The aim of the research of the Advanced Drug Delivery and Biomaterials research group is to develop new delivery systems for unmet medical and pharmaceutical needs that address two main challenges for the pharmaceutical industry, namely the delivery of i) biotech-based drugs (proteins, vaccines, nucleic acids) and ii) poorly water soluble drugs. After the definition of an unmet need, a hypothesis or mechanism‐driven approach is used to design new drug delivery systems. A proof of concept is provided. The scientific approach is multidisciplinary and mechanistic-driven.
It involves: i) the development of new formulations, ii) their physico‐chemical characterization, iii) the demonstration of their efficacy and safety in vitro and in vivo, iv) the understanding of their mechanisms of action and of their interactions with biological systems.
Based on the expertise of the different principal investigators within the group, we develop the following research themes.
Our research mainly focuses on (i) intravenous delivery of drug-loaded of nanoparticles targeting the tumoral endothelium and cancer cells ii) local delivery of anticancer drugs.
Several mechanisms of delivery of drug-loaded nanoparticles to tumors have been investigated (Figure 2): (i) passive targeting through leaky vasculature surrounding the tumors, described as the enhanced permeability and retention effect (EPR) (ii) “active” targeting by grafting specific ligands of cancer cells or angiogenic endothelial cells to the surface of the nanocarrier (iii) magnetic targeting of SPIO (small paramagnetic iron oxides) loaded nanoparticles. We formulated various nanocarriers (micelles and untargeted or targeted nanoparticles) loaded with several anti-cancer drugs to specifically target tumors and improve the therapeutic index of anti-cancer drugs by nanomedicines. Exploiting the αvβ3 integrin overexpression by tumoral endothelium and tumor cells, we designed PLGA-based nanoparticles grafted with the RGD peptide and demonstrated the “active” targeting of these PLGA-based nanoparticles. We formulated multi-functional nanoparticles for the encapsulation of a therapeutic drug and a contrast agent (SPIO) that can be targeted by magnets and significantly enhanced drug biodistribution in colorectal cancer and glioblastoma (GBM).
GBM is a particularly aggressive brain cancer associated with high recurrence and poor prognosis. The local delivery of active agents within the tumor resection cavity has emerged as an attractive means to initiate oncological treatment immediately post-surgery. Anticancer drug-loaded nanomedicines are developed for the local treatment of glioblastoma. (Figure 3) In particular lauroyl gemcitabine lipid nanocapsules forming hydrogel or PEGDMA photomerizable hydrogel loaded with anticancer nanomedicine significantly improved the survival of glioblastoma bearing mice when perisurgically injected in the resection cavity.
Our current projects are focussed on the mechanisms of action of nanomedicines, in particular their effect on the tumor microenvironment.
Figure 1: Passive, active and magnetic targeting of anticancer drug-loaded nanomedicines
We aim to develop formulation (nanoparticles, polymer conjugates) and physical methods (electroporation) for the delivery of DNA and RNA with a particular interest in vaccination and cancer treatments. Optimised patented plasmids encoding tumor antigens elicited humoral and cellular immune response and induced tumor control or regression. Our current research focuses on the combination of optimized anticancer DNA vaccines (DNA vaccine and antigen-polymer conjugates) and immunomodulators.
Figure 2: Schematic representation of the local treatment of GBM (From Bastiancich et al. Adv Drug Del Rev, 2021)
The oral route is the most preferred route of drug administration. It is easy to administer, pain free and cheaper compared to other routes of administration. However, this route is sometimes inefficient due to the partial/inadequate absorption of the drug, first-pass metabolism, the instability of the drug in harsh gastrointestinal conditions (such as intestinal pH or enzyme degradation). There is an unmet need for the administration of biologics via the oral route of administration, especially in the treatment of chronic diseases where a daily painful administration is often required.
