Encapsulation and Covalent Binding of Molecular Payload in Enzymatically Activated Micellar NanocarriersJ. Am. Chem. Soc.


Ido Rosenbaum, Assaf J. Harnoy, Einat Tirosh, Marina Buzhor, Merav Segal, Liat Frid, Rona Shaharabani, Ram Avinery, Roy Beck, Roey J. Amir
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Encapsulation and Covalent Binding of Molecular Payload in

Enzymatically Activated Micellar Nanocarriers

Ido Rosenbaum,†,§ Assaf J. Harnoy,†,§ Einat Tirosh,‡,§ Marina Buzhor,†,§ Merav Segal,†,§ Liat Frid,†,§

Rona Shaharabani,‡,§ Ram Avinery,∥,§ Roy Beck,∥,§ and Roey J. Amir*,†,§ †Department of Organic Chemistry, School of Chemistry, Faculty of Exact Sciences, ‡Department of Physical Chemistry, School of

Chemistry, Faculty of Exact Sciences, §Tel Aviv University Center for Nanoscience and Nanotechnology, and ∥School of Physics and

Astronomy, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel *S Supporting Information

ABSTRACT: The high selectivity and often-observed overexpression of specific disease-associated enzymes make them extremely attractive for triggering the release of hydrophobic drug or probe molecules from stimuli-responsive micellar nanocarriers. Here we utilized highly modular amphiphilic polymeric hybrids, composed of a linear hydrophilic polyethylene glycol (PEG) and an esterase-responsive hydrophobic dendron, to prepare and study two diverse strategies for loading of enzyme-responsive micelles. In the first type of micelles, hydrophobic coumarin-derived dyes were encapsulated noncovalently inside the hydrophobic core of the micelle, which was composed of lipophilic enzyme-responsive dendrons. In the second type of micellar nanocarrier the hydrophobic molecular cargo was covalently linked to the end-groups of the dendron through enzyme-cleavable bonds. These amphiphilic hybrids self-assembled into micellar nanocarriers with their cargo covalently encapsulated within the hydrophobic core. Both types of micelles were highly responsive toward the activating enzyme and released their molecular cargo upon enzymatic stimulus. Importantly, while faster release was observed with noncovalent encapsulation, higher loading capacity and slower release rate were achieved with covalent encapsulation. Our results clearly indicate the great potential of enzyme-responsive micellar delivery platforms due to the ability to tune their payload capacities and release rates by adjusting the loading strategy. ■ INTRODUCTION

Micellar nanocarriers composed of stimuli-responsive amphiphilic block copolymers are of interest due to their potential utilization in the field of controlled drug delivery.1−4 Such smart micelles were shown to encapsulate hydrophobic molecular cargo (e.g., fluorescent dyes or drugs) within their hydrophobic cores and release them upon external stimuli that alter their amphiphilic nature.5,6 Various types of stimuliresponsive polymers, which can switch their amphiphilic nature upon stimuli, have been reported to respond to irradiated light,7−9 reduction,10,11 changes in temperature12−14 and pH,15,16 or their combinations.17−19 Enzymatic responsive block copolymers possess several significant advantages over polymers that respond to other stimuli for biomedical applications.20−24 The catalytic nature, often-observed overexpression, and activity of specific disease-associated enzymes make them extremely attractive for selectively triggering the release of hydrophobic drug or probe molecules from such smart micellar nanocarriers. Yet, to date, there have been few reports on synthetic amphiphilic block copolymers that can self-assemble into stimuli-responsive micelles and disassemble upon enzymatic stimuli.25−28

We have recently reported on enzyme-responsive amphiphilic block copolymers composed of a hydrophilic polyethylene glycol (PEG) block and a dendron with enzymatically cleavable lipophilic end-groups as the hydrophobic block.29

Cleavage of the hydrophobic end-groups by penicillin G amidase revealed primary amines that were protonated under physiological pH. This enzymatically induced decrease in amphiphilicity of the PEG-dendron hybrids results in destabilization and disassembly of the micelles and release of encapsulated Nile red molecules. Here, we demonstrate the modularity of these hybrids systems by introducing esterase cleavable end-groups and utilize this enzyme-responsive platform to study two distinct types of esterase-responsive carriers: noncovalently and covalently encapsulating micelles. A schematic representation of these two diverse loading strategies is presented in Figure 1.

In the first type of micelles, the hydrophobic guests are noncovalently encapsulated within the hydrophobic cores of the micelles. Enzymatic cleavage of the hydrophobic endgroups from the dendrons will lead to the formation of

Received: September 30, 2014

Article pubs.acs.org/JACS © XXXX American Chemical Society A DOI: 10.1021/ja510085s

J. Am. Chem. Soc. XXXX, XXX, XXX−XXX hydrophilic PEG-dendron hybrids and result in disassembly of the micelles and subsequent release of the encapsulated molecules. In the second type of micelles, the hydrophobic molecular payload is covalently attached to the end-groups of the dendron block through enzymatically cleavable linkages.

These covalently functionalized amphiphilic PEG-dendron hybrids are expected to self-assemble into micellar nanocarriers that covalently encapsulate their molecular cargo. In this case, the enzymatic cleavage of the hydrophobic end-groups would lead directly to the release of the “active” end-groups and simultaneously to the switching of the amphiphilicity of the hybrids as they become hydrophilic. Preliminary comparison of the loading capacities, stabilities, and release rates of these two diverse loading approaches demonstrates their potential application for drug delivery, revealing interesting insights into the unique features and challenges associated with each of these two loading strategies. ■ MOLECULAR DESIGN

To allow the direct comparison between the two loading approaches, we used 7-diethylamino-3-carboxycoumarin (coumarin acid) and its alkyl ester derivatives as model cargo molecules. Adjusting the length of the alkyl chains should enable fine-tuning of the hydrophobicity of the dyes, which is a crucial parameter for noncovalent encapsulation. Furthermore, the carboxylic acid moiety can be used for conjugation to the dendron through an ester bond in order to allow its covalent encapsulation. Taking advantage of the modularity of our molecular design, we prepared two PEG-dendron hybrids, 1 and 2, containing either four nonfluorescent phenyl acetate or four fluorescent coumarin-derived end-groups, respectively (Scheme 1). Hybrid 1 is expected to self-assemble into micelles that will noncovalently encapsulate dye molecules, whereas hybrid 2 is expected to self-assemble into micellar nanocarriers that covalently bind the cargo molecules in their cores. ■ RESULTS AND DISCUSSION