Stem cells from human apical papilla decrease neuro-inflammation and stimulate oligodendrocyte progenitor differentiation via activin-A secretion

Secondary damage following spinal cord injury leads to non-reversible lesions and hampering of the reparative process. The local production of pro-inflammatory cytokines such as TNF-α can exacerbate these events. Oligodendrocyte death also occurs, followed by progressive demyelination leading to significant tissue degeneration. Dental stem cells from human apical papilla (SCAP) can be easily obtained at the removal of an adult immature tooth. This offers a minimally invasive approach to re-use this tissue as a source of stem cells, as compared to biopsying neural tissue from a patient with a spinal cord injury. We assessed the potential of SCAP to exert neuroprotective effects by investigating two possible modes of action: modulation of neuro-inflammation and oligodendrocyte progenitor cell (OPC) differentiation. SCAP were co-cultured with LPS-activated microglia, LPS-activated rat spinal cord organotypic sections (SCOS), and LPS-activated co-cultures of SCOS and spinal cord adult OPC. We showed for the first time that SCAP can induce a reduction of TNF-α expression and secretion in inflamed spinal cord tissues and can stimulate OPC differentiation via activin-A secretion. This work underlines the potential therapeutic benefits of SCAP for spinal cord injury repair

Injectable alginate hydrogel loaded with GDNF promotes functional recovery in a hemisection model of spinal cord injury

We hypothesized that local delivery of GDNF in spinal cord lesion via an injectable alginate hydrogel gelifying in situ would support spinal cord plasticity and functional recovery. The GDNF release from the hydrogel was slowed by GDNF encapsulation in microspheres compared to non-formulated GDNF (free GDNF). When injected in a rat spinal cord hemisection model, more neurofilaments were observed in the lesion when the rats were treated with free GDNF-loaded hydrogels. More growing neurites were detected in the tissues surrounding the lesion when the animals were treated with GDNF microsphere-loaded hydrogels. Intense GFAP (astrocytes), low III tubulin (neural cells) and RECA-1 (endothelial cells) stainings were observed for non-treated lesions while GDNF-treated spinal cords presented less GFAP staining and more endothelial and nerve fiber infiltration in the lesion site. The animals treated with free GDNF-loaded hydrogel presented superior functional recovery compared with the animals treated with the GDNF microsphere-loaded hydrogels and non-treated animals

An improved in vitro model of human M cells as a useful tool to study nanoparticle transport across the intestinal epithelium

Recently, the progresses in the field of biotechnologies have led to the development of many drugs based on peptides and proteins. These new drugs have been traditionally delivered by parenteral administration. However, aiming to improve the patient quality of life, as well as the compliance, more and more efforts have been dedicated to the development of mucosal formulations as an alternative to parenteral administration. The most popular mucosal route is the oral administration. The intestinal mucosa is composed of an epithelium which consists in an effective barrier against foreign material (particles, microorganisms) and Organized Mucosa Associated Lymphoid Tissues (O-MALT) called also GALT (Gut Associated Lymphoid Tissues) which are specialized antigen sampling sites. The GALT function is mainly performed by membranous epithelial M cells. The most interesting characteristic of these M cells is their increased ability to transcytose particles. The objective of this thesis was to develop a more robust and reliable M cell model and to study how nanoparticles with different surface characteristics and a therapeutic peptide in free and nanoparticulate form were transported in this model as compared to a reference model of absorptive cells of the intestinal epithelium. An in vitro model of M like cells in the Follicle Associated Epithelium (FAE) was used. The importance of the nanoparticle size, concentration, surface properties, as well as the influence of proteins in the apical medium has been first enlightened. The optimal nanoparticle size is about 200 nm, and hydrophobic nanoparticles are better transported than less hydrophobic ones. The transport is concentration-dependent and the presence of some proteins in the apical medium seems to inhibit the nanoparticle transport. Then, the in vitro model has been improved, being more physiological (presence of B lymphocytes within the cell monolayers), more reproducible and more robust (90 % of model functional). The presence of M-like cells has been demonstrated, using different techniques and the percentage of M-like cells in cell monolayers has been estimated, for the first time in an in vitro model of the FAE, at 15-30%. Mechanistic studies performed with 0.2 µm polystyrene model nanoparticles allowed to conclude that the transport of these nanoparticles occurred via a transcellular, energy-dependent mechanism, probably by macropinocytosis. The transport of helodermin, a model therapeutic peptide, through intestinal barrier was investigated. Encapsulation in PEG-PLA:PLGA {poly(ethylene glycol)-co-poly(lactic acid): ploy(lactic-co-glycolic acid)} nanoparticles protected the peptide from degradation. M cells increased the transport of nanoparticles containing helodermin. At last, the transport of different type of nanoparticles by M cells was compared. Chitosan (CS) nanoparticles appeared as the most efficient delivery system but were not M cells specific. Although less efficient than CS nanoparticles, pegylated nanoparticles were more transported by M-like cells than PLGA nanoparticles. Therefore, even it remains difficult to elicit the optimal formulation for M cells uptake, pegylated nanoparticles seem the optimal choice. In conclusion, this thesis has contributed to produce, settle and characterize a functional and reliable in vitro model of the human FAE, providing a tool to evaluate the role of M cells on drug delivery. This model has allowed to underline the influence of various parameters on the nanoparticle transport by M-like cells, as well as to compare transcytosis of different carrier. It has also put in evidence the influence of M-like cells on the delivery of therapeutic peptide across the epithelial barrier. In the future, this model could be used in the context of local drug delivery and oral vaccination studies.Thèse de doctorat en sciences pharmaceutiques (FARM 3)--UCL, 200

