Name: manufacture and drug delivery applications of silk nanoparticles
Uploaded: 2016-10-08T15:00:00
Description: Watch this Scientific Journal Video about Manufacture and Drug Delivery Applications of Silk Nanoparticles at JoVE.com

This research was supported by a Marie Curie FP7 Career Integration Grant 334134 within the seventh European Union Framework Program.

1. Udarbejdelse af en Reverse-manipuleret Silk Solution fra Bombyx mori Kokoner BEMÆRK: Denne metode er baseret på protokoller beskrevet andetsteds 12,27. <ol><li> Skær 5 g tørrede kokoner med en saks i 5 mm x 5 mm stykker. Fjern eventuelle snavsede lag. </li><li> Afvej 4,24 g natriumcarbonat og tilføje denne omhyggeligt til 2 l kogende destilleret vand. BEMÆRK: Dette giver en 0,02 M natriumcarbonatopløsning. </li><li> Tilsæt cut kokon brikkerne til det kogende natriumcarbona...

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Forskellige fremgangsmåder er tilgængelige til at producere silke nanopartikler, herunder polyvinylalkohol blande 20, spraytørring 21, udsaltning 22, kapillær microdot udskrivning 23, superkritisk CO2 udfældning 24 og nanoprecipitation 16,25 (gennemgået i henvisning 26). Imidlertid nanoprecipitation, på grund af sin samlede enkelhed, er den mest populære metode til generering silke nanopartikler. Derfor er formålet med denne...

Nanostørrelse lægemiddeladministrationssystemer anvendes ofte til at styre lægemiddelfrigivelse og levere et bredt sæt af nyttevirkning - for eksempel proteiner, peptider og lille molekylvægt lægemidler - at målrette celler og væv. Disse terapeutiske nyttelast er ofte medtaget i forskellige makromolekylære lægemiddelbærere såsom liposomer, vandopløselige polymerer (herunder dendrimerer) og mikro- og nanopartikler 1. Nanopartikler (typisk i en størrelsesorden på 1 nm til 1000 nm) bliver meget undersøgt som potentielle lægemiddelbærere, især for anticancer drug delivery 2. Den vellykkede indførelse af Abraxane (120 nm størrelse albumin-nanopartikler fyldt med paclitaxel) i almindelig klinisk praksis 3 har katalyseret marken, så mange flere nanopartikler til lægemiddeladministration går nu ind kliniske forsøg 4. Solide tumorer viser generelt dårlig lymfedrænage og har utætte blodkar, som betyder, at nanoparticles på op til 200 nm, vil blive passivt rettet mod disse tumorer efter intravenøs administration. Denne passive targeting fænomen kaldes den forbedrede permeabilitet og retention (EPR) virkning og blev først rapporteret i 1986 5. EPR effekt kan føre til en 50- til 100-fold stigning i medikamentkoncentrationer i tumormikromiljøet for en given dosis lægemiddel, når lægemidlet nyttelast afgives ved anvendelse af en makromolekylær lægemiddel carrier tilgang frem for den frie lægemiddel uden bæreren. Drug nanopartikler designet til anticancer drug delivery skal nå tumormikromiljøet og ofte skal indtaste en bestemt intracellulært, sædvanligvis ved endocytisk optagelse, for lægemidlet for at opnå den ønskede terapeutiske virkning 3. Nanopartikler er konstrueret til intracellulær lægemiddelafgivelse udnytte endocytose som en gateway i cellen samt en rute til at overvinde drug resistensmekanismer. Lægemiddelfrigivelse fra nanopartikler ofte specielt designet til at occur i lysosomer (dvs. lysosomotropiske drug delivery) 6, hvor pH reaktionsevne nanopartikel luftfartsselskab (lysosomal pH ca. 4,5) kan tjene som udløser for narkotika release eller lysosomale enzymer, som frigør nyttelasten fra bæreren 7. Mange forskellige klasser af materialer kan anvendes til at generere nanopartikler (f.eks, metaller og mange organiske og uorganiske materialer). Imidlertid er biopolymerer fremstår som attraktive materialer på grund af deres kendte biokompatibilitet, bionedbrydelighed og lav toksicitet 8. Mange biopolymerer bliver undersøgt, herunder albumin, alginat, chitosan og silke. Heraf har silke dukket op som en lovende udfordrer til udvikling i drug delivery-systemer 9. Silke af forskellige typer produceres af en række leddyr, herunder edderkopper (f.eks Nephila clavipes) og silkeorme (f.eks Bombyx mori). Avlen silke bruges langt mere EXTENkende end edderkop silke fordi avlen er helt tamme og silke repræsenterer således en reproducerbar udgangsmateriale. Avlen silke er en Food and Drug Administration (FDA) godkendt materiale til humant brug, især som suturmateriale; det har en robust sikkerhed rekord i mennesker, og er kendt for at nedbryde in vivo 10. Nedbrydningen profil silke kan finjusteres til at variere fra time (lav krystallinsk silke) til 12 måneder eller mere (høj krystallinsk silke). Silke nedbrydningsprodukter er ugiftige og metaboliseres i legemet 10. Den silke struktur bibringer evnen til at binde små molekylvægt forbindelser og makromolekylære protein narkotika 11, hvilket gør det et godt materiale til kontrolleret frigivelse af lægemiddel. Proteinlægemidler (f.eks antistoffer) er modtagelige for denaturering, aggregering, proteolytisk spaltning og clearance af immunsystemet. Imidlertid silke stabiliserer terapeutiske proteiner grundet bufferkapacitet dens nanokrystallinske reregioner og dens evne til at skræddersy vandindhold på nanoskala 11. Disse unikke funktioner giver fysisk beskyttelse og reducerer nyttelasten mobilitet 11 og er typisk ikke set med andre (bio) polymerer. Mange anticancer lægemiddelafgivelsessystemer, f.eks silke-baserede hydrogeler 12, film 13-15 og nanopartikler 16,17, er nu blevet udviklet til at udnytte disse funktioner (revideret i referencer 18,19) Her blev silke nanopartikler karakteriseret ved at bestemme deres størrelse og ladning over et forlænget tidsrum. Doxorubicin, en klinisk relevant anticancer narkotika, blev brugt som model lægemiddel til lægemiddel lastning og cytotoksicitet studier i tredobbelt negative humane brystkræftceller behandlet med narkotika-loaded silke nanopartikler.