The aim of our research is developing improved alternative drug delivery systems to fulfill the potential of the oral route of administration. For this purpose, we are exploiting the unique pathophysiology of the gut towards the development of novel drug delivery strategies, focusing on the treatment of two main chronic diseases: type 2 diabetes mellitus (T2DM) and inflammatory bowel diseases (IBD). Our recent results describe a novel nanosystem compatible with human use that synergizes its own biological effect with the effects of increasing the bioavailability of a GLP-1 analogue. The effects of the formulation were comparable to the results observed for the marketed subcutaneous formulation. This nanocarrier-based strategy represents a novel promising approach for oral peptide delivery in incretin-based diabetes treatment. A schematic representation of our strategy is depicted in Figure 1.
In the context of IBD, we are also developing innovative drug delivery systems for the local treatment of IBD via oral delivery of biologics (e.g. siRNA, mAb).
Figure 1: A schematic representation of the dual effect attained with our novel nanoformulation (from Xu et al., J Control Release, 2020)
Unraveling the mechanisms of nanoparticle transport across the intestinal barrier is essential for designing more efficient nanoparticles for oral administration. For this purpose, we have development in vitro models of the intestinal epithelium and follicle-associated epithelium containing M cells to evaluate the mechanisms of transport of our drug delivery systems at the intestinal site. In concrete, we study the physicochemical parameters that dictate the fate of the drug delivery systems across the intestinal barrier. This includes evaluating targeting strategies that could potentially ameliorate the transport of our drug delivery systems across the intestinal epithelium (Figure 2).
Figure 2: Targeting strategies towardsthe intestinal barrier(from Xu et al., J Control Release, 2020)
The research aims at improving the treatment or prophylaxis of severe respiratory diseases by designing nanomedicines to enhance the local efficacy of drugs. Our approaches include i) the preparation of polyethylene glycol (PEG)-drug conjugates to sustain drug release within the lung, and ii) the formulation of nanocarriers to target vaccines to lung dendritic cells.
Inhalation of recombinant human deoxyribonuclease I (rhDNase) is a gold-standard therapy in the management of cystic fibrosis. Yet, the rapid elimination of the mucolytic from the lungs requires its daily administration and rhDNase contributes to the high therapy burden of patients with cystic fibrosis. We have prepared a long-acting PEGylated version of rhDNase that could be delivered once weekly instead of once daily. Conjugation of rhDNase to a PEG chain sustained its presence and mucolytic activity within the murine lungs for more than 15 days. One single dose of PEGylated rhDNase was as effective as 1 daily dose of unconjugated rhDNase during 5 days to decrease the DNA content in the lungs of β-ENaC mice, a model of the CF lung disease. PEGylated rhDNase was stable to jet nebulization. Multiple high-dose administrations of PEGylated rhDNase for up to three months did not cause any significant pulmonary or systemic toxicity, nor accumulation of the rhDNase or PEG moieties in biological fluids.
We elucidated the biodistribution and elimination pathways of native and PEGylated rhDNase after intratracheal instillation in mice. In vivo fluorescence imaging revealed that PEGylated rhDNase was retained in mouse lungs for a significantly longer period of time than native rhDNase. Confocal microscopy confirmed the presence of PEGylated rhDNase in lung airspaces for at least 7 days (Figure 1). In contrast, the unconjugated rhDNase was cleared from the lung lumina within 24 hours and was only found in the lung parenchyma and alveolar macrophages thereafter. Systemic absorption of intact rhDNase and PEG-rhDNase was observed. However, this was significantly lower for the latter. Catabolism, primarily in the lungs and secondarily systemically followed by renal excretion of byproducts were the predominant elimination pathways for both native and PEGylated rhDNase. On the other hand, mucociliary clearance appeared to play a less prominent role in the clearance of those proteins after pulmonary delivery.
Figure 1: Localization of PEGylated rhDNase in mouse lungs by confocal imaging 4 days after intratracheal instillation. 1 nmol of Alexa488-PEG-rhDNase (green) was administered to NMRI mice by intratracheal instillation. Four days later mice were sacrificed and lung slices were imaged with Cell Observer Spinning Disk. Images were recorded in green (Alexa488-PEG30-rhDNase), red (tissue, MitoTracker Red CMXRos), and blue (nuclei, Draq5). Signal from Alexa488-PEG-rhDNase is indicated by arrows in alveolar macrophages and stars in alveolar spaces. Scale bars are 50 μm.From Guichard et al, Advanced Therapeutics, in press.
