Young investigator award

LogoSANOFIThe « APGI YOUNG INVESTIGATOR AWARD" sponsored by Sanofi grants each year a doctoral thesis completed in Drug delivery Science. The award (with a check) is given during an APGI meeting.

APGI thesis award 2018/2019


Dr Karen Peynshaert

Faculty of Pharmaceutical Sciences, Ghent University

Promotors: Prof. Katrien Remaut and Prof. Stefaan De Smedt

Titre: What you seen is what you get: The physicochemistry of nanoparticles dictates how they cross ocular delivery barriers and alter autophagy

Download the thesis abstract



APGI thesis award 2016/2017


Laboratory for General Biochemistry and Physical Pharmacy,

Faculty of Pharmaceutical Sciences, Ghent University

Promoters: Prof. S. De Smedt, Prof. K. Raemdonck, Prof. K. Braeckmans

has been awarded for his Ph.D. thesis entitled:

"Exploring extracellular vesicles for siRNA delivery and Raman-based diagnostics"




Extracellular vesicles (EVs) are membranous structures that are released by cells in the surrounding biofluid. EVs consist of a lipid and protein shell that encapsulates an aqueous core containing, among others, proteins and nucleic acids. It is believed that the molecular composition of EVs is in part actively regulated by the producing cell and, once released, it has been demonstrated that EVs are able to interact with other cells. As they are composed of numerous, potentially bioactive molecules, this interaction can induce phenotypic alternations in the recipient cell. In this respect, EVs are increasingly considered as important mediators of intercellular communication, enabling the functional transfer of (macro)molecules from one cell to another. Their inherent physiological effects can be exploited in a therapeutic context for which numerous examples are provided and discussed in chapter 1 (e.g. cell free vaccination, MSC surrogate therapy, etc.). Interestingly, it is believed that part of the induced alterations are due to the EV’s ability to fuse with the cell and/or endosomal membrane, thus allowing subsequent delivery of their nucleic acid cargo (e.g. miRNAs and mRNAs) to the receptive cell’s cytoplasm. This is a very interesting feature that attracted the attention of the drug delivery community, given that efficient cytoplasmic delivery of macromolecular biotherapeutics (including nucleic acids and proteins) is currently one of the major hurdles hampering clinical translation of biologics with an intracellular target.

In this thesis the ability of EVs to functionally deliver small interfering RNA (siRNA) was explored. Despite some interesting earlier reports in the literature on the value of EVs as bio-inspired drug carriers, many fundamental biological questions, pertaining to the EV biodistribution, cell uptake specificity and cargo release, remain largely unanswered to date. Additionally, technical hurdles such as inadequate purification strategies and the lack of an efficient loading strategy for macromolecular therapeutics should be overcome to reliably assess the true advantage EVs might have over current state-of-the-art delivery strategies (e.g. liposomes and viral vectors).

A first step in pursuit of harnessing EVs for siRNA delivery is the development of a method to obtain purified vesicles. It is important to realize that EVs represent only a fraction of the cell’s secretome. Different methods to isolate and purify EVs out of conditioned cell medium and biological fluids have been suggested. These approaches rely on the EV’s typical size, density, solubility, surface components or a combination of the above. Currently, no consensus on a gold standard protocol exists, which hampers unambiguous comparison of different studies and increases the risk of misconceptions due to residual impurities when using insufficiently stringent purification protocols. In chapter 3 a number of commonly used techniques to purify EVs from endogenous (e.g.protein complexes) and exogenous (e.g. fluorescent dyes) components were compared. Protocols based on a density gradient and size-exclusion chromatography outperformed differential centrifugation- and precipitation-based approaches. In combination with a better understanding of the influence of the respective isolation procedures on the EV functionality, these observations can contribute to the implementation of a more standardized purification protocol.

A second technical hurdle that was addressed in this thesis, is the loading of isolated EVs with exogenous siRNA. One of the strategies suggested in the literature is the electroporation of EVs in the presence of the siRNA of interest. Despite the fact that this technique has already been adopted by different groups, the underlying biophysical loading mechanism was never thoroughly investigated. In chapter 2 an in-depth study on this process revealed that electric pulses in electroporation buffers result in extensive precipitation of siRNA into salt aggregates. This phenomenon was a consequence of metal ions, released from the cuvette electrodes, forming insoluble aggregates with the hydroxide ions present in pH neutral buffers. During this aggregate formation process, siRNAs (and EVs) are co-precipitated. As a result, the encapsulation efficiency for siRNA is easily overestimated when commonly used electroporation conditions and quantification techniques are employed. When preventing aggregation, e.g. by using chelating acidic buffers or polymer-based cuvettes, the measured encapsulation of siRNA into EVs decreased to negligible amounts.

