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Bio-ingénierie

Et tripeptid-stabiliseret Nanoemulsion af oliesyre

Published: February 27, 2019
doi:
10.3791/59034
, , , ,
1Department of Chemistry,Brooklyn College, The City University of New York, 2Ph.D. Program in Chemistry,The Graduate Center of The City University of New York, 3Ph.D. Program in Biochemistry,The Graduate Center of The City University of New York, 4Department of Genetics and Genomic Sciences,Icahn School of Medicine at Mount Sinai, 5Women’s Health Research Institute,Icahn School of Medicine at Mount Sinai, 6Laboratory for Translational Research,Western Connecticut Health Network

Summary

Denne protokol beskriver en effektiv metode til at syntetisere en nanoemulsion af en oliesyre acids-platinum(II) konjugat stabiliseret med en lysin-tyrosin-fenylalanin (KYF) tripeptid. Nanoemulsion formularer på mild syntetiske betingelser via samlesæt af KYF og konjugation.

Abstract

Vi beskriver en metode til at producere en nanoemulsion bestående af en oliesyre acids-Pt(II) kerne og en lysin-tyrosin-fenylalanin (KYF) belægning (KYF-Pt-NE). KYF-Pt-NE indkapsler Pt(II) på 10 wt. %, har en diameter på 107 ± 27 nm og en negativ overflade ladning. KYF-Pt-NE er stabilt i vand og i serum, og er biologisk aktive. Konjugation af en fluorophore til KYF tillader syntesen af en fluorescerende nanoemulsion, der egner sig til biologiske billeddannelse. Syntesen af nanoemulsion er udført i et vandigt miljø og KYF-Pt-NE former via samlesæt for en kort KYF peptid og en oliesyre acids-platinum(II) konjugat. Samlesæt proces afhænger af temperaturen af løsningen, det molære forholdet mellem substraterne, og strømningshastigheden af substrat tilsætning. Afgørende skridt omfatter opretholde den optimale omrøring sats under syntesen, tillader tilstrækkelig tid til samlesæt og pre at koncentrere nanoemulsion gradvist i en centrifugal koncentrator.

Introduction

I de seneste år har der været en voksende interesse for Ingeniørvidenskab af nanopartikler til sådanne biomedicinske anvendelser som medicinafgivelse og bioimaging1,2,3,4. Landbrugets multifunktionalitet nanopartikel-baserede systemer kræver ofte inkorporerer flere komponenter inden for én formulering. De byggesten, der er baseret på lipider eller polymerer ofte adskiller sig med hensyn til deres fysisk-kemiske egenskaber samt deres biokompatibilitet og biologiske nedbrydelighed, som i sidste ende kan påvirke funktionen af nanostrukturer1, 5,6. Biologisk afledte materialer, såsom proteiner og peptider, har længe været anerkendt som lovende komponenter af multifunktionelle nanostrukturer på grund af deres sekvens fleksibilitet7,8. Peptider selv samle til stærkt bestilte Supramolekylær arkitekturer danner spiralformet bånd9,10, fibrøst stilladser11,12, og mange flere, dermed bane vejen til bygning biomolekyle-baserede hybrid nanostrukturer ved hjælp af en bottom-up tilgang13.

Peptider er udtømt for applikationer i medicin og bioteknologi, især til anticancer behandling14 og hjerte-kar-sygdomme15 såvel som for antibiotika udvikling16,17, metaboliske lidelser18, og infektioner19. Der er over hundrede af små-peptid therapeutics gennemgår kliniske forsøg20. Peptider er lette at ændre og hurtig til at syntetisere til en lav pris. Derudover er de biologisk nedbrydelige, som i høj grad letter deres biologiske og farmaceutiske anvendelser21,22. Brugen af peptider som strukturelle komponenter omfatter engineering af modtagelig, peptid-baserede nanopartikler og hydrogel depoter for kontrolleret frigivelse23,24,25,26 , 27, peptid-baserede biosensorer28,29,30,31, eller bio-elektroniske enheder32,33,34. Vigtigere, selv korte peptider med to eller tre amino-syre rester, der omfatter phenylalanin blev fundet til at guide den samlesæt behandler35,36,37 og oprette stabiliseret emulsioner38 .

