Name: evaluation of antimicrobial activities of nanoparticles and nanostructured surfaces in vitro
Uploaded: 2023-04-21T14:40:00
Description: Watch this Scientific Journal Video about Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro at JoVE.com

Forfatterne sætter pris på den økonomiske støtte fra US National Science Foundation (NSF CBET-pris 1512764 og NSF PIRE 1545852), National Institutes of Health (NIH NIDCR 1R03DE028631), University of California (UC) Regents Faculty Development Fellowship, Udvalget for Research Seed Grant (Huinan Liu) og UC-Riverside Graduate Research Mentorship Program Grant tildelt Patricia Holt-Torres. Forfatterne sætter pris på den hjælp, der ydes af Central Facility for Advanced Microscopy and Microanalysis (CFAMM) ved UC-Riverside til brug af SEM / EDS og Dr. Perry Cheung til brug af XRD. Forfatterne vil også gerne takke Morgan Elizabeth Nator og Samhitha Tumkur for deres hjælp med eksperimenterne og dataanalyserne. Eventuelle meninger, resultater, konklusioner eller anbefalinger udtrykt i denne artikel er forfatternes og afspejler ikke nødvendigvis synspunkterne fra National Science Foundation eller National Institutes of Health.

For at præsentere metoderne til direkte samkultur og direkte eksponering bruger vi magnesiumoxidnanopartikler (nMgO) som modelmateriale til at demonstrere bakterielle interaktioner. For at præsentere den direkte kultur og fokuserede kontakteksponeringsmetoder bruger vi en Mg-legering med nanostrukturerede overflader som eksempler.
1. Sterilisering af nanomaterialer
BEMÆRK: Alle nanomaterialer skal steriliseres eller desinficeres inden mikrobiel kul...

