JoVE Journal > Chemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Chemistry

Anionisk polymerisation af en amfifil Copolymer for Udarbejdelse af blokcopolymer Miceller stabiliseret af π-Tr Stacking Interaktioner

Published: October 10, 2016
doi:
10.3791/54422
, ,
1Leslie Dan Faculty of Pharmacy,University of Toronto

Summary

De vigtigste trin i levende anioniske polymerisering af phenylglycidylether (PheGE) på methoxy-polyethylenglycol (mPEG b -PPheGE) er beskrevet. De resulterende blokcopolymer miceller (BCMS) blev fyldt med doxorubicin 14% (vægt%) og vedvarende afgivelse af lægemidlet over 4 dage under fysiologisk relevante betingelser blev opnået.

Abstract

I denne undersøgelse blev en amfifil copolymer, der indbefatter en kerne-dannende blok med phenylgrupper syntetiseret ved levende anionisk polymerisation af phenyl glycidylether (PheGE) på methoxy-polyethylenglycol (mPEG B -PPheGE). Karakterisering af copolymeren afslørede en smal molekylær fordeling (PDI <1,03) og bekræftede polymerisationsgrad mPEG 122 – b – (PheGE) 15. Den kritiske micellekoncentration af copolymeren blev vurderet ved anvendelse af en etableret fluorescensmetode med aggregeringen adfærd vurderes ved dynamisk lysspredning og transmission elektronisk mikroskopi. Potentialet af copolymeren til brug i lægemiddeltilførselsanvendelser blev evalueret i en foreløbig måde, herunder in vitro biokompatibilitet, lastning og frigivelse af den hydrofobe anti-cancer stof doxorubicin (DOX). En stabil micelle formulering af DOX blev fremstillet med medikamentloading niveauer op til 14% (vægt-%), lægemiddelfyldning efficiencies> 60% (vægt / vægt) og vedvarende afgivelse af lægemidlet over 4 dage under fysiologisk relevante betingelser (surt og neutralt pH, tilstedeværelse af albumin). Det høje medikamentbelastning niveau og langvarig frigivelse tilskrives stabilisere n-π interaktioner mellem DOX og kernedannende blok af micellerne.

Introduction

I vandige medier, amfifile blokcopolymerer samles for at danne nanostørrelse blokcopolymer miceller (BCMS), der består af en hydrofob kerne omgivet af en hydrofil skal eller corona. Den micelle kerne kan tjene som et reservoir for inkorporering af hydrofobe stoffer; mens den hydrofile corona giver en grænseflade mellem kernen og det ydre medium. Poly (ethylenglycol) (PEG) og derivater deraf er en af de vigtigste klasser af polymerer og en af de mest udbredte i lægemiddelformulering. 1-3 BCMS har vist sig at være en værdig drug delivery platform med flere formuleringer afhængige af dette teknologi nu i sen klinisk udvikling. 4 Mest almindeligt, den hydrofobe blok af copolymeren består af polycaprolacton, poly (D, L-lactid), poly (propylenoxid) eller poly (β-benzyl-L-aspartat). 5 -9

Kataoka gruppe undersøgte sfæriske miceller dannet af PEO- b </em> -PBLA Og poly (ethylenoxid) – b -. (Polyasparaginsyre-konjugeret doxorubicin) til levering af doxorubicin (DOX) 10,11 i deres rapporter, de fremførte, at π-Tr interaktioner mellem polymer-konjugeret lægemiddel eller PBLA og gratis DOX handle for at stabilisere micelle kerne resulterer i stigninger i medikamentifyldning og fastholdelse. Det er fastslået, at kompatibilitet eller interaktioner mellem et lægemiddel og kernen-dannende blok er determinanter for key performance parametre. 12 Ud over DOX, en række kræftmedicin omfatter aromatiske ringe inden for deres kerne struktur (fx methotrexat, olaparib, SN -38).

Som følge heraf er der betydelig interesse i syntesen af ​​copolymerer, der omfatter benzyl ringe i deres kernedannende blokke. Anionisk ringåbnende polymerisation af PEG og dets derivater muliggøre kontrol over molekylvægten og resulterer i materialer med lav polydispersitet i godt udbytte. 13,14 Ethylene oxid med phenylglycidylether (PheGE) eller styren oxid (SO) kan være (co) polymeriseres til at danne blokcopolymerer, der danner miceller til solubilisering af hydrofobe stoffer. 15-18 Den aktuelle rapport beskriver de nødvendige skridt for at leve anioniske polymerisering af phenyl glycidylether monomer på mPEG-OH som makroinitiator (figur 1). Den resulterende blokcopolymer og dets aggregater derpå karakteriseret i form af egenskaberne relevante at bruge i lægemiddeladministration.

