No abstract
Cell cytosol and the different subcellular organelles house the most important biochemical processes that control cell functions. Effective delivery of bioactive agents within cells is expected to have an enormous impact on both gene therapy and the future development of new therapeutic and/or diagnostic strategies based on single-cell-bioactive-agent interactions. Herein a biomimetic nanovector is reported that is able to enter cells, escape from the complex endocytic pathway, and efficiently deliver actives within clinically relevant cells without perturbing their metabolic activity. This nanovector is based on the pH-controlled self-assembly of amphiphilic copolymers into nanometer-sized vesicles (or polymersomes). The cellular-uptake kinetics can be regulated by controlling the surface chemistry, the polymersome size, and the polymersome surface topology. The latter is controlled by the extent of polymer-polymer phase separation within the external envelope of the polymersome.
We have recently achieved efficient cytosolic delivery by using pH-sensitive poly(2-(methacryloyloxy)ethylphosphorylcholine)-co-poly(2-(diisopropylamino)ethylmethacrylate) (PMPC-PDPA) diblock copolymers that self-assemble to form vesicles, known as polymersomes, in aqueous solution. It is particularly noteworthy that these diblock copolymers form stable polymersomes at physiological pH but rapidly dissociate below pH 6 to give molecularly-dissolved copolymer chains (unimers). These PMPC-PDPA polymersomes are used to encapsulate nucleic acids for efficient intracellular delivery. Confocal laser scanning microscopy and fluorescence flow cytometry are used to quantify cellular uptake and to study the kinetics of this process. Finally, we examine how PMPC-PDPA polymersomes affect the viability of primary human cells (human dermal fibroblasts (HDF)), paying particular regard to whether inflammatory responses are triggered.
Nature has the exquisite ability to design specific surface patterns and topologies on both the macro- and nanolength scales that relate to precise functions. Following a biomimetic approach, we have engineered fully synthetic nanoparticles that are able to self-organize their surface into controlled domains. We focused on polymeric vesicles or "polymersomes"; enclosed membranes formed via self-assembly of amphiphilic block copolymers in water. Exploiting the intrinsic thermodynamic tendency of dissimilar polymers to undergo phase separation, we mixed different vesicle-forming block copolymers in various proportions in order to obtain a wide range of polymersomes with differing surface domains. Using a combination of confocal laser scanning microscopy studies of micrometer-sized polymersomes, and electron microscopy, atomic force microscopy, and fluorescence spectroscopy on nanometer-sized polymersomes, we find that the domains exhibit similar shapes on both the micro- and nanolength scales, with dimensions that are linearly proportional to the vesicle diameter. Finally, we demonstrate that such control over the surface "patchiness" of these polymersomes determines their cell internalization kinetics for live cells.
A series of amphiphilic ABC triblock copolymers are synthesized by atom transfer radical polymerization, wherein the ‘A’ and ‘C’ blocks are hydrophilic and the pH‐sensitive ‘B’ block can be switched from hydrophilic in acidic solution to hydrophobic at pH 7. Careful addition of base to the molecularly dissolved copolymer in acidic solution readily induces the self‐assembly of such triblock copolymers at around neutral pH to form pH‐sensitive polymersomes (a.k.a. vesicles) with asymmetric membranes. By systematic variation of the relative volume fractions of the ‘A’ and ‘C’ blocks, the chemical nature of the polymer chains expressed at the interior or exterior corona of the polymersomes can be selected. Treatment of primary human dermal fibroblast cells with these asymmetric polymersomes demonstrates the biological consequences of such spatial segregation, with both polymersome cytotoxicity and endocytosis rates being dictated by the nature of the polymersome surface chemistry. The pH‐sensitive nature of the polymersomes readily facilitates their dissociation after endocytosis due to the relatively low endosomal pH, which results in the rapid release of an encapsulated dye. Selective binding of anionic substrates such as DNA within the inner cationic polymersome volume, coupled with a biocompatible exterior, leads to potential gene delivery applications for these pH‐sensitive asymmetric nanovectors.
BackgroundMicroscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery.Principal FindingsWe show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots.SignificanceWe show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation.
There is an emerging need both in pharmacology and within the biomedical industry to develop new tools to target intracellular mechanisms. The efficient delivery of functionally active proteins within cells is potentially a powerful research strategy, especially through the use of antibodies. In this work, we report on a nanovector for the efficient encapsulation and delivery of antibodies into live cells with no significant loss of cell viability or any deleterious effect on cell metabolic activity. This delivery system is based on poly[2-(methacryloyloxy)ethyl phosphorylcholine]-block-[2-(diisopropylamino)ethyl methacrylate] (PMPC-PDPA), a pH-sensitive diblock copolymer that self-assembles to form nanometer-sized vesicles, also known as polymersomes, at physiological pH. Polymersomes can successfully deliver relatively high antibody payloads within different types of live cells. We demonstrate that these antibodies can target their respective epitope showing immunolabeling of γ-tubulin, actin, Golgi protein, and the transcription factor NF-κB in live cells. Finally, we demonstrate that intracellular delivery of antibodies can control specific subcellular events, as well as modulate cell activity and proinflammatory processes.
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