Platinum-baserede lægemidler på grund af deres høje effekt, der anvendes i mange kræft behandlingsregimer, både alene og i kombination med andre agenter39,40. Platin forbindelser inducerer DNA-skader ved at danne monoadducts og intrastrand eller interstrand cross-links. Pt-DNA læsioner er anerkendt af den cellulære maskiner, og hvis ikke repareret, føre til cellulært apoptose. Den vigtigste mekanisme, hvorved Pt(II) bidrager til kræft celledød, er hæmning af DNA transkription41,42. Fordele ved platinum terapi er dog faldet med systemisk toksicitet af Pt(II), der udløser alvorlige bivirkninger. Dette fører til lavere kliniske dosering af Pt(II)43, hvilket ofte resulterer i sub terapeutiske koncentrationer af platin nå DNA’ET. Som følge heraf bidrager DNA reparation, der følger til kræft celle overlevelse og erhverve Pt(II) modstand. Platin kemo-modstand er et stort problem i anticancer terapi og den vigtigste årsag til behandling fiasko44,45.

Vi har udviklet en stabil nanosystem, der indkapsler Pt(II) agent for at give en afskærmning virkning i systemisk cirkulation og mindske bivirkninger Pt II-induceret. Systemet er baseret på en oliesyre acids-Pt(II) kerne stabiliseret med en KYF tripeptid til at danne en nanoemulsion (KYF-Pt-NE)46. Byggesten af KYF-Pt-NE, aminosyrer tripeptid samt oliesyre, har generelt anerkendt som sikre (GRAS) status med Food and Drug Administration (FDA). KYF-Pt-NE er udarbejdet ved hjælp af en nanoprecipitation metode47. Kort sagt, er oliesyre acids-Pt(II) konjugat opløst i et organisk opløsningsmiddel og derefter tilsættes dråbevis til en vandig KYF løsning (figur 1) ved 37 ° C. Løsningen rørte i flere timer at tillade samlesæt af KYF-Pt-NE. Nanoemulsion er koncentreret i 10 kDa centrifugal koncentratorer og vaskes tre gange med vand. Den kemiske ændring af KYF med en fluorophore tillader syntesen af fluorescerende FITC-KYF-Pt-NE egnet til biomedicinsk billeddannelse.

Protocol

1. Sammenfatning af oliesyre Acids–Platinum(II) konjugat Aktivering af cisplatin Suspendere 50 mg (0.167 mmol) af cisplatin i 4 mL vand (fx nanopure) ved 60 ° C. Tilsættes dråbevis 55,2 mg (0,325 mmol) af AgNO3 i 0,5 mL vand til løsningen af cisplatin og omrøres reaktion i mindst 2 timer ved 60 ° C. Den hvide bundfald af AgCl vil danne, der angiver status for reaktionen. For at bestemme, hvis aktivisering reaktionen er afsluttet, udføre test med 10% HCl for tils…

Representative Results

Repræsentant TEM billede af KYF-Pt-NE udarbejdet ved hjælp af denne protokol er vist i figur 2A. KYF-Pt-NEs er sfærisk i morfologi, godt spredt, og ensartede i størrelse. Kerne diameter af KYF-Pt-NEs, målt direkte fra tre TEM billeder med et minimum af 200 målinger udført, er 107 ± 27 nm. Den hydrodynamiske diameter af KYF-Pt-NE, analyseret ved hjælp af dynamiske lys spektroskopi (DLS), fandtes for at være 240 nm med en polydispersity indeks af 0.15…

Discussion

Kritiske trin i nanoemulsion syntese omfatter justering kindtand forholdet mellem substraterne, vedligeholdelse temperatur og flow sats kontrol under oliesyre acids–Pt(II) tilsætning, giver tilstrækkelig tid til samlesæt og rensning af produktet ved hjælp af en centrifugal koncentrator kolonne. Disse parametre påvirke størrelse og morfologi af KYF-Pt-NE; således er det især vigtigt at fastholde de rette molære forhold og justere de syntetiske betingelser korrekt.

Forholdet mellem su…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Vi taknemmeligt anerkender støtte fra National Cancer Institute, give SC2CA206194. Ingen konkurrerende finansielle interesser er erklæret.