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			<td height="40" style="height:40px;width:216px;">Acetone</td>
			<td style="width:235px;">VWR International, Radnor, PA, USA</td>
			<td style="width:119px;">20066.33</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Automated Critical Point Dryer</td>
			<td style="width:235px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:119px;">EM CPD300</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Balancing&#160;</td>
			<td style="width:235px;">Mettler Toledo, Greifensee, Switzerland</td>
			<td style="width:119px;">NewClassic MS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Black polystyrene microplate , 96 well</td>
			<td style="width:235px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:119px;">3991</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Capillary cell (DTS 1070)</td>
			<td style="width:235px;">Malvern Instrument, Worcestershire, UK</td>
			<td style="width:119px;">DTS107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Carbon adhesive disc</td>
			<td style="width:235px;">Agar Scientific, Essex, UK</td>
			<td style="width:119px;">G3347N</td>
			<td></td>
		</tr>
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			<td height="40" style="height:40px;width:216px;">Centrifuge&#160;</td>
			<td style="width:235px;">Hermle Labortechnik, Wehingen, Germany</td>
			<td style="width:119px;">Z323K</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Centrifuge&#160;</td>
			<td style="width:235px;">Beckman Coulter, Brea, CA, USA</td>
			<td style="width:119px;">Avanti J-E, Rotor: J20</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">Centrifuge&#160;</td>
			<td style="width:235px;">Beckman Coulter, Brea, CA, USA</td>
			<td style="width:119px;">Optima L-70K, Rotor: 50.2 Ti, Adaptor 303392</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Coater, low vacuum</td>
			<td style="width:235px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:119px;">EM ACE200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cuvettes, polystyrene, disposable</td>
			<td style="width:235px;">Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">FB55147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Doxorubixin&#160;</td>
			<td style="width:235px;">LC Laboratories, Boston, MA, USA</td>
			<td style="width:119px;">D4000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electronic pipetting, Easypet&#160;</td>
			<td style="width:235px;">Eppendorf, Hamburg, Germany</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">FE-SEM</td>
			<td style="width:235px;">Hitachi High-Technologies, Krefeld, Germany</td>
			<td style="width:119px;">SU6600</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Freeze dryer</td>
			<td style="width:235px;">Martin Christ, Osterode, Germany</td>
			<td style="width:119px;">Epsilon 2-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Heat inactivated Bombyx mori silk cocoons</td>
			<td style="width:235px;">Tajima Shoji, Kanagawa, Japan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Hotplate with Stirrer</td>
			<td style="width:235px;">Bibby Scientific, Stanffordshire, UK</td>
			<td style="width:119px;">US 152</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Incubator</td>
			<td style="width:235px;">Memmert, Schwabach, Germany</td>
			<td style="width:119px;">INB 200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Insulin, human recombinant, zinc solution</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">12585-014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Lithium bromide</td>
			<td style="width:235px;">Acros Organics, Geel, Belgium</td>
			<td style="width:119px;">AC199870025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MDA-MB-231</td>
			<td style="width:235px;">ATCC, Manassas, VA, U.S.A</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Micropipette and tips</td>
			<td style="width:235px;">Eppendorf, Hamburg, Germany</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Microplate Reader</td>
			<td style="width:235px;">Molecular devices, Sunnyvale, CA, USA</td>
			<td style="width:119px;">SpectraMax M5</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Oak Ridge High-Speed Centrifuge Tubes, 50 ml</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Open-Top Thickwall Polycarbonate tube, 4 ml</td>
			<td style="width:235px;">Beckman Coulter, Brea, CA, USA</td>
			<td style="width:119px;">355645</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Penicilin/streptomycin&#160;</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">RPMI medium</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Serological pipettes, 5 ml</td>
			<td style="width:235px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Silicon wafers</td>
			<td style="width:235px;">Agar Scientific, Essex, UK</td>
			<td style="width:119px;">G3391</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Slide-A-Lyzer Dialysis cassettes, 3.5K MWCO, 15 ml</td>
			<td style="width:235px;">Thermo Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">87724</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium carbonate anhydrous</td>
			<td style="width:235px;">Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">S/2840/62</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Specimen stubs for SEM</td>
			<td style="width:235px;">Agar Scientific, Essex, UK</td>
			<td style="width:119px;">G301</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Ultrasonic homogenizer</td>
			<td style="width:235px;">Bandelin, Berlin, Germany</td>
			<td style="width:119px;">Sonoplus HD 2070</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">UV transparent microplate, 96 well</td>
			<td style="width:235px;">Sigma-Aldrich, St. Louis, MO, USA</td>
			<td style="width:119px;">3635</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Vortex</td>
			<td style="width:235px;">IKA, Staufen, Germany</td>
			<td style="width:119px;">Genius 3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Zetasizer</td>
			<td style="width:235px;">Malvern Instrument, Worcestershire, UK</td>
			<td style="width:119px;">Nano ZS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">Zetasizer Software version 7.11</td>
			<td style="width:119px;"></td>
			<td>DLS software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Micro Modulyo</td>
			<td style="width:235px;">&#160;Thermo Fisher</td>
			<td style="width:119px;">230</td>
			<td>Freeze drying system&#160;</td>
		</tr>
	</tbody>
</table>