<ol>
	<li>Haque, M., Sartelli, M., McKimm, J., Abu Bakar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+care-associated+infections+-+An+overview.">Health care-associated infections - An overview.</a> Infection and Drug Resistance. 11, 2321-2333 (2018).</li><li>O'Connell, K. M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combating+multidrug-resistant+bacteria:+Current+strategies+for+the+discovery+of+novel+antibacterials.">Combating multidrug-resistant bacteria: Current strategies for the discovery of novel antibacterials.</a> Angewandte Chemie. 52 (41), 10706-10733 (2013).</li><li>Li, B., Webster, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+antibiotic+resistance:+New+challenges+and+opportunities+for+implant-associated+orthopedic+infections.">Bacteria antibiotic resistance: New challenges and opportunities for implant-associated orthopedic infections.</a> Journal of Orthopaedic Research. 36 (1), 22-32 (2018).</li><li>Yung, D. B. Y., Sircombe, K. J., Pletzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friends+or+enemies?+The+complicated+relationship+between+Pseudomonas+aeruginosa+and+Staphylococcus+aureus.">Friends or enemies? The complicated relationship between Pseudomonas aeruginosa and Staphylococcus aureus.</a> Molecular Microbiology. 116 (1), 1-15 (2021).</li><li>Adams, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rifamycin+antibiotics+and+the+mechanisms+of+their+failure.">Rifamycin antibiotics and the mechanisms of their failure.</a> The Journal of Antibiotics. 74 (11), 786-798 (2021).</li><li>Harding, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+global+progress+report+on+tuberculosis+elimination.">WHO global progress report on tuberculosis elimination.</a> The Lancet. Respiratory Medicine. 8 (1), 19 (2020).</li><li>Baptista, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-strategies+to+fight+multidrug+resistant+bacteria-&amp;#34;A+battle+of+the+titans&amp;#34.">Nano-strategies to fight multidrug resistant bacteria-&amp;#34;A battle of the titans&amp;#34.</a> Frontiers in Microbiology. 9, 1441 (2018).</li><li>Seong, M., Lee, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticles+against+Salmonella+enterica+serotype+typhimurium:+Role+of+inner+membrane+dysfunction.">Silver nanoparticles against Salmonella enterica serotype typhimurium: Role of inner membrane dysfunction.</a> Current Microbiology. 74 (6), 661-670 (2017).</li><li>Dasgupta, N., Ramalingam, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticle+antimicrobial+activity+explained+by+membrane+rupture+and+reactive+oxygen+generation.">Silver nanoparticle antimicrobial activity explained by membrane rupture and reactive oxygen generation.</a> Environmental Chemistry Letters. 14, 477-485 (2016).</li><li>Su, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+disruption+of+bacterial+membrane+integrity+through+ROS+generation+induced+by+nanohybrids+of+silver+and+clay.">The disruption of bacterial membrane integrity through ROS generation induced by nanohybrids of silver and clay.</a> Biomaterials. 30 (30), 5979-5987 (2009).</li><li>Cui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+action+of+bactericidal+gold+nanoparticles+on+Escherichia+coli.">The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli.</a> Biomaterials. 33 (7), 2327-2333 (2012).</li><li>Ranjan, S., Ramalingam, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titanium+dioxide+nanoparticles+induce+bacterial+membrane+rupture+by+reactive+oxygen+species+generation.">Titanium dioxide nanoparticles induce bacterial membrane rupture by reactive oxygen species generation.</a> Environmental Chemistry Letters. 14, 487-494 (2016).</li><li>Kadiyala, U., Turali-Emre, E. S., Bahng, J. H., Kotov, N. A., VanEpps, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+insights+into+antibacterial+activity+of+zinc+oxide+nanoparticles+against+methicillin+resistant+Staphylococcus+aureus+(MRSA).">Unexpected insights into antibacterial activity of zinc oxide nanoparticles against methicillin resistant Staphylococcus aureus (MRSA).</a> Nanoscale. 10 (10), 4927-4939 (2018).</li><li>Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+bioresorbable+Mg-Zn-Ca+alloy+for+bone+repair+in+a+comparison+study+with+Mg-Zn-Sr+alloy+and+pure+Mg.">Antimicrobial bioresorbable Mg-Zn-Ca alloy for bone repair in a comparison study with Mg-Zn-Sr alloy and pure Mg.</a> ACS Biomaterials Science and Engineering. 6 (1), 517-538 (2020).</li><li>Lock, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+and+antibacterial+properties+of+magnesium+alloys+in+artificial+urine+for+potential+resorbable+ureteral+stent+applications.">Degradation and antibacterial properties of magnesium alloys in artificial urine for potential resorbable ureteral stent applications.</a> Journal of Biomedical Materials Research. Part A. 102 (3), 781-792 (2014).</li><li>Lin, J., Nguyen, N. -. Y. T., Zhang, C., Ha, A., Liu, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+properties+of+MgO+nanostructures+on+magnesium+substrates.">Antimicrobial properties of MgO nanostructures on magnesium substrates.</a> ACS Omega. 5 (38), 24613-24627 (2020).</li><li>Nguyen, N. -. Y. T., Grelling, N., Wetteland, C. L., Rosario, R., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activities+and+mechanisms+of+magnesium+oxide+nanoparticles+(nMgO)+against+pathogenic+bacteria,+yeasts,+and+biofilms.">Antimicrobial activities and mechanisms of magnesium oxide nanoparticles (nMgO) against pathogenic bacteria, yeasts, and biofilms.</a> Scientific Reports. 8 (1), 16260 (2018).</li><li>Bindhu, M. R., Umadevi, M., Micheal, M. K., Arasu, M. V., Al-Dhabi, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+morphological+and+optical+properties+of+MgO+nanoparticles+for+antibacterial+applications.">Structural morphological and optical properties of MgO nanoparticles for antibacterial applications.</a> Materials Letters. 166, 19-22 (2016).</li><li>He, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+mechanism+of+antibacterial+action+of+magnesium+oxide+nanoparticles+against+foodborne+pathogens.">Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens.</a> Journal of Nanobiotechnology. 14 (1), 54 (2016).</li><li>Zhang, K., An, Y., Zhang, L., Dong, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+controlled+nano-MgO+and+investigation+of+its+bactericidal+properties.">Preparation of controlled nano-MgO and investigation of its bactericidal properties.</a> Chemosphere. 89 (11), 1414-1418 (2012).</li><li>Hayat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antibiofilm+and+anti-adhesion+effects+of+magnesium+oxide+nanoparticles+against+antibiotic+resistant+bacteria.">In vitro antibiofilm and anti-adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria.</a> Microbiology and Immunology. 62 (4), 211-220 (2018).</li><li>Dong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Mg(OH)2+nanoparticles+as+an+antibacterial+agent.">Investigation of Mg(OH)2 nanoparticles as an antibacterial agent.</a> Journal of Nanoparticle Research. 12, 2101-2109 (2010).</li><li>Halbus, A. F., Horozov, T. S., Paunov, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+antimicrobial+action+of+surface+modified+magnesium+hydroxide+nanoparticles.">Controlling the antimicrobial action of surface modified magnesium hydroxide nanoparticles.</a> Biomimetics. 4 (2), 41 (2019).</li><li>Pan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+antibacterial+activity+and+related+mechanism+of+a+series+of+Nano-Mg(OH)2.">Investigation of antibacterial activity and related mechanism of a series of Nano-Mg(OH)2.</a> ACS Applied Materials and Interfaces. 5 (3), 1137-1142 (2013).</li><li>Ipe, D. S., Kumar, P. T. S., Love, R. M., Hamlet, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticles+at+biocompatible+dosage+synergistically+increases+bacterial+susceptibility+to+antibiotics.">Silver nanoparticles at biocompatible dosage synergistically increases bacterial susceptibility to antibiotics.</a> Frontiers in Microbiology. 11, 1074 (2020).</li><li>Parvekar, P., Palaskar, J., Metgud, S., Maria, R., Dutta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimum+inhibitory+concentration+(MIC)+and+minimum+bactericidal+concentration+(MBC)+of+silver+nanoparticles+against+Staphylococcus+aureus.">The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles against Staphylococcus aureus.</a> Biomaterial Investigations in Dentistry. 7 (1), 105-109 (2020).</li><li>Loo, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+antimicrobial+activity+of+green+synthesized+silver+nanoparticles+against+selected+gram-negative+foodborne+pathogens.">In vitro antimicrobial activity of green synthesized silver nanoparticles against selected gram-negative foodborne pathogens.</a> Frontiers in Microbiology. 9, 1555 (2018).</li><li>Ali, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+accelerated+green+synthesis+of+stable+silver+nanoparticles+with+Eucalyptus+globulus+leaf+extract+and+their+antibacterial+and+antibiofilm+activity+on+clinical+isolates.">Microwave accelerated green synthesis of stable silver nanoparticles with Eucalyptus globulus leaf extract and their antibacterial and antibiofilm activity on clinical isolates.</a> PLoS One. 10 (7), e0131178 (2015).</li><li>Al-Jumaili, A., Alancherry, S., Bazaka, K., Jacob, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+the+antimicrobial+properties+of+carbon+nanostructures.">Review on the antimicrobial properties of carbon nanostructures.</a> Materials. 10 (9), 1066 (2017).</li><li>MTI Corporation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 80L NTRL Certified Convection Drying Oven (18"x16"x18", 250°C) with Digital Temperature Controller (SSP) - BPG-7082. , (2022).</li><li>CHEBI. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tris (CHEBI:9754). , (2023).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+Spring+Harbor+Protocols.+Phosphate+buffer.">Cold Spring Harbor Protocols. Phosphate buffer.</a> Cold Spring Harbor Laboratory Press. , (2016).</li><li>Barat, R., Montoya, T., Seco, A., Ferrer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+biological+and+chemically+induced+precipitation+of+calcium+phosphate+in+enhanced+biological+phosphorus+removal+systems.">Modelling biological and chemically induced precipitation of calcium phosphate in enhanced biological phosphorus removal systems.</a> Water Research. 45, 3744-3752 (2011).</li><li>Carlsson, H., Aspegren, H., Lee, N., Hilmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+phosphate+precipitation+in+biological+phosphorus+removal+systems.">Calcium phosphate precipitation in biological phosphorus removal systems.</a> Water Research. 31 (5), 1047-1055 (1997).</li><li>Gonzalez, J., Hou, R. Q., Nidadavolu, E. P. S., Willumeit-R&#246;mer, R., Feyerabend, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnesium+degradation+under+physiological+conditions+-+Best+practice.">Magnesium degradation under physiological conditions - Best practice.</a> Bioactive Materials. 3 (2), 174-185 (2018).</li><li>Oyane, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+assessment+of+revised+simulated+body+fluids.">Preparation and assessment of revised simulated body fluids.</a> Journal of Biomedical Materials Research. Part A. 65 (2), 188-195 (2003).</li><li>Xia, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+histology+stain+for+rapid+bacterial+endospore+imaging.">Amyloid histology stain for rapid bacterial endospore imaging.</a> Journal of Clinical Microbiology. 49 (8), 2966-2975 (2011).</li><li>Pankey, G. A., Sabath, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+of+bacteriostatic+versus+bactericidal+mechanisms+of+action+in+the+treatment+of+Gram-positive+bacterial+infections.">Clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram-positive bacterial infections.</a> Clinical Infectious Diseases. 38 (6), 864-870 (2004).</li><li>Ribeiro, M., Monteiro, F. J., Ferraz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+orthopedic+implants+with+emphasis+on+bacterial+adhesion+process+and+techniques+used+in+studying+bacterial-material+interactions.">Infection of orthopedic implants with emphasis on bacterial adhesion process and techniques used in studying bacterial-material interactions.</a> Biomatter. 2 (4), 176-194 (2012).</li></ol>