Innovative drug delivery strategies to the CNS for the treatment of multiple sclerosis.

Disorders of the central nervous system (CNS), such as multiple sclerosis (MS) represent a great emotional, financial and social burden. Despite intense efforts, great unmet medical needs remain in that field. MS is an autoimmune, chronic inflammatory demyelinating disease with no curative treatment up to date. The current therapies mostly act in the periphery and seek to modulate aberrant immune responses as well as slow down the progression of the disease. Some of these therapies are associated with adverse effects related partly to their administration route and show some limitations due to their rapid clearance and inability to reach the CNS. The scientific community have recently focused their research on developing MS therapies targeting different processes within the CNS. However, delivery of therapeutics to the CNS is mainly limited by the presence of the blood-brain barrier (BBB). Therefore, there is a pressing need to develop new drug delivery strategies that ensure CNS availability to capitalize on identified therapeutic targets. Several approaches have been developed to overcome or bypass the BBB and increase delivery of therapeutics to the CNS. Among these strategies, the use of alternative routes of administration, such as the nose-to-brain (N2B) pathway, offers a promising non-invasive option in the scope of MS, as it would allow a direct transport of the drugs from the nasal cavity to the brain. Moreover, the combination of bioactive molecules within nanocarriers bring forth new opportunities for MS therapies, allowing and/or increasing their transport to the CNS. Here we will review and discuss these alternative administration routes as well as the nanocarrier approaches useful to deliver drugs for MS

Mechanisms of transport of polymeric and lipidic nanoparticles across the intestinal barrier.

Unraveling the mechanisms of nanoparticle transport across the intestinal barrier is essential for designing more efficient nanoparticles for oral administration. The physicochemical parameters of the nanoparticles (e.g., size, surface charge, chemical composition) dictate nanoparticle fate across the intestinal barrier. This review aims to address the most important findings regarding polymeric and lipidic nanoparticle transport across the intestinal barrier, including the evaluation of critical physicochemical parameters of nanoparticles that affect nanocarrier interactions with the intestinal barrier

Fibrin hydrogels for non-viral vector delivery in vitro

Fibrin based hydrogels have been employed in vitro as a scaffold to promote tissue formation and investigate underlying molecular mechanisms. These hydrogels support a variety of cellular processes, and are being developed to enhance the presentation of biological cues, or to tailor the biological cues for specific tissues. The presentation of these cues could alternatively be enhanced through gene delivery, which can be employed to induce the expression of tissue inductive factors in the local environment. This report investigates gene delivery within fibrin hydrogels for two in vitro models of tissue growth: i) cell encapsulation within and ii) cell seeding onto the hydrogel. Naked plasmid and lipoplexes can be efficiently entrapped within the hydrogel, and after 1 day in solution more than 70% of the entrapped DNA is retained within the gel, with a sustained release observed for at least 19 days. Encapsulated lipoplexes did not aggregate and retained their original size. Transgene expression in vitro by delivery of lipoplexes was a function of the fibrinogen and DNA concentration. For encapsulated cells, all cells had intracellular plasmid and transgene expression persisted for at least 10 days, with maximal levels achieved at day 1. For cell infiltration, expression levels were less than those observed for encapsulation, and expression increased throughout the culture period. The increasing expression levels suggest that lipoplexes retain their activity after encapsulation; however, interactions between fibrin and the lipoplexes likely limit internalization. The inclusion of non-viral vectors into fibrin-based hydrogels can be employed to induce transgene expression of encapsulated and infiltrating cells, and may be employed with in vitro models of tissue growth to augment the intrinsic bioactivity of fibrin

