Marzia Massignani

Block copolymer nanostructures

One of the most important classes of synthetic systems for creating self-assembled nanostructures is amphiphilic block copolymers. By controlling the architecture of individual molecules, it is possible to generate nanostructures either in an undiluted melt or in solution. These ordered nanostructures are tunable over a broad variety of morphologies, ranging from discrete micelles and vesicles to continuous network structures. Their synthetic nature allows the design of interfaces with different chemical functional groups and geometrical properties. This, in combination with molecular architecture, determines the levels of ordering in self-organizing polymeric materials. For these and other reasons, block copolymer micelles, vesicles, and mesophases are finding applications in several areas, ranging from nanocomposites to biomedical devices.

Polymersomes: nature inspired nanometer sized compartments

Provided the right hydrophilic/hydrophobic balance can be achieved, amphiphilic block copolymers are able to assemble in water into membranes. These membranes can enclose forming spheres with an aqueous core. Such structures, known as polymer vesicles or polymersomes (from the Greek “-some” = “body of”), have sizes that vary from tens to thousands of nanometers. The wholly synthetic nature of block copolymers affords control over parameters such as the molar mass and composition which ultimately determine the structure and properties of the species in solution. By varying the copolymer molecular mass it is possible to adjust the mechanical properties and permeability of the polymersomes, while the synthetic nature of copolymers allows the design of interfaces containing various biochemically-active functional groups. In particular, non-fouling and non-antigenic polymers have been combined with hydrophobic polymers in the design of biocompatible nano-carriers that are expected to exhibit very long circulation times. Stimulus-responsive block copolymers have also been used to exploit the possibility to trigger the disassembly of polymersomes in response to specific external stimuli such as pH, oxidative species, and enzyme degradation. Such bio-inspired ‘bottom-up’ supramolecular design principles offer outstanding advantages in engineering structures at a molecular level, using the same long-studied principles of biological molecules. Thanks to their unique properties, polymersomes have already been reported and studied as delivery systems for both drugs, genes, and image contrast agents as well as nanometer-sized reactors.

Controlling Cellular Uptake by Surface Chemistry, Size, and Surface Topology at the Nanoscale

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.

Non-cytotoxic polymer vesicles for rapid and efficient intracellular delivery

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.

Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes

Background Microscopic 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 Findings We 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. Significance We 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.

Fully synthetic polymer vesicles for intracellular delivery of antibodies in live cells

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.—Canton, I., Massignani, M., Patikarnmonthon, N., Chierico, L., Robertson, J., Renshaw, S. A., Warren, N. J., Madsen, J. P., Armes, S P., Lewis, A. L., Battaglia, G. Fully synthetic polymer vesicles for intracellular delivery of antibodies in live cells. FASEB J. 27, 98–108 (2013). www.fasebj.org

Polymersomes: A Synthetic Biological Approach to Encapsulation and Delivery

Compartmentalization, i.e. the ability to create controlled volumes and separate molecules one from another is possibly the most important requisite for complex manipulations. Indeed, compartmentalization has been the first step to isolate the building blocks of life and ensure the dynamic nature that today makes the complexity of any living system. For decades scientists have tried using many synthetic approaches to imitate such ability and one the most successful comes from mimicking the biological component responsible for the compartmentalization: the phospholipid. We are now able to synthesize macromolecular analogues of the phospholipid using advanced co-polymerization techniques. Copolymers that comprise hydrophilic and hydrophobic components (i.e. amphiphilic) can be designed to self assemble into membrane enclosed structures. The simplest of those is represented by a sac resulting from the enclosure of a membrane into a sphere: the vesicle. Vesicles made of amphiphilic copolymers are commonly known as polymersomes and are now one of the most important nanotechnological tool for many applications spanning from drug delivery, gene therapy, medical imaging, electronics and nanoreactors. Herein we review the molecular properties, the fabrication processes and the most important applications of polymersomes.

Internalization and biodistribution of polymersomes into oral squamous cell carcinoma cells in vitro and in vivo.

The prognosis for oral squamous cell carcinoma (OSCC) is not improving despite advances in surgical treatment. As with many cancers, there is a need to deliver therapeutic agents with greater efficiency into OSCC to improve treatment and patient outcome. The development of polymersomes offers a novel way to deliver therapy directly into tumor cells. Here we examined the internalization and biodistribution of two different fluorescently labeled polymersome formulations; polyethylene oxide (PEO)-poly 2-(diisopropylamino)ethyl methacrylate (PDPA) and poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)-PDPA, into SCC4 OSCC cells in vitro and in vivo. In vitro SCC4 monolayers internalized PMPC-PDPA and PEO-PDPA at similar rates. However, in vivo PMPC-PDPA polymersomes penetrated deeper and were more widely dispersed in SCC4 tumors than PEO-PDPA polymersomes. In the liver and spleen PMPC-PDPA mainly accumulated in tissue macrophages. However, in tumors PMPC-PDPA was found extensively in the nucleus and cytoplasm of tumor cells as well as in tumor-associated macrophages. Use of PMPC-PDPA polymersomes may enhance polymersome-mediated antitumor therapy.

Wet Nanoscale Imaging and Testing of Polymersomes

Polymeric vesicles, a.k.a. “polymersomes”, are enclosed membranes formed by the self-assembly of amphiphilic block copolymers in water.[1] In recent years polymersomes have attracted much attention due to their unique features, such as improved mechanical properties, high stability, and long circulation half-lives in the body compared to liposomes, as well as their ability to incorporate both hydrophilic compounds in the aqueous core and hydrophobic compounds in the membrane.[1a] Furthermore, amphiphilic block copolymers can be designed to be noncytotoxic, efficiently internalized by cells, etc.[2] Polymersomes can be decorated with proteins and/or antibodies, either by chemically attaching the active moieties to the hydrophilic brushes[3] or by inserting membrane proteins across the hydrophobic membrane.[4] Recently, fine control over polymersome surface topology and its consequences on cell internalization kinetics has been demonstrated by the authors.[5] Thus the ability to examine the surface properties of polymersomes, as well as other water-borne nanoparticles, in situ on the nanoscale is becoming increasingly important in several fields. Imaging of wet nanoparticles is normally performed by transmission or scanning electron microscopy (EM). However, the high vacuum conditions necessary for such imaging require either dried or frozen (e.g., cryogenic EM) samples, potentially causing artefacts. Wet imaging can be performed by optical microscopy and recently the problem of diffraction-limited spatial resolution has been overcome by new fluorescence-based microscopy, such as scanning near-field optical microscopy (SNOM), photo-activated localization microscopy (PALM), stimulated emission depletion microscopy (STED), and structured illumination microscopy (SIM).[6] Atomic force microscopy (AFM) is another valuable analytical tool that has been used for both imaging and also for assessing mechanical, electrical, and surface properties.[7]

A micro-incubator for cell and tissue imaging

A low-cost micro-incubator for imaging dynamic processes in living cells and tissues has been developed. This micro-incubator provides a tunable environment that can be altered to study responses of cell monolayers for several days as well as relatively thick tissue samples and tissue-engineered epithelial tissues in experiments lasting several hours. Samples are contained in a sterile cavity closed by a gas-permeable membrane. The incubator can be positioned in any direction and used on an inverted or upright microscope. Temperature is regulated using a Peltier module controlled by a sensor positioned close to the sample, enabling compensation for any changes in temperature. Rapid changes in a samples surrounding environment can be achieved due to the fast response of the Peltier module. These features permit monitoring of sample adaptation to induced environmental changes.