Vi har præsenteret fire in vitro metoder (A-D) til at karakterisere nanopartiklers og nanostrukturerede overfladers antibakterielle aktiviteter. Mens hver af disse metoder kvantificerer bakterievækst og levedygtighed over tid som reaktion på nanomaterialer, findes der en vis variation i de metoder, der anvendes til at måle den oprindelige bakterielle såtæthed, vækst og levedygtighed over tid. Tre af disse metoder, metoden med direkte samkultur (A)17, metoden med direkte kultur (C)1...

I USA alene udvikler 1,7 millioner individer en hospitalserhvervet infektion (HAI) årligt, hvor en ud af hver 17 af disse infektioner resulterer i død1. Derudover anslås det, at behandlingsomkostningerne for HAI'er varierer fra $ 28 milliarder til $ 45 milliarder årligt 1,2. Disse HAI'er domineres af methicillinresistente Staphylococcus aureus (MRSA)3,4 og Pseudomonas aeruginosa4, som almindeligvis isoleres fra kroniske sårinfektioner og normalt kræver omfattende behandling og tid for at producere et gunstigt patientresultat.
I løbet af de sidste årtier er der udviklet flere antibiotikaklasser til behandling af infektioner relateret til disse og andre patogene bakterier. For eksempel er rifamycinanaloger blevet anvendt til behandling af MRSA, andre gram-positive og gram-negative infektioner og Mycobacterium spp. infektioner5. I 1990'erne, for effektivt at behandle et stigende antal M. tuberculosis-infektioner, blev yderligere lægemidler kombineret med rifamycinanaloger for at øge deres effektivitet. Imidlertid forbliver ca. 5% af M. tuberculosis-tilfældene resistente over forrifampicin5,6, og der er stigende bekymring for multiresistente bakterier7. I øjeblikket er brugen af antibiotika alene muligvis ikke tilstrækkelig til behandling af infektioner erhvervet i sundhedsvæsenet, og dette har fremkaldt en løbende søgning efter alternative antimikrobielle behandlingsformer1.
Tungmetaller, såsom sølv (Ag)8,9,10 og guld (Au)11, og keramik, såsom titandioxid (TiO 2)12 og zinkoxid (ZnO)13, i nanopartikel (NP) form (henholdsvis AgNP, AuNP, TiO2 NP og ZnONP) er blevetundersøgt for deres antimikrobielle aktiviteter og er blevet identificeret som potentielle antibiotikaalternativer. Derudover bioresorberbare materialer, såsom magnesiumlegeringer (Mg-legeringer)14,15,16, magnesiumoxidnanopartikler 17,18,19,20,21 og magnesiumhydroxidnanopartikler [henholdsvis nMgO og nMg(OH)2]22,23,24, er også blevet undersøgt. De tidligere antimikrobielle undersøgelser af nanopartikler anvendte imidlertid inkonsekvente materialer og forskningsmetoder, hvilket resulterede i data, der er vanskelige eller umulige at sammenligne og undertiden er modstridende i naturen18,19. For eksempel varierede den mindste hæmmende koncentration (MIC) og den minimale bakteriedræbende koncentration (MBC) af sølvnanopartikler signifikant i forskellige undersøgelser. Ipe et al.25 evaluerede de antibakterielle aktiviteter af AgNP'er med en gennemsnitlig partikelstørrelse på ~ 26 nm for at bestemme MIC'erne mod gram-positive og gram-negative bakterier. De identificerede MIC for P. aeruginosa, E. coli, S. aureus og MRSA var henholdsvis 2 μg/ml, 5 μg/ml, 10 μg/ml og 10 μg/ml. I modsætning hertil evaluerede Parvekar et al.26 AgNP'er med en gennemsnitlig partikelstørrelse på 5 nm. I dette tilfælde viste AgNP MIC og en MBC på 0,625 mg / ml sig at være effektive mod S. aureus. Derudover evaluerede Loo et al.27 AgNP'er med en størrelse på 4,06 nm. Når E. coli blev eksponeret for disse nanopartikler, blev MIC og MBC rapporteret ved 7,8 μg/ml. Endelig undersøgte Ali et al.28 de antibakterielle egenskaber af sfæriske AgNP'er med en gennemsnitlig størrelse på 18 nm. Når P. aeruginosa, E. coli og MRSA blev udsat for disse nanopartikler, blev MIC identificeret ved henholdsvis 27 μg / ml, 36 μg / ml, 27 μg / ml og 36 μg / ml, og MBC blev identificeret ved henholdsvis 36 μg / ml, 42 μg / ml og 30 μg / ml.
Selvom nanopartiklers antibakterielle aktivitet er blevet grundigt undersøgt og rapporteret i de seneste årtier, er der ingen standard for de materialer og forskningsmetoder, der anvendes til at muliggøre direkte sammenligninger på tværs af undersøgelser. Af denne grund præsenterer vi to metoder, den direkte samkulturmetode (metode A) og den direkte eksponeringsmetode (metode B), til at karakterisere og sammenligne nanopartiklers antimikrobielle aktiviteter, samtidig med at materialerne og metoderne holdes konsistente.
Ud over nanopartikler er nanostrukturerede overflader også blevet undersøgt for antibakterielle aktiviteter. Disse omfatter kulstofbaserede materialer, såsom grafennanoark, kulstofnanorør og grafit29 samt rene Mg- og Mg-legeringer. Hvert af disse materialer har udvist mindst en antibakteriel mekanisme, herunder fysisk skade på cellemembraner af kulstofbaserede materialer og skade på metaboliske processer eller DNA gennem frigivelse af reaktive iltarter (ROS), når Mg nedbrydes. Når zink (Zn) og calcium (Ca) kombineres i dannelsen af Mg-legeringer, forbedres forfiningen af Mg-matrixkornstørrelsen, hvilket fører til en reduktion i bakteriel vedhæftning til substratoverflader sammenlignet med Mg-only-prøver14. For at demonstrere antibakteriel aktivitet præsenterer vi den direkte kulturmetode (metode C), som bestemmer bakteriel vedhæftning på og omkring nanostrukturerede materialer over tid gennem kvantificering af bakteriekolonidannende enheder (CFU'er) med direkte og indirekte overfladekontakt.