manufacture and drug delivery applications of silk nanoparticles

Silke er en lovende biopolymer til biomedicinske og farmaceutiske anvendelser på grund af dets enestående mekaniske egenskaber, biokompatibilitet og bionedbrydelighed, samt dets evne til at beskytte og efterfølgende frigive sit nyttelast som svar på en udløser. Mens silke kan formuleres i forskellige materialer formater, er silke nanopartikler fremstår som lovende lægemiddeltilførselssystemer. Derfor er denne artikel dækker procedurerne for reverse engineering silke kokoner at give en regenereret silke løsning, der kan bruges til at generere en stabil silke nanopartikler. Disse nanopartikler efterfølgende karakteriseret, lægemiddel fyldte og udforsket som en potentiel anticancer lægemiddelafgivelsessystem. Kort fortalt silkekokoner revers manipuleret først ved degumming kokoner fulgt af opløsning silke og rydde op, hvilket gav en vandig silke opløsning. Dernæst den regenererede silke opløsning underkastes nanoprecipitation til opnåelse silke nanopartikler - en simpel men kraftfuld metodeder genererer ensartede nanopartikler. Silke nanopartikler karakteriseres efter deres størrelse, zetapotentialet, morfologi og stabilitet i vandige medier, samt deres evne til at indeslutte et kemoterapeutisk nyttelast og dræbe humane brystcancerceller. Samlet set den beskrevne metode giver ensartede silke nanopartikler, der let kan udforskes til et utal af applikationer, herunder deres anvendelse som en potentiel nanomedicin.

Data blev statistisk analyseret som beskrevet tidligere 17. Den t-test blev anvendt til prøvepar og envejs variansanalyse (ANOVA) efterfulgt af Bonferronis multipel sammenligning post hoc test for multiple prøver. En stjerne angiver statistisk signifikans således: * P &lt;0,05 og ** P &lt;0,001. Alle data præsenteres som middelværdier ± standardafvigelse (SD) og tallene i parentes angiver antallet af uafhængige forsøg. <p class="jove_content" fo:keep-together.withi...

Watch this Scientific Journal Video about Manufacture and Drug Delivery Applications of Silk Nanoparticles at JoVE.com

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Nanopartikler dukker op som lovende drug delivery systemer til en bred vifte af indikationer. Her beskriver vi en simpel endnu kraftfulde metode til at fremstille silke nanopartikler anvendelse af omvendt manipuleret Bombyx mori silke. Disse silke nanopartikler kan let fyldes med en terapeutisk nyttelast og efterfølgende undersøgt for lægemiddeltilførselsanvendelser.

Fremstilling and Drug Delivery Anvendelser af Silk Nanopartikler

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Research

JoVE Journal

Bioengineering

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

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Selvrapportering Stilladser for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Modeller og metoder til evaluering Transport af Drug Delivery Systems over cellulære Barrierer

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Designing Silke-silkeprotein legering materialer til biomedicinske anvendelser

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Drug-induceret sensibilisering af adenylylcyclase: Assay Strømlining og Minituriasering for Small Molecule og siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Biofunctionalized Prussian Blue Nanopartikler til multimodal Molekylære billedbehandling

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Udviklet 3D Silke-collagen-baserede model for polariseret nervevæv

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Solid lipidnanopartikler (SLNs) til intracellulær målretning Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Mesenchymale stromacelle Kultur og Levering i Autologe Betingelser: En smart tilgang til Ortopædisk Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Syntese af Functionalized 10-nm Polymer-belagt guld partikler til endotelet målretning og Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...