The shortcomings of electroporation and the current lack of alternatives to load hydrophilic macromolecules into EVs prompted us to explore new approaches. In chapter 4 we developed a generally applicable method to attach siRNA to the surface of isolated EVs by means of a cholesterol anchor. Moreover, given the complexity and heterogeneity of EV isolates and the previously described loading artifacts with electroporation, here we used a combination of three complementary assays to confirm and quantify siRNA loading (i.e. a gel retention assay, an antibody capture assay and a density gradient co-localization assay). As this approach was also able to load pre- formed liposomes with siRNA with comparable efficiency, a direct comparison between EVs and synthetic liposomes with regard to siRNA delivery could be made. To this end, we selected negatively charged, fusogenic liposomes with a size distribution comparable to EVs. Unfortunately, under the tested in vitro conditions, EVs underperformed compared to the liposomes for their ability to functionally deliver the siRNA therapeutic, which could be attributed to the lack of an intrinsic mechanism to induce endosomal escape prior to trafficking to lysosomes for degradation. Likewise, the endogenously present miRNAs were not functionally delivered to recipient cells. These observations question the efficiency and universal applicability of EVs as a gene therapy nanocarrier.

Besides therapeutic applications, EVs have also been the subject of investigation in a diagnostic context. The EV architecture and part of the molecular composition are common among EVs isolated from different cells. However, some EV-associated components are unique for the producing cell type and even cellular status. Moreover, upon in vivo release, part of the EVs end up in neighboring biological fluids making them available for liquid biopsies. In this respect, EVs can be considered as easy accessible windows on otherwise difficult to reach (diseased) cells. These features make them ideal biomarker candidates for early disease detection and treatment monitoring.

Yet, as contextualized in chapter 1, to optimally exploit EVs in a diagnostic setting, there is a need for new characterization techniques which can attain high sensitivity on a single vesicle level. In an attempt to address this need, in chapter 5 a nanotechnological platform relying on enhanced Raman spectroscopy for individual EV characterization, was developed. The signal enhancement was evoked by decorating the surface of each individual vesicle with a gold nanoparticle-based plasmonic substrate, which allowed to obtain a Raman spectrum with acceptable acquisition speed. Subsequently, the acquired spectra could be subjected to downstream analysis using dedicated multivariate statistical models allowing to discriminate between EVs derived from red blood cells and EVs derived from melanoma cells. Furthermore, due to the single vesicle approach, this technique was able to quantify the relative abundance of each EV type in a mixture.


In conclusion, in a first part of this dissertation the potential of EVs as a drug delivery carrier for siRNA was assessed. We could obtain pure EVs by means of a density gradient purification protocol and load them by exploiting the hydrophobic interaction between the EV membrane and a cholesterol tag covalently attached to one of the siRNA strands. However, under the experimental conditions EVs were unable to bypass the endolysosomal degradation pathway and hence were unable to functionally deliver siRNA upon cellular internalization. To a certain extent, our observations temper the high expectations linked to exploiting EVs as a drug delivery carrier and call for a more in-depth biological understanding of the EV’s cellular delivery mechanism and related cell type specificity. Nonetheless, other therapeutic applications of EVs, as discussed in chapter 6, are very promising and are already developed up to market level (e.g. EV- based immunotherapy). In the second part of this dissertation, we developed a new nanotechnological platform that allows the fast characterization of individual EVs via surface enhanced Raman spectroscopy. As EVs are very promising biomarkers, the high sensitivity inherent to the developed technology makes this an attractive platform to explore further in a diagnostic setting. 



APGI thesis award 2015/2016


Institut Galien Paris-Sud


University of Paris-Sud

School of Pharmacy

Promoters: Prof. P. Couvreur and Prof. K. Andrieux

has been awarded for her Ph.D. thesis entitled:

« Squalenoyl Adenosine Nanoparticles and Cerebral Ischemia: evaluation of the passage of the Blood-Brain Barrier, pharmacological efficiency and theranostic »


This PhD work, as a part of the ERC Advanced Grant project «TERNANOMED», aimed at developing a squalenoyl nanomedicine of adenosine (SQAd NAs), for the treatment of stroke and spinal cord injury (SCI). The first part of this research was dedicated to the preparation and characterization of SQAd NAs, and highlighted their dramatic therapeutic activity in pre-clinical models of cerebral ischemia and SCI. To further understand the mechanism of action of these NAs, the second part of this thesis was devoted to the detailed study of their transcytosis across the Blood- Brain Barrier. It was shown that the NAs were disassembled inside the endothelial cells, confirming that the pharmacological mechanism of the SQAd NAs action appeared to be a primary vascular protection via the improvement of microcirculation, leading to a secondary neuronal preservation, likely thanks to neurovascular coupling and to the pleiotropic multitargeted abilities of adenosine. The third part of this work aimed to describe the pharmacokinetic profile and issue distribution of SQAd NAs, thanks to innovative techniques of radiolabeling. Finally, the fourth part presented preliminary results on the development of a theranostic tool, by incorporating USPIO as a MRI contrast agent inside the SQAd NAs. Overall, this PhD work established the foundation to the extension of the squalenoylation platform to the treatment of neurological diseases.


Keywords: nanomedicine, squalene, adenosine, stroke, spinal cord injury, blood-brain barrier