Nanostrukturernes geometri på overflader, herunder størrelse, form og orientering, kan påvirke materialernes bakteriedræbende aktiviteter. For eksempel fremstillede Lin et al.16 forskellige nanostrukturerede MgO-lag på overfladerne af Mg-substrater gennem anodisering og elektroforetisk aflejring (EPD). Efter en periode med eksponering for den nanostrukturerede overflade in vitro blev væksten af S. aureus væsentligt reduceret sammenlignet med ubehandlet Mg. Dette indikerede en større styrke af den nanostrukturerede overflade mod bakteriel vedhæftning versus den ubehandlede metalliske Mg-overflade. For at afsløre de forskellige mekanismer for de antibakterielle egenskaber ved forskellige nanostrukturerede overflader diskuteres en fokuseret kontakteksponeringsmetode (metode D), der bestemmer celle-overfladeinteraktionerne inden for interesseområdet, i denne artikel.
Formålet med denne artikel er at præsentere fire in vitro-metoder, der kan anvendes på forskellige nanopartikler, nanostrukturerede overflader og mikrobielle arter. Vi diskuterer vigtige overvejelser for hver metode for at producere konsistente, reproducerbare data til sammenlignelighed. Specifikt anvendes metoden med direkte samkultur17 og metoden med direkte eksponering til undersøgelse af nanopartiklers antimikrobielle egenskaber. Gennem den direkte co-kulturmetode kan de mindste hæmmende og minimale bakteriedræbende koncentrationer (henholdsvis MIC og MBC90-99,99) bestemmes for individuelle arter, og den mest potente koncentration (MPC) kan bestemmes for flere arter. Gennem metoden med direkte eksponering kan nanopartiklers bakteriostatiske eller bakteriedræbende virkninger ved minimale hæmmende koncentrationer karakteriseres ved realtidsaflæsninger af optisk densitet over tid. Direct Culture14-metoden er velegnet til at undersøge bakterier direkte og indirekte i kontakt med nanostrukturerede overflader. Endelig præsenteres metoden med fokuseret kontakteksponering16 for at undersøge den antibakterielle aktivitet af et specifikt område på en nanostruktureret overflade gennem direkte anvendelse af bakterier og karakterisering af bakterievækst ved celle-nanostrukturgrænsefladen. Denne metode er modificeret fra den japanske industristandard JIS Z 2801:200016 og har til formål at fokusere på mikrobe-overfladeinteraktioner og udelukke virkningerne af nedbrydning af bulkprøver i mikrobiel kultur på antimikrobielle aktiviteter.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Milipore Sigma</td>
			<td>Z336777</td>
			<td></td>
		</tr>
		<tr>
			<td>80 L NTRL Certified Convection Drying Oven&#160;</td>
			<td>MTI Corporation</td>
			<td>BPG-7082</td>
			<td>https://www.mtixtl.com/BPG-7082.aspx</td>
		</tr>
		<tr>
			<td>(hydroxymethyl) aminomethane buffer pH 8.5; Tris buffer&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>42457</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSpec&#160;THIOFLAVIN T ULTRAPURE GRADE</td>
			<td>Fisher Scientific</td>
			<td>50-850-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron-multiplying charge-coupled device digital camera&#160;</td>
			<td>Hamamatsu</td>
			<td>C9100-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluteraldehyde</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brightline, Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>Inductively coupled plasma - optical emission spectrometry (ICP-OES)</td>
			<td>PerkinElmer</td>
			<td>8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverse microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma Life Science&#160;</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Bertani Broth + agar</td>
			<td>Sigma Life Science&#160;</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>MacroTube 5.0&#160;&#160;</td>
			<td>Benchmark Scientific</td>
			<td>C1005-T5-ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium oxide nanoparticles</td>
			<td>US Research Nanomaterials, Inc</td>
			<td>Stock #:&#160;US3310&#160;&#160;&#160;M</td>
			<td>MgO, 99+%, 20 nm</td>
		</tr>
		<tr>
			<td>MS Semi-Micro Balance</td>
			<td>Mettler Toledo</td>
			<td>MS105D</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose paper</td>
			<td>Fisherbrand</td>
			<td>09-801A</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 12-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351143</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 48-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351178</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-tissue treated 96-well polystyrene plate</td>
			<td>Falcon Corning Brand&#160;</td>
			<td>351172</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 100 mm</td>
			<td>VWR</td>
			<td>470210-568</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 15 mm</td>
			<td>Fisherbrand</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>VWR</td>
			<td>SP70P</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscopy (SEM)</td>
			<td>TESCAN&#160;</td>
			<td>Vega3 SBH</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>VWR</td>
			<td>97043-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Fisher Scientific</td>
			<td>accuSpin Micro 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Table top centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5430</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>MP</td>
			<td>1010617</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>Sigma-Aldrich</td>
			<td>22092-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer&#160;</td>
			<td>Tecan</td>
			<td>Infinite 200 PRO</td>
			<td>https://lifesciences.tecan.com/plate_readers/infinite_200_pro</td>
		</tr>
		<tr>
			<td>VWR Benchmark Incu-shaker 10L</td>
			<td>VWR</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray power defraction</td>
			<td>&#160;Panalytical</td>
			<td>N/A</td>
			<td>PANalytical Empyrean Series 2</td>
		</tr>
	</tbody>
</table>