The general scientific objective of Anne des Rieux’s research is the development of new drug and stem cell delivery systems to address neurological unmet medical needs. Her interest focuses mainly on new delivery systems that potentiate the therapeutic action of bioactive molecules and cells. Over the years, Anne des Rieux has developed a strong expertise in nanomedicines for central nervous system (CNS) and tissue engineering.
Nanomedicines for Multiple Sclerosis
We develop nanomedicines for the central nervous system (CNS) repair. Our objective is to stimulate CNS repair by different strategies. So far, we were able to stimulate the repopulation of a brain white matter lesion by new oligodendrocytes with one ventricular injection of retinoic acid loaded lipid nanocapsules (LNC) (Thesis of D. Carradori) (Figure 1).
Figure 1: Targeted drug delivery for CNS repair.
We also designed SDF-1-loaded PLGA nanoparticles that, once implanted at the site of a traumatic brain injury, were able to recruit NSC at the damaged area (L. Zamproni, Universidade Federal de Sao Paulo, BR).
Our ongoing projects focus on new nanomedicines aiming at stimulating the differentiation of oligodendrocyte progenitor cells and resolving inflammation in the brain, more particularly in the scope of multiple sclerosis (MS) (Figure 2). This work is performed in collaboration with Prof. Muccioli (BPBL). In that scope, we were able to graft an antibody (anti-PDGFRa) aiming to target oligodendrocyte progenitor cells at LNC surface (PhD project of Y. Labrak). We also optimized LNC by modification of their surface with a cell penetrating peptide (TAT) to enhance their Nose-to-Brain transport and to deliver to the CNS a potent anti-inflammatory lipid (PhD project of A. Mwema).
We recently started to develop nanomedicines based on extra cellular vesicles (EV), still in the scope of MS. We set a new protocol in the lab for their isolation and characterization and we are currently working on drug encapsulation in EV (PhD projects of V. Gratpain and M. Auquière). Together with Prof. Muccioli and Prof. van Pesch we obtained an ARC grant focused on EV and MS.
Figure 2: New drug delivery strategies for the treatment of multiple sclerosis.
Therapeutic potential of stem cells of the apical papilla for SCI repair.
Human dental stem cells of the apical papilla (SCAP) have been increasingly studied as an alternative source of mesenchymal stem cells due to their accessibility, their neural crest origin and their high proliferation rate. We have then selected them for our cell therapy projects as they have a high translational potential. This part of our research focuses mostly on spinal cord injury (Figure 3).
Figure 3: Therapeutic potential of SCAP for spinal cord repair.
We aim to develop new strategies for the delivery of stem cells that would ensure and support their viability and therapeutic efficiency (Figure 4).
Figure 4: Scap delivery strategies.
SCAP reduce inflammation and stimulate remyelination: when co-cultured with activated microglia cells (BV2 cells) and rat spinal cord organotypic cultures, SCAP decreased TNF secretion, increased oligodendrocyte survival and oligodendrocyte OPC differentiation, partially through activin A secretion (Thesis of P. De Berdt).
Impact of SCAP encapsulation in hydrogels:incorporation in hydrogels maintained SCAP viability, proliferation and neurodifferentiation in vitro but also proliferation, collagen secretion and angiogenesis in vivo (collaboration with Prof. Dupont, UCLouvain). Encapsulation in extracellular matrix-derived hydrogels also maintained their viability and proliferation while stimulating the expression of neural markers (collaboration with Dr. White, Nottingham University, UK). It did not affect SCAP ability to secrete immunomodulatory molecules like indoleamine-pyrrole 2,3-dioxygenase (IDO) upon pro-inflammatory stimulation nor to induce the decrease of the iNOS/Arginase ratio stimulation) (Thesis of N. Tatic)
To further enhance SCAP viability after transplantation we used an alternative cell delivery system termed pharmacologically active microcarriers (PAMs) loaded with brain derived neurotrophic factor (BDNF)18 (collaboration with Prof. Montero-Menei, Angers University, FR). We observed a significant increase of TSG-6, VEGF, activin A and IL10 gene expression when SCAP were grown on PAMs, as well as an improved locomotor function of rats subjected to a spinal cord contusion treated with BDNF-loaded PAMs, compared to non-treated animals (Thesis of S. Kandalam).
