Antioxidant capacity of an ethanolic extract of Elaeagnus x submacrophylla Servett. leaves.

In this study, we investigated the ethanolic extraction of the leaves of a very common but little studied plant species, Elaeagnus x submacrophylla Servett. and the opportunity of generating an antioxidant ingredient. The phytochemical profile of an ethanolic extract is also described here using gas chromatography and ultra-performance liquid chromatography, both combined with mass spectrometry (GC-MS and UPLC-MS), highlighting the presence of flavonoids, saponins, triterpenoids and a set of volatile compounds. Through in vitro assays (DPPH, ABTS, ORAC), the free radical scavenging capacity of the ingredient was then investigated (from 0.25 to 1.75 mmol TE/g) and compared with well-known standard antioxidants (BHT, gallic acid, quercetin, Trolox and vitamin C). In addition, in cellulo antioxidant capacity was performed using mice fibroblasts, revealing an activity equivalent to 50 mg/L of quercetin when tested the ethanolic extract in the concentration range of 50-300 mg/L, suggesting a synergistic combination effect of the identified phytochemicals. These results support the use of Elaeagnus x submacrophylla as a source of antioxidant ingredients.