evaluation of antimicrobial activities of nanoparticles and nanostructured surfaces in vitro

De antimikrobielle aktiviteter af nanopartikler og nanostrukturerede overflader, såsom sølv, zinkoxid, titandioxid og magnesiumoxid, er tidligere blevet undersøgt i kliniske og miljømæssige omgivelser og i forbrugsvarer. Manglende konsistens i de anvendte forsøgsmetoder og materialer har imidlertid kulmineret i modstridende resultater, selv blandt undersøgelser af de samme nanostrukturtyper og bakteriearter. For forskere, der ønsker at anvende nanostrukturer som additiv eller belægning i et produktdesign, begrænser disse modstridende data deres udnyttelse i kliniske omgivelser. 
For at konfrontere dette dilemma præsenterer vi i denne artikel fire forskellige metoder til at bestemme nanopartiklers og nanostrukturerede overfladers antimikrobielle aktiviteter og diskuterer deres anvendelighed i forskellige scenarier. Tilpasning af konsistente metoder forventes at føre til reproducerbare data, der kan sammenlignes på tværs af undersøgelser og implementeres for forskellige nanostrukturtyper og mikrobielle arter. Vi introducerer to metoder til at bestemme nanopartiklers antimikrobielle aktiviteter og to metoder til nanostrukturerede overfladers antimikrobielle aktiviteter. 
For nanopartikler kan metoden med direkte samdyrkning anvendes til at bestemme de mindste hæmmende og minimale bakteriedræbende koncentrationer af nanopartikler, og dyrkningsmetoden med direkte eksponering kan anvendes til at vurdere bakteriostatisk versus bakteriedræbende aktivitet i realtid som følge af nanopartikeleksponering. For nanostrukturerede overflader anvendes metoden med direkte dyrkning til at bestemme levedygtigheden af bakterier, der indirekte og direkte er i kontakt med nanostrukturerede overflader, og metoden med fokuseret kontakteksponering anvendes til at undersøge antimikrobiel aktivitet på et bestemt område af en nanostruktureret overflade. Vi diskuterer vigtige eksperimentelle variabler, der skal overvejes til in vitro-undersøgelsesdesign , når de antimikrobielle egenskaber af nanopartikler og nanostrukturerede overflader bestemmes. Alle disse metoder er relativt billige, anvender teknikker, der er relativt lette at mestre og gentages for konsistens, og er anvendelige på en bred vifte af nanostrukturtyper og mikrobielle arter.

Identifikationen af den antibakterielle aktivitet af magnesiumoxidnanopartikler og nanostrukturerede overflader er blevet præsenteret ved hjælp af fire in vitro-metoder , der kan anvendes på tværs af forskellige materialetyper og mikrobielle arter.
Metode A og metode B undersøger bakterieaktiviteter, når de udsættes for nanopartikler i en lagfase (metode A) og logaritmisk fase (metode B) i en varighed på 24 timer eller længere. Metode A giver resultater vedrørende MIC og MBC...

Watch this Scientific Journal Video about Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro at JoVE.com

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

Vi introducerer fire metoder til at evaluere nanopartiklers og nanostrukturerede overfladers antimikrobielle aktiviteter ved hjælp af in vitro-teknikker . Disse metoder kan tilpasses til at studere interaktionerne mellem forskellige nanopartikler og nanostrukturerede overflader med en bred vifte af mikrobielle arter.

Evaluering af antimikrobielle aktiviteter af nanopartikler og nanostrukturerede overflader in vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