Apical papilla stimulates spinal cord repair:when implanted in a rat spinal cord hemisection model, the human apical papilla allowed the restoration of several gait parameters (Catwalk™) and the reduction of microglia activation (6 weeks post-lesion). One week after implantation, the apical papilla also decreased the concentration of pro-inflammatory cytokines in the spinal cord (collaboration with Prof. Diogenes, San Antonio University, USA). We observed a significant decrease of CD68, Iba-1 and GFAP (markers of activated glia) and increase of 5HT (marker for motoneurons (serotonin)) (Thesis of P. De Berdt).
Figure 1: Current approaches for the treatment of tooth decay
1) In case of tooth decay and a healthy pulp, a resin-based composite is used. 2) In case of tooth decay with an inflamed pulp, a calcium-silicate cement is applied on the pulp before the resin-based composite. 3) When the pulp is partially necrotic, this part is removed and replaced by a combination of calcium-silicate cement and resin-based composite. 4) When the complete pulp is necrotic, it is completely removed and replaced by inert filling material. Novel approaches for the treatment of tooth decay. 3’) When the pulp will be partially removed, it will be replaced by a hydrogel loaded with growth factors in order to attract stem cells from the remaining pulp. 4’) When the pulp will be completely removed, it will be replaced by a hydrogel loaded with stem cells in order to re-create a new dental pulp tissue.
In the treatment of tooth decay, restorative dental materials are required to exhibit excellent mechanical, biological properties and most uniquely, display good aesthetics. The research carried out focuses 1) on the characterization of currently available commercial materials, in relation with clinical requirements and 2) on developing new biomaterials for tooth restoration, from a conventional conservative but also from a more advanced regenerative standpoint.
Conservative approach The use of restorative materials allows for relatively fast treatments as they may be implemented directly in the oral cavity in a matter of minutes. They are also highly versatile. However several concerns exist with regards to the suitability of some materials in terms of mechanical or biological properties. Additionally the very mechanisms responsible for the setting of materials or interactions with the biological are little understood.
a) In vitro-methods We are continuously invested in determining the most suited set of characterization methods to properly analyze both mechanical and biological properties of commercial materials, leading to innovative experimental research. Our previous results describe the setting kinetics and mechanical properties of ultra-fast polymerizing resin composites, based on a monoacylphosphate photoinitiator and bioactive calcium silicate cements. In collaboration with Pr. Möginger (University of Bonn-Rhein, Germany) and Pr. Will Palin (University of Birmingham, UK) an innovative combination of characterization techniques was set up, allowing for a precise analysis of polymerization kinetics in heavily filled composites. Moreover, the group has been recently awarded a grant to acquire a Raman spectrometer, to enable chemometric analyses, which nicely complements the previous developments.
b) In vitro-material development The formulation of resin composites is fine-tuned (photoinitiator, resin composition, etc) to quicken kinetic, increase longevity and bring mechanical properties close to that of hard tissues. The use of micro hydroxyapatite particles and amorphous CaP nano particles is investigated for the release of Ca²⁺ and PO₄²ˉ with antibacterial and re-mineralizing potential. The impact of their introduction in model formulations on kinetics and mechanical properties is studied. Additionally, ceramics are investigated for their use as alternatives of resin composites following root canal treatment (Figure 1, item 4). Finally, we are currently working on the incorporation of anti-inflammatory drugs in tricalcium silicate cements (Figure 1, item 2) to modulate pulp inflammation and push the borders of vital pulp therapy.
