Cellular delivery of antibodies: effective targeted subcellular imaging and new therapeutic tool

It is already more than a century since the pioneering work of the Nobel Laureate Ehrlich gave birth to the side chain theory1, which helped to define antibodies and their ability to target specific biological sites. However, the use of antibodies is still restricted to the extracellular space due to the lack of a suitable delivery vehicle for the efficient transport of antibodies into live cells without inducing toxicity. In this work, we report the efficient encapsulation and delivery of antibodies into live cells with no significant loss of cell viability or any deleterious affect on the 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. These polymersomes can successfully deliver relatively high antibody payloads within live cells. Once inside the cells, we demonstrate that these antibodies can target their epitope by immune-labelling of cytoskeleton, Golgi, and transcription factor proteins in live cells. We also demonstrate that this effective antibody delivery mechanism can be used to control specific subcellular events, as well as modulate cell activity and pro-inflammatory process

A micro-incubator for cell and tissue imaging

International audienceA low-cost micro-incubator for the imaging of dynamic processes in living cells and tissues has been developed. This micro-incubator provides a tunable environment which can be altered to study the response of cell monolayers for several days as well as relatively thick tissue samples and tissue engineered epithelial tissues in experiments lasting several hours. Samples within the incubator 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 as well as on an upright microscope. The temperature is regulated with a Peltier system controlled with a sensor positioned close to the sample to be able to compensate for any changes in temperature. Rapid changes in the environment can be applied to the sample because of the fast response of the Peltier system and the sample's adaptations to induced changes in the environment can be monitored. To evaluate the performance of the micro-incubator we report on studies using cultured cells in monolayers, on monolayers of cells stretched to breaking point on a distensible membrane, on cells in open 3D fibrous scaffolds and on fluorescently labelled polymersome penetration into 3D tissue engineered oral mucosa

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

Polymersomes for intracellular delivery : mechanism of action and applications

The 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. The main aim of this project was the evaluation of pH sensitive polymersomes made of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2- (diisopropylamino)ethyl methacrylate) (PMPC-PDPA) block copolymer as a potential vector for intracellular delivery applications. Upon internalization through endocytosis, polymersomes were demonstrated to disassemble, triggering an increase in osmotic pressure within the endosomal compartments. This increase in pressure temporally destabilizes the endosomal membrane and facilitated the release of the polymersome payload within the cell cytosol. Biocompatibility of polymersomes and their uptake kinetics by different cells (both primary cells and cell lines) were assessed by Confocal Laser Scanning Microscopy (CLSM), Transmission Electron Microscopy (TEM), Flow Cytometry and Fluorescence Spectroscopy. The cellular-uptake kinetics was strongly dependent on the polymersomes surface chemistry, size and surface topology. The latter is controlled by the extent of polymer-polymer phase separation within the external envelope of the polymersome. Polymersomes were also successfully used as efficient vectors for the delivery of DNA, functional proteins and different imaging probes.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

HDF cells seeded in a pre-cast fibrin clot gel stained using Rhodamine B octadecyl ester-loaded polymersomes and subsequently monitored using fluorescent microscopy.

<p>(<b>a</b>) Imaged after 7 days at the initial cell seeding area. (<b>b</b>) Imaged after 7 days at the interface between the initial cell seeding area and the pre-cast gel. (<b>c</b>) Imaged after 14 days at the initial cell seeding area. (<b>d</b>) Imaged after 14 days at the interface between the initial cell seeding area and the pre-cast gel. Figure bar = 0.1 mm. 3D volumetric reconstruction of HDF cells migrating within fibrin-clot gels imaged at the migration front areas (<b>e</b>) and at the initial cell seeding area (<b>f</b>). Details of single cells and their filipodia imaged at the seeding area (<b>g</b>) and the migration front (<b>h</b>). Figure bar = 0.01 mm.</p

Selected examples of cells treated with Rhodamine-loaded polymersomes.

<p>(<b>a</b>) Live primary human dermal fibroblast (HDF), primary human epidermal keratinocytes (HEK), primary human endothelial cells (HE), primary human monocytes (HMC), primary human macrophages (HMP), primary human mesenchymal stem cells (HMSc), primary rabbit limbal epithelial (RLE) cells, primary rat cortical neurons (RCN), primary rat motor neurons (RMN), Chinese hamster ovary (CHO) cells, rat Shawnoma (nemap22) cell, preosteocytes (MLO-A5) cells, human melanoma (A375SM) cell, human head & neck cancer (KB) and (SCC4) cells have all been exposed to Rhodamine-loaded polymersomes and successfully stained. 3D reconstruction from confocal laser scanning optical stacks of HDF cells (<b>b</b>), HE cells (<b>c</b>), SCC4 cells (<b>c</b>), and RMN cells (<b>d</b>). Figure bar = 0.02 mm.</p

Polymersomes preparation.

<p>(a) Chemical structure and solution behaviour of the PMPC-PDPA copolymer in water at different pHs. (b) Process of encapsulations for both hydrophilic, hydrophobic and amphiphilic molecules.</p

Tracking period and cytotoxicity induced by polymersome-mediated staining compared with commercial method.

<p>(<b>a</b>) Cell viability determined by MTT assay at different times for HDF cells exposed to either Rhodamine-loaded polymersomes or CellTracker (n = 3, error bar = SEM; *p<0.05). (<b>b</b>) Fluorescence intensity from HDF cells plated at different initial densities and exposed to daily dose of Rhodamine-loaded polymersomes (0.005 mM) (n = 3, error bar = SEM). (<b>c</b>) Fluorescence intensity exhibited by HDF cells after a single dose of polymersomes loaded with varying amounts of Rhodamine or CellTracker (n = 3, error bar = SEM). (<b>d</b>) Fluorescence micrographs of HDF cells recorded for the same exposure at different days after a single dose of Rhodamine-loaded polymersomes. (<b>e</b>) Fluorescence micrographs of primary HDF cells after 24 h incubation with PMPC-PDPA polymersomes loaded with membrane-staining amphiphilic Rhodamine B octadecyl ester perchlorate, (<b>f</b>) BODIPY TR ceramide, (<b>g</b>) fluorescein 1,2-dihexadecylphosphatidylethanolamine (DHPE), (<b>h</b>) labelled NBD cholesterol, (<b>i</b>) DNA staining membrane-impermeable propidium iodide, (<b>l</b>) FITC-labelled antibody anti α-tubulin, (<b>m</b>) labelled nucleic acids, (<b>n</b>) or large quantum dots. Figure bar = 0.02 mm.</p