Protokollerne giver en metode til at undersøge nanopartiklers og nanostrukturerede overfladers antimikrobielle aktiviteter fra indledende undersøgelser til mere komplekse overfladeundersøgelser. I modsætning til tidligere beskrevne metoder, der resulterede i uforlignelige data på tværs af undersøgelser, producerer disse metoder konsistente og sammenlignelige resultater på tværs af forskellige nanopartikelmaterialer, nanostrukturerede materialer og mikrobielle arter. Det kan være vanskeligt at opretholde en homogen fordeling af nanopartikler i metode A og B.Vortexing prøver grundigt vil sikre nanopartikel suspension, og pipettering tre til fire gange mellem alikvoter vil opretholde suspensioner Begynd at indsamle dyrkede bakterier ved aliquoting en milliliter af den natten over dyrkede bakteriekultur i 1,5 milliliter mikrocentrifugerør. Efter at have skabt et tilstrækkeligt antal alikvoter, centrifugeres de for at nå den ønskede siddetæthed. Supernatanten samles i en opsamlingsbeholder.Og genophæng cellepelleten i 0,5 ml frisk vækstmedium. Kombiner suspensionen fra to rør, og gentag centrifugaliseringen. Efter centrifugaliseringen gives endnu en vask med frisk vækstmedium, og indholdet fra to glas gentages til et, før glassene centrifugeres.Giv den tredje vask med 0,33 ml trisbuffer eller hydroxyethylaminomethanbuffer. Kombiner de tre suspensioner, hver på 0,33 ml volumen, i en 1,5 ml mikrocentrifuge. Efter centrifugalisering fjernes supernatanten fra de pelleterede celler, og cellepelleten resuspenderes i en milliliter frisk trisbuffer, der vil blive kendt som cellesuspensionen eller en lagfase bakteriel såkultur.For at danne bakterie- og nanopartikelkulturerne alikvotes de to milliliter bakteriesåningskultur i hver brønd i en 12 brønd ikke-vævsbehandlet polystyrenplade. For hvert fem milliliter mikrocentrifugerør indeholdende forvejede magnesiumoxidpartikler tilsættes tre ml bakteriesåningskultur. Og hvirvel røret kort for at blande magnesiumoxid nanopartikler med bakterierne.Efter blanding alikvote en milliliter magnesiumoxidnanopartikler suspension til tre separate brønde for at skabe de tredobbelte prøver af hver forudmålt vægt af magnesiumoxid nanopartikler. Prøven inkuberes natten over ved 37 grader Celsius og 120 o / min. Og overfør en prøve på tre milliliter til et individuelt 15 ml konisk rør ved hjælp af en pipette.Udfør derefter en til 10 seriel fortynding i en 96 brøndplade ved at tilføje 180 mikroliter trisbuffer til hvert hul i række B til række G for det passende antal kolonner. Kort hvirvel et 15 ml konisk rør indeholdende bakterieprøver, og tilsæt 50 mikroliter bakterieprøve til en individuel brønd i række A.Derefter overføres 20 mikroliter bakterieprøve fra række A til den tilsvarende brønd i række B med kort blanding. Ved hjælp af en steril pipettespids overføres 20 mikroliter fra brønden i række B til det tilsvarende hul i række C.Fortsæt dette indtil række G for at afslutte den serielle fortynding fra 10 til den negative i række B til 10 til de negative seks i række G.Når det er gjort, pipetteres 100 mikroliter af hvert hul i en passende vækstagarplade. og spredes over pladen for at sprede cellekulturen.Placer brøndpladerne i en inkubatorryster natten over ved 37 grader Celsius, og inkuber agarpladerne ved 37 grader Celsius i 24 timer. Undersøg pladen, og tæl pladerne med ca. 25 til 300 kolonier. Vælg om muligt plader med samme fortyndingsværdi.For hvert materiale, der skal testes, fortyndes bakterieprøverne natten over ved at tilsætte tre 200 mikroliter alikvoter til vækstmediet i tre huller og tre 200 mikroliter alikvoter af bakteriekulturen i yderligere huller. Placer 96-brøndpladen i pladelæseren, og scan den. Fortsæt om nødvendigt med at tilføje bouillon eller bakteriekultur natten over, indtil den optiske densitet ved 600 nanometer eller OD 600 er nået ca. 0,01.For at forberede bakterielle nanopartikelsuspensioner og sterile medier nanopartikelsuspensioner fjernes en milliliter bakteriekultur eller medium fra et tredobbelt sæt af 15 ml koniske rør. Og tilsæt det i et fem milliliter centrifugerør indeholdende forudmålte nanopartikler. Vortex rørene for at blande opløsningen.Nanopartiklerne fordeles homogent ved at overføre en milliliter alikvoter fra det fem milliliter centrifugerør til det 15 ml koniske rør. Når det er gjort, alikvote 200 mikroliter af hver prøve i de enkelte brønde i en 96 brøndplade. Efter bestemmelse af den tidligere beskrevne siddetæthed tilsættes prøverne og kontrollen i de enkelte brønde i en 48 brønds ikke-vævsbehandlet polystyrenplade.Derefter alikvote 0,75 ml af cellesuspensionen i hvert hul, der indeholder prøverne og kontrollerne. Placer 48 brøndpladen i en inkubatorryster i 24 timer ved 37 grader Celsius med omrystning ved 120 omdr./min. Efter inkubation indsamles to prøver fra hver gruppe og overføres individuelt til mærkede fem milliliter mikrocentrifugerør.Tilsæt to milliliter revideret simuleret kropsvæske eller RSBF til hvert rør. Forbered steriliseret nitrous cellulosepapir ved at trimme dem til en centimeter diameter. Det skårne nitrocellulosepapir anbringes på en agarplade, der indeholder det passende