c) The interactions with pulp tissues and oral commensal bacteria are also researched. The potential of apatite-loaded resin composites is being evaluated, aiming both at S.mutans/S.gordonii biofilm reduction and increased dental pulp stem cells (DPSC) viability (induced osteo-differentiation is also analyzed). Further, as resin composites do not polymerize completely, the toxicity of monomers and un-reacted compounds on DPSC is investigated. Even in the absence of toxicity, some monomers may still induce oxidative stress and genotoxic effects. Methods are being developed to quantify ROS production and osteo-differentiation inhibition on a large number of samples. Again, the addition of the new Raman spectrometer will help characterize the resulting modifications in mineralized matrix produced by the DPSCs and/or the odontoblasts.
d) Clinical work As a result of strong collaborations with the dental clinics, several studies are currently under way, focusing on the analysis of the suitability of resin composites for the treatment of large cavities, in a retrospective manner. Another study underway was designed to investigate prospectively the suitability of a pulpotomy strategy (more conservative approach) as permanent treatment in molars with irreversible pulpitis (Figure 1, item 3), which are currently treated by root canal therapy. Regenerative approach
In modern dentistry, there is currently a paradigm shift from restorative procedures to strategies based on regenerative medicine. In this context, alternatives to current clinical restorative strategies where pulp tissue is partially or completely lost (irreversibly inflamed and necrotic dental pulps) must we designed by combining bioactive matrices and dental stem cells in a clinically relevant way.
Dental stem cells are mesenchymal stem cells that may be collected in large amounts from dental tissues. Such cells display a higher proliferation rate than bone marrow stem cells and have better neural and epithelial properties as they originate from the neural crests. Additionally dental stem cells can differentiate in multiple cells types, like osteo- odonto-, adipo-, neuro-, chondroblast-like cells… Among dental stem cells, we selected dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAP) for their potential. While we have worked with SCAP (RP89 cell line), originating from one patient and obtained from Dr. Diogenes (University of Texas, USA), we recently created a pool of DPSC and SCAP from 10 different patients. These cell pools will be fully characterized by cell-surface markers analysis, by differentiation potential and by stem cell gene expression and used as an internal standard for all of our work. Such efforts will allow us to have a much genetically diverse and relevant cell source.
For the regenerative approach, cells must be properly delivered. The design of an “ideal” bioactive matrix is thus necessary. This one would be biocompatible, injectable and would ideally resemble the native pulp tissues in terms of mechanical properties and allow cell invasion, survival and proliferation. Therefore, we will test in vitro different hydrogels, which will be provided through different collaborations (Prof. Anne des Rieux, UCL; Prof. Berit Strand, NTNU, Norway; Prof. Patrick Henriet, UCL; Prof. Christine Dupont, UCL).
Fibrine/Alginate hydrogels are currently being investigated, testing for DPSC attachment and viability on the medium-term. Once an « ideal » bioactive matrix is designed, it will be implemented in two different regenerative strategies and tested in vitro/in vivo:
Dental pulp stem cell homing from residual dental pulp tissue in case of partial pulp tissue removal, through the injection of a bioactive scaffold loaded with factors like SDF1, bFGF and TGF-B (Figure 1, item 3’)
Exogenous dental pulp stem cell delivery in case of complete pulp tissue loss, to regenerate the lost tissue volume into a vascularized, innervated and functional de-novo dentin-pulp complex (Figure 1, item 4’).
The tools currently available to the dentists for diagnostics purposes are limited. The extent of pulp and periapical inflammation are currently evaluated using mechanical and thermal stimuli, which are not enough reliable and have low level of evidence. A promising approach to better diagnose the inflammatory conditions of the pulp and periapical tissues in vital pulp therapy and endodontic treatments is to quantify the level of expression of pro-inflammatory and pro-resolution molecules. We are developing an in vitro and in vivo model to achieve these goals, in collaboration with Pr. Yusuke Takahashi (University of Osaka, Japan). Future strategies could be planned based on in-situ readings of such levels, leading to improved diagnostics and better patient care.