Protocol

Figur 1. Skematisk viser de ni vigtigste skridt i forberedelsen af mPEG B -PPheGE copolymer. Klik her for at se en større version af dette tal. 1. Forberedelse af reagenser under tørre forhold Fremstilling af reagenserne. Afvej 15 g mPEG-5K (M n = 5.40…

Representative Results

Figur 3. Illustration af den anioniske polymerisation af phenylglycidylether på mPEG makroinitiator til frembringelse mPEG b – (PheGE) 15 til fremstilling af blokcopolymer-miceller til lastning af doxorubicin Den skematiske illustrerer deprotonering af hydroxylgruppen i mPEG hjælp naphthalen kalium. som en radik…

Discussion

På grund af den gode kontrol, anionisk polymerisation giver over molekylvægten er det en af ​​de mest anvendte processer i industrien til fremstilling af polymerer baseret på oxiran monomerer (PEG og PPG). Optimale og stringente betingelser skal anvendes for vellykket polymerisation skal opnås. Streng oprensning af alle reagenser og passende apparat er essentielle for levende karakter af syntesen. Begrænsninger i den nuværende setup er for det meste forbundet med overførslen teknik, der er afhængig af kanyle…

Disclosures

The authors have nothing to disclose.

Acknowledgements

CA acknowledges a Discovery grant from the Natural Sciences and Engineering Research Council of Canada. CA acknowledges a Chair in Pharmaceutics and Drug Delivery from GSK. The authors declare no competing financial interest.

Materials

DMEM/HAMF12 Gibco, Life Technologies 12500 Supplemented with 10%FBS. Warm in 37 °C water bath
                          
Trypsin-EDTA(0.25%) Sigma-Aldrich T4049 Warm in 37 °C water bath 
Fetal bovine serum (FBS) Sigma-Aldrich F1051 Canada origin
MDA-MB-468 cell line ATCC HTB-132
MTS tetrazolium reagent PROMEGA G111B
Phenazine ethosulfate (PES) Sigma-Aldrich P4544 >95%
mPEG5K (Mn 5400 g/mol) Sigma-Aldrich 81323 PDI=1.02
Dimethylsolfoxide (DMSO) Sigma-Aldrich D4540 >99.5%
Naphthalene Sigma-Aldrich 147141 >99%
Phenyl glycidyl ether Sigma-Aldrich A32608 >85%
Benzophenone Sigma-Aldrich 427551 >99%
Potassium Sigma-Aldrich 451096 >98%
Tetrahydrofuran Caledon Laboratory Chemicals 8900 1 ACS
Hexane Caledon Laboratory Chemicals 5500 1 ACS
Calcium hydride (CaH2) ACP C-0460 >99.5%
Diethyl Ether Caledon Laboratory Chemicals 1/10/4800 ACS
Microplate reader BioTek Instruments
Differential scanning calorimetry (DSC) TA Instruments Inc DSC Q100
Gel permeation chromatography (GPC) Waters 2695 separation moldule / 2414 detector  2 Columns: Agilent Plgel 5µm Mixed-D
NMR spectroscopy Varian Mercury 400MHz
Chloroform-d Sigma-Aldrich 151858 99.96%
DMSO-d Sigma-Aldrich 156914 99.96%
Vaccum pump  Gardner Denver Welch Vacuum Tech, Inc. Ultimate  pressure 1.10-4 torr
Drierit with indicator, 8 mesh Sigma-Aldrich 238988 Regenerated at 230°C for 2 hrs
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Tags

Anionic PolymerizationAmphiphilic CopolymerBlock CopolymerMicellesπ-π StackingRing Opening PolymerizationDMSOPhenyl Glycerol EtherMacroinitiatorDrug DeliveryPolymeric Drug Delivery

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Le Dévédec, F., Houdaihed, L., Allen, C. Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by π-π Stacking Interactions. J. Vis. Exp. (116), e54422, doi:10.3791/54422 (2016).
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