medium.Derefter tilsættes 50 mikroliter af den fortyndede bakteriekultur på filterpapiret, inden der tilsættes 50 mikroliter af et passende medium til midten af hver prøveoverflade. Brug steriliseret pincet til at hente salpetercellosepapiret fra sneglens overflade. Vend forsigtigt salpetercellulosepapiret og læg det på prøveoverfladen, så bakterierne er i kontakt med 50 mikroliter medium og den nanostrukturerede overflade af interesse.Hvis prøven er nedbrydelig i kultur, skal du sætte en holder under den for at løfte den for at undgå kontakt med trisbufferen. Bevar fugtigheden ved at tilsætte en milliliter trisbuffer til en prøve, der indeholder godt. Efter inkubation natten over opsamles nitrocellulosepapiret fra hver prøveoverflade og anbringes i fem ml trisbuffer.Vortex de indsamlede filterpapirer og nanooverfladematerialeprøver i fem sekunder. Tris-stødpudesuspensionerne opsamles fra prøven og anbringes i individuelle friske opsamlingsrør. De antimikrobielle virkninger ved anvendelse af metode A identificerede minimums- og hæmmende koncentration eller MIC på et milligram pr. milliliter magnesiumoxidnanopartikler og minimale bakteriocide koncentrationer eller MBC 99,9 på henholdsvis 1,0 og 1,6 milligram pr. milliliter magnesiumoxidnanopartikler for gramnegative Escherichia coli og pseudomonas aeruginosa.Den gram-positive stafylokokker epidermis, S.Aureus, og methicillinresistente staphylococcus aureus, eller MRSA, demonstrerede MIC's værdier på henholdsvis 0,5, 0,7 og en milligram pr. milliliter magnesiumoxid nanopartikler. NBC 99,99 værdier på 1,6 og 1,2 milligram per milliliter blev identificeret for henholdsvis S.epidermis og S.aureus. Mens MRSA ikke blev reduceret ud over NBC 90.MIC'er på 1,2 og et milligram pr. milliliter blev identificeret i lægemiddelfølsomme og lægemiddelresistente candida-arter, henholdsvis C-albicaner og C-albicaner, fluconazolresistente eller FR. I modsætning hertil viste magnesiumoxidnanopartikler MIC-værdier på en og 0,7 milligram pr. milliliter eller henholdsvis C. glabrata og C glabrata echinocandin resistent eller ER. Hver kandidatart nåede en NBC 90 på 0,7 til 1,2 milligram pr. milliliter, men kun C.glabrata ER blev reduceret til NBC 99,9 ved 1,2 milligram pr. milliliter.I metode B voksede MRSA eksponeret for nanopartikler eksponentielt til en OD på 0,85. Eksponering for 1,2 milligram pr. milliliter magnesiumoxidnanopartikler og 6,25 milligram pr. milliliter trimethoprim resulterede i henholdsvis 80,2% og 81,6% reduceret bakterievækst. Tilsvarende reducerede 2.9 milligram pr. milliliter magnesiumhydroxidnanopartikler bakterievækst til 70.3%Eksponering for en mikroliter pr. milliliter vancomycin resulterede i en 99.99% reduktion i væksten af MRSA, hvilket tyder på den bakteriostatiske aktivitet af magnesiumoxid og magnesiumhydroxidnanopartikler.Metode C viste ingen hæmning af bakterievækst ved indirekte kontakt. Resultaterne indikerede, at ZC21 havde den stærkeste antibakterielle aktivitet mod vedhæftning og vækst af MRSA for alle de testede prøver. I metode D blev der ikke identificeret nogen levedygtig staph aureus i 1.9A-, 1.9 AA- og EPD-prøverne eller deres parrede filterpapir.Eksponering for EPD-prøverne efter en knælende reducerede imidlertid bakterievæksten til nogle få celler på den nanostrukturerede overflade og det parerede filterpapir. Identifikation af antimikrobielle aktiviteter på nanopartikler og nanostrukturerede overflader har ført til yderligere undersøgelser inden for bioteknologi, herunder dem, der er relateret til kroniske sår og implantatrelaterede infektioner.

evaluation-antimicrobial-activities-nanoparticles-nanostructured

Research

JoVE Journal

Bioengineering

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Virale Nanopartikler for<em&gt; In vivo</em&gt; Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Evaluering af Polymeric Gene Delivery nanopartikler ved Nanopartikel Tracking Analyse og Højkapacitetsforskning Flowcytometri

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

Fremstilling af en funktionaliseret Magnetic Bakteriel Nanocellulose med Iron Oxide Nanopartikler

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Øjenirritation Test (EIT) for Hazard Identifikation af Eye Irriterer Kemikalier hjælp rekonstrueret human Cornea-lignende Epithelial (RHCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Udarbejdelse af termoresponsiv nanostrukturerede overflader for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Fremstilling af metal nanopartikler ved pulserende laserablation i væsker: Et værktøj til at studere de antibakterielle egenskaber ved nanopartikler

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

en<em&gt; In vitro</em&gt; Orgel Kultur Model af muse diskusprolaps

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

Syntese af Multi-bjergomgivne kulstofnanorør modificeret med sølv nanopartikler og evaluering af deres antibakterielle aktiviteter og cytotoksiske egenskaber

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

Antimikrobiel karakterisering af avancerede materialer til bioteknologi applikationer

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...