Rapid Serum-Free Isolation of Oligodendrocyte Progenitor Cells from Adult Rat Spinal Cord.

Oligodendrocyte progenitor cells (OPCs) play a pivotal role in both health and disease within the central nervous system, with oligodendrocytes, arising from resident OPCs, being the main myelinating cell type. Disruption in OPC numbers can lead to various deleterious health defects. Numerous studies have described techniques for isolating OPCs to obtain a better understanding of this cell type and to open doors for potential treatments of injury and disease. However, the techniques used in the majority of these studies involve several steps and are time consuming, with current culture protocols using serum and embryonic or postnatal cortical tissue as a source of isolation. We present a primary culture method for the direct isolation of functional adult rat OPCs, identified by neuron-glial antigen 2 (NG2) and platelet derived growth factor receptor alpha (PDGFrα) expression, which can be obtained from the adult spinal cord. Our method uses a simple serum-free cocktail of 3 growth factors - FGF2, PDGFAA, and IGF-I, to expand adult rat OPCs in vitro to 96% purity. Cultured cells can be expanded for at least 10 passages with very little manipulation and without losing their phenotypic progenitor cell properties, as shown by immunocytochemistry and RT-PCR. Cultured adult rat OPCs also maintain their ability to differentiate into GalC positive cells when incubated with factors known to stimulate their differentiation. This new isolation method provides a new source of easily accessible adult stem cells and a powerful tool for their expansion in vitro for studies aimed at central nervous system repair

Fate of polymeric nanocarriers for oral drug delivery

This review will focus on two polymeric nanocarriers: nanoparticles and micelles that have been studied for oral drug delivery at preclinical level. Their potential for oral drug delivery will first be illustrated. Then their mechanisms of uptake and their fate after oral delivery will be discussed. Future directions for oral delivery with nanocarriers will be analyzed with a special emphasis on optimal properties. The recent advances highlight the need to tune and to control their design with a good balance in their physicochemical properties and suggest that more sophisticated nanosystems will be developed for the oral delivery of drugs, biopharmaceuticals and vaccines, thanks to (i) the development of biocompatible polymers with tailored properties for oral drug delivery and formulation of nanocarriers, (ii) the understanding of cellular uptake mechanisms of polymeric nanocarriers, (iii) the novel techniques to study the fate of nanocarriers, polymers and drugs in the body and (iv) the identification of new ligands for targeted oral delivery. Major recent advances Recent advances in the (i) development of biocompatible polymers with tailored properties for oral drug delivery and nanocarrier formulation, (ii) the understanding of cellular uptake mechanisms of polymeric nanocarriers (iii) the new techniques to study fate of nanocarriers, polymers and drugs in the body and (iv) the identification of new ligands for targeted oral delivery have promoted the development of novel polymeric carriers for the oral delivery of drugs, biopharmaceuticals and vaccines

Mechanical properties and lipase-catalyzed biodegradation of alpha-methyl, epsilon-caprolactone/epsilon-caprolactone copolymers obtained by chemical modification of poly(epsilon-caprolactone)

Novel (epsilon-caprolactone)-based copolymers of different compositions were synthesized by allowing methyl iodide to react with the polycarbanion that resulted from the action of lithium diisopropylamide on poly(epsilon-caprolactone) in THF at 70°C under argon atmosphere. The copolymers were characterized by various techniques, namely 1H nuclear magnetic resonance, size exclusion chromatography, differential scanning calorimetry, x-ray diffraction and viscoelasticimetry. They were submitted to hydrolytic and lipase-catalyzed enzymatic degradation in comparison with genuine PCL. The Young modulus depended on the degree of methylation. In contrast, loss angle and glass transition temperature did not depend on this parameter. It is shown that the lipase-catalyzed degradation of methylated PCL is much slower than in the case of genuine PCL