Marina Buzhor

Encapsulation and covalent binding of molecular payload in enzymatically activated micellar nanocarriers.

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.

Modular Synthetic Approach for Adjusting the Disassembly Rates of Enzyme-Responsive Polymeric Micelles

Self-assembled nanostructures and their stimuli-responsive degradation have been recently explored to meet the increasing need for advanced biocompatible and biodegradable materials for various biomedical applications. Incorporation of enzymes as triggers that can stimulate the degradation and disassembly of polymeric assemblies may be highly advantageous owing to their high selectivity and natural abundance in all living organisms. One of the key factors to consider when designing enzyme-responsive polymers is the ability to fine-tune the sensitivity of the platform toward its target enzyme in order to control the disassembly rate. In this work, a series of enzyme-responsive amphiphilic PEG-dendron hybrids with increasing number of hydrophobic cleavable end-groups was synthesized, characterized, and compared. These hybrids were shown to self-assemble in aqueous media into nanosized polymeric micelles, which could encapsulate small hydrophobic guests in their cores and release them upon enzymatic stimulus. Utilization of dendritic scaffolds as the responsive blocks granted ultimate control over the number of enzymatically cleavable end-groups. Remarkably, as we increased the number of end-groups, the micellar stability increased significantly and the range of enzymatic sensitivity spanned from highly responsive micelles to practically nondegradable ones. The reported results highlight the remarkable role of hydrophobicity in determining the micellar stability toward enzymatic degradation and its great sensitivity to small structural changes of the hydrophobic block, which govern the accessibility of the cleavable hydrophobic groups to the activating enzyme.

Molecular Precision and Enzymatic Degradation: From Readily to Undegradable Polymeric Micelles by Minor Structural Changes

Studying the enzymatic degradation of synthetic polymers is crucial for the design of suitable materials for biomedical applications ranging from advanced drug delivery systems to tissue engineering. One of the key parameters that governs enzymatic activity is the limited accessibility of the enzyme to its substrates that may be collapsed inside hydrophobic domains. PEG-dendron amphiphiles can serve as powerful tools for the study of enzymatic hydrolysis of polymeric amphiphiles due to the monodispersity and symmetry of the hydrophobic dendritic block, which significantly simplifies kinetic analyses. Using these hybrids, we demonstrate how precise, minor changes in the hydrophobic block are manifested into tremendous changes in the stability of the assembled micelles toward enzymatic degradation. The obtained results emphasize the extreme sensitivity of self-assembly and its great importance in regulating the accessibility of enzymes to their substrates. Furthermore, the demonstration that the structural differences between readily degradable and undegradable micelles are rather minor, points to the critical roles that self-assembly and polydispersity play in designing biodegradable materials.

Supramolecular Translation of Enzymatically Triggered Disassembly of Micelles into Tunable Fluorescent Responses.

The need for advanced fluorescent imaging and delivery platforms has motivated the development of smart probes that change their fluorescence in response to external stimuli. Here a new molecular design of fluorescently labeled PEG-dendron hybrids that self-assemble into enzyme-responsive micelles with tunable fluorescent responses is reported. In the assembled state, the fluorescence of the dyes is quenched or shifted due to intermolecular interactions. Upon enzymatic cleavage of the hydrophobic end-groups, the labeled polymeric hybrids become hydrophilic, and the micelles disassemble. This supramolecular change is translated into a spectral response as the dye-dye interactions are eliminated and the intrinsic fluorescence is regained. We demonstrate the utilization of this molecular design to generate both Turn-On and spectral shift responses by adjusting the type of the labeling dye. This approach enables transformation of non-responsive labeling dyes into smart fluorescent probes.

Fluorinated smart micelles as enzyme-responsive probes for 19F-magnetic resonance

Labeling of smart PEG-dendron hybrids with fluorinated groups transformed their assemblies into enzyme-responsive micellar probes for 19F-magnetic resonance (MR). Two distinct labeling approaches were used to study the ability of these smart hybrids to be turned OFF at the assembled micellar state and to turn ON their 19F-MR signal upon enzymatic activation.

Micellar Stability in Biological Media Dictates Internalization in Living Cells

The dynamic nature of polymeric assemblies makes their stability in biological media a crucial parameter for their potential use as drug delivery systems in vivo. Therefore, it is essential to study and understand the behavior of self-assembled nanocarriers under conditions that will be encountered in vivo such as extreme dilutions and interactions with blood proteins and cells. Herein, using a combination of fluorescence spectroscopy and microscopy, we studied four amphiphilic PEG-dendron hybrids and their self-assembled micelles in order to determine their structure-stability relations. The high molecular precision of the dendritic block enabled us to systematically tune the hydrophobicity and stability of the assembled micelles. Using micelles that change their fluorescent properties upon disassembly, we observed that serum proteins bind to and interact with the polymeric amphiphiles in both their assembled and monomeric states. These interactions strongly affected the stability and enzymatic degradation of the micelles. Finally, using spectrally resolved confocal imaging, we determined the relations between the stability of the polymeric assemblies in biological media and their cell entry. Our results highlight the important interplay between molecular structure, micellar stability, and cell internalization pathways, pinpointing the high sensitivity of stability-activity relations to minor structural changes and the crucial role that these relations play in designing effective polymeric nanostructures for biomedical applications.