Materials

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU)		 ANASPEC INC.: AS-20376 SPPS
4-well chamber confocal dish Lab-Tek II, Thermo Fisher Scientific 154526 For imaging
6-bromohexanoic acid Chem-Impex INT’L INC. 24477 Click modification for peptide
A2780 Generously doanted by professor John Martignetti from The Mount Sinai Hospital Ovarian cancer cell line
Barnstead Nanopure Thermo Fisher D11901 water filtration system
BUCHI rotavapor R-3 Buchi Z568090 For solvent removal and sample drying
Centrifuge 5810 R eppendorf 5811F For platinum complex separation
Cis-dichlorodiamineplatinum (II) 99% Acros Organics 19376-0050 in vitro tests
CP70 Generously doanted by professor John Martignetti from The Mount Sinai Hospital Ovarian cancer cell line
Digital water bath VWR 97025-134 For warming up media for cell culture
Dynamic Light Scattering (DLS) Brookhaven Instrument Corporation For nanoparticle size measurments
ES-2 ATCC CRL-1978 ovarian cancer cell line
Fmoc-L-Lys(Boc)-OH 99.79% Chem-Impex INT’L INC. 00493 SPPS
Fmoc-L-Phe 4-alkoxybenzyl alcohol resin (0.382 meq/g), Chem-Impex INT’L INC. 01914 SPPS
Fmoc-LTyr(tBu)-OH 98% Alfa Aesar H59730 SPPS
HERACELL 150i CO2 incubator Thermo Scientific Fisher incubator
High pressure syringe pump New Era 1010-US For platinum complex addition in nanoparticle synthesis
Hotplate/stirrer VWR 12365-382 For sample stirring and heating
LAMP-1 Antibody(cojugated with Alexa Fluor 647) Santa Cruz Biotechnology sc-18821 AF647 For imaging
N,N-diisopropylethylamine (DIPEA) Oakwood Chemical 005027 SPPS
Ninhydrin 99% Alfa Aesar A10409 Kaiser test
Oleic acid Chem-Impex INT’L INC. 01421 For platinum complex synthesis
OV90 ATCC CRL-11732 Ovarian cancer cell line
PBS Corning 21-031-CV For cell wash
Permount mounting medium Fisher Chemical SP15-100 For imaging
Phenol Fisher Chemical A92500 Kaiser test
Phosphotungstic acid Fisher Chemical A248-25 negative stain for TEM
Piperidine 99% BTC 219260-2.5L SPPS
Platinum AAS standard soultion Alfa Aesar 88086 1000ug/ml for calibration curve
Propargyl bromide 97% Alfa Aesar L10595 For alkyne modification of fluoresceine
Scientific biological cabinet Thermo Scientific Fisher 1385 Bio-hood for cell culture
Self-Cleaning Vacuum System Welch 2028 Vacuum pump for rotavapor
Silver nitrate Acros Organics 19768-0250 Cisplatin activation
SKOV3 ATCC HTB-77 Ovarian cancer cell line
Sodium hydroxide Fisher Scientific S313-1 For platinum complex synthesis
Tin (II) chloride Sigma Aldrich 208256 Test for Platinum presence
TOV21G ATCC CRL-11730 Ovarian cancer cell line
Trifluoroacetic acid 99% (TFA) Alfa Aesar L06374 SPPS
Triisopropylsilane (TIPS) Chem-Impex INT’L INC. 01966 SPPS
Triton-X Sigma Aldrich T8787-100ML For imaging
Uranine powder 40% Fisher Scientific S25328A For alkyne modification of fluoresceine
Vivaspin 20 (10000 MWCO) Sartorious VS2001 For Nanoparticle wash and condensation
VWR Inverted Microscope VWR 89404-462 For cell culture monitoring
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Tags

TripeptideNanoemulsionOleic AcidCisplatinPlatinumOvarian CancerPeptide ModificationDiagnosticPharmaceuticalIn-vivo StudiesFmocL-phenylalanineL-tyrosineTBTUDIPEA

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Dragulska, S. A., Wlodarczyk, M. T., Poursharifi, M., Martignetti, J. A., Mieszawska, A. J. A Tripeptide-Stabilized Nanoemulsion of Oleic Acid. J. Vis. Exp. (144), e59034, doi:10.3791/59034 (2019).
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