beam radiationis very effective and robust in inactivating all the viruses tested.

doi:10.1016/j.jbiotec.2010.09.670

[P-M.83]

Comparative study of skin penetration of hGH

Rogayyeh shakory 1,2, Mahvash Khodabandeh 2,∗, TayebehToliyat 3, Sadegh Azimzadeh 1,4, Zohreh-Azita Sadigh 5, LeilaBadiefar 2

1 Centeral Payam Noor of Tehran, Islamic Republic of, Iran2 National Institute of Genetic Engineering and Biotechnology, IslamicRepublic of, Iran3 Tehran University of Medical Sciences, Islamic Republic of, Iran4 Zanjan University, Islamic Republic of, Iran5 Razi Vaccine and Serum Research Institute, Islamic Republic of, IranKeywords: Skin absorption; Penetration enhancer; Growth hor-mone

Polyvinil pyrolidin (PVP) is a synthetic polymer that has beencreated from binding of the 1 vinil to 2 pyrolidin. This substanceis soluble in the water and the other solvents. The difference of itspolymerization degree causes the change in the molecular weight

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S457

and the variation of the molecular weight causes the change of theviscosity at the water environment.

PVP is used as a chemical penetration enhancer for the largedrug molecules. Since large molecules such as polypeptides andproteins are usually injected to the body, a lot of attempts havebeen done so as to find the uninvasive ways for the prescription ofthese drugs, one of these methods is skin absorption. In this study,the penetration enhancer effect of PVP and hydroxyl � cyclodextrinon permeation of the growth hormone through skin was comparedto each other. The growth hormone recognizes as an antiaging fac-tor. In the previous work (under publication), the family membersof the cyclodextrin were compared to each other in related to theenhancing skin absorption and the results indicated that hydroxy �cyclodextrin had the most efficiency. We measured the amount ofpermeated hGH through the rat abdominal skin using Franz Diffu-sion cell. The amount of permeated hGH through the rat abdominalskin was measured using growth hormone ELIZA test. The resultsshow that PVP has the less efficiency of the growth hormone perme-ation through membrane than hydroxy � cyclodextrin. The amountof the protein transmission has gradually increased from 1/2 hourto 6th hour, after that remains with no variation to 24 hours. Theamount of the protein permeation using PVP is less than hydroxy �cyclodextrin at the first hour (the first 4 hours) but at the followingtimes the effect on the protein transmission are relatively equal.Therefore with regard to the less toxic effect of hp�CD in compar-ison to the other chemical penetration enhancer, the use of it isprefered to the other evaluated factors.

doi:10.1016/j.jbiotec.2010.09.671

[P-M.84]

Involvement of MAPKs in Prevention of Hydrogen Peroxide-Induced Apoptosis in PC12 Neuron-like Cells by Chitosan

G. Joodi ∗, F. Khodagholi

Neuroscience Research Center, Shahid Beheshti University of MedicalSciences, Tehran, Iran, Islamic Republic of IranKeywords: Chitosan; Mitogen Activated Protein Kinase; HydrogenPeroxide; PC12

Introduction: There is mounting evidence implicating the roleof oxidative stress induced by reactive oxygen species (ROS) in neu-rodegenerative disease, including Alzheimer’s disease. Accordinglycompounds with antioxidant characteristics have become focus ofattention in treating such diseases. In this study we investigatedthe possible protective effects of Chitosan, an antioxidant oligosac-charide, on hydrogen peroxide induced cell death.

Methods: Using NGF-differentiated rat PC12 adrenal pheochro-mocytoma cells, the levels of Caspase-3, p38 Mitogen ActivatedProtein Kinase (p38MAPK),C-jun N-terminal Kinase (JNK), Extracel-lular signal-regulated Kinase (ERK), NF-E2-Related Factor 2(Nrf2),Heat Shock Protein 70 (HSP70), intracellular reactive oxygenspecies (ROS) and ionized calcium (Ca2+) were determined afterexposure to H2O2 in the presence and/or absence of Chitosan.

Results and Discussion: H2O2 exposure induced apopto-sis which was associated with increased ROS generation andintracellular Ca 2+. In addition, H2O2 induced an increase in phos-phorylation of p38 and JNK without any significant change in theirtotal levels. Pretreatment of PC12 cells with different concentra-tions of Chitosan protected cells from apoptosis as determinedby MTT and Caspase-3. Intracellular generation of ROS and Ca2+in response to H2O2 were also diminished by Chitosan. In orderto identify the underlying molecular mechanism, we measuredlevels of different MAPKs, Nrf2 and HSP70. We observed that Chi-

tosan blocked H2O2-induced p38 and JNK phosphorylation, butit increased the amount of phosphorylated ERK within cell. Con-comitantly, Chitosan caused a dose-dependent induction of Nrf2,an essential transcription factor for the antioxidant responsiveelement (ARE)-mediated induction of phase II detoxifying andoxidative stress enzyme genes. Pretreatment with Chitosan alsoresulted in upregulation of HSP70 as a protective response againstoxidative stress.

Conclusion: Our results suggest that Chitosan protects PC12cells from oxidative stress by regulating MAPKs.

doi:10.1016/j.jbiotec.2010.09.672

[P-M.85]

Inhibition evaluation of angiotensin converting enzyme byproanthocyanidins extracted from Vitis vinífera L red grapes

Gonzalo Eriz, Veronica Sanhueza, Marlene Roeckel, KatherinaFernández ∗

Universidad de Concepcion, ChileKeywords: Grapes; Angiotensin converting enzyme; Inhibition;Molecular size

Introduction: Proanthocyanidins (PAs), oligomers of flavan-3-oles, are associated to diverse bioactivities: antioxidants, anticancer and anti-hypertensives, the latter of interest for thisresearch. The objective of this study was to evaluate the influence oftwo kinds of extracts rich in PAs on the inhibition of the angiotensinconverting enzyme (ECA), and to relate the inhibition power withthe type and number of subunits which compose the polymeric PAschain. Extracts were obtained from skin and seeds of Vitis vinífera LChilean black grapes (uva país) harvested in three locations, namelyGuarilihüe, Nipas and Quillón from Itata Valley, VIII Región, Chile.

Methodology: Grape seeds and skins were manually separated,and PAs extracted with an acetone/water mixture. Size exclusionchromatography was used for purification and characterizationwas made by acid catalysis depolymerization. ECA activity wasmeasured by hyppuric acid determination from the hydrolysis ofhippuryl-histidyl-leucine by ECA with quantitative HPLC separa-tion.

Results and discussion: Structural compositions differ signifi-cantly between both extracts; indeed, those from skin do notexhibit epicatechin (EC) and epicatechin-gallate (ECG), whereas inseed extracts epigalocatechin (EGC) was not detected. Also, skinextracts have higher mean degree of polymerization (mDP) thanseed extracts.

Skin extract exhibited a higher inhibition powerIC50 = 0.14 ± 0.03 �M if compared to seed extract IC50 = 0.480 ±0.03 �M, where IC50 = concentration to reduce 50% the enzymeactivity. Monomers of catechin (IC50 = 1495 ± 90 �M) andepicatechin(IC50 = 1772 ± 121 �M) exhibit lower inhibition thannaturals extracts. Commercial natural extracts that inhibit ECAactivity are not available. Both mDP and EGC increase content inskin extract increase inhibition power of the extract.

Conclusions: Structural differences between skin and seed PAsfrom Chilean black grapes influence the ECA inhibition capacity.The larger inhibitory power of skin extract was associated to largerOH availability, mDP increase and EGC presence.

doi:10.1016/j.jbiotec.2010.09.673