Enzyme loading of
polymersomes requires permeability to enable
them to interact with the external environment, typically requiring
addition of complex functionality to enable porosity. Herein, we describe
a synthetic route toward intrinsically permeable polymersomes loaded
with functional proteins using initiator-free visible light-mediated
polymerization-induced self-assembly (photo-PISA) under mild, aqueous
conditions using a commercial monomer. Compartmentalization and retention
of protein functionality was demonstrated using green fluorescent
protein as a macromolecular chromophore. Catalytic enzyme-loaded vesicles
using horseradish peroxidase and glucose oxidase were also prepared
and the permeability of the membrane toward their small molecule substrates
was revealed for the first time. Finally, the interaction of the compartmentalized
enzymes between separate vesicles was validated by means of an enzymatic
cascade reaction. These findings have a broad scope as the methodology
could be applied for the encapsulation of a large range of macromolecules
for advancements in the fields of nanotechnology, biomimicry, and
nanomedicine.
Zwitterionic polymers, including polyampholytes and polybetaines, are polymers with both positive and negative charges incorporated into their structure.
Covalent PEGylation
of biologics has been widely employed to reduce
immunogenicity, while improving stability and half-life in
vivo. This approach requires covalent protein modification,
creating a new entity. An alternative approach is stabilization by
encapsulation into polymersomes; however this typically requires multiple
steps, and the segregation requires the vesicles to be permeable to
retain function. Herein, we demonstrate the one-pot synthesis of therapeutic
enzyme-loaded vesicles with size-selective permeability using polymerization-induced
self-assembly (PISA) enabling the encapsulated enzyme to function
from within a confined domain. This strategy increased the proteolytic
stability and reduced antibody recognition compared to the free protein
or a PEGylated conjugate, thereby reducing potential dose frequency
and the risk of immune response. Finally, the efficacy of encapsulated l-asparaginase (clinically used for leukemia treatment) against
a cancer line was demonstrated, and its biodistribution and circulation
behavior in vivo was compared to the free enzyme,
highlighting this methodology as an attractive alternative to the
covalent PEGylation of enzymes.
Advanced applications of polymeric self-assembled structures require a stringent degree of control over such aspects as functionality location, morphology and size of the resulting assemblies. A loss of control in the polymeric building blocks of these assemblies can have drastic effects upon the final morphology or function of these structures. Gaining precise control over various aspects of the polymers, such as chain lengths and architecture, blocking efficiency and compositional distribution is a challenge and, hence, measuring the intrinsic mass and size dispersity within these areas is an important aspect of such control. It is of great importance that a good handle on how to improve control and accurately measure it is achieved. Additionally dispersity of the final structure can also play a large part in the suitability for a desired application. In this Tutorial Review, we aim to highlight the different aspects of dispersity that are often overlooked and the effect that a lack of control can have on both the polymer and the final assembled structure.
We demonstrate that the PISA of identical block copolymers by either a photo or thermally initiated approach leads to structures that are both chemically and morphologically distinct.
Water-soluble and amphiphilic polymers are of great interest to industry and academia, as they can be used in applications such as biomaterials and drug delivery. Whilst ring-opening metathesis polymerization (ROMP) is a fast and functional group tolerant methodology for the synthesis of a wide range of polymers, its full potential for the synthesis of water-soluble polymers has yet to be realized. To address this, we report a general strategy for the synthesis of block copolymers in aqueous milieu using a commercially available ROMP catalyst and a macroinitiator approach. This allows for excellent control in the preparation of block copolymers in water. If the second monomer is chosen such that it forms a water-insoluble polymer, polymerization-induced self-assembly (PISA) occurs and a variety of self-assembled nano-object morphologies can be accessed.
Photoinitiated polymerization-induced
self-assembly (photo-PISA)
is an efficient approach to predictably prepare polymeric nanostructures
with a wide range of morphologies. Given that this process can be
performed at high concentrations and under mild reaction conditions,
it has the potential to have significant industrial scope. However,
given that the majority of industrial (and more specifically biotechnological)
formulations contain mixtures of polymers and surfactants, the effect
of such surfactants on the PISA process is an important consideration.
Thus, to expand the scope of the methodology, the effect of small
molecule surfactants on the PISA process, specifically for the preparation
of unilamellar vesicles, was investigated. Similar to aqueous photo-PISA
findings in the absence of surfactant molecules, the originally targeted
vesicular morphology was retained in the presence of varying concentrations
of non-ionic surfactants, while a diverse set of lower-order morphologies
was observed for ionic surfactants. Interestingly, a critical micelle
concentration (CMC)-dependent behavior was detected in the case of
zwitterionic detergents. Additionally, tunable size and membrane thickness
of vesicles were observed by using different types and concentration
of surfactants. Based on these findings, a functional channel-forming
membrane protein (OmpF porin), stabilized in aqueous media by surfactant
molecules, was able to be directly inserted into the membrane of vesicles
during photo-PISA. Our study demonstrates the potential of photo-PISA
for the direct formation of protein–polymer complexes and highlights
how this method could be used to design biomimicking polymer/surfactant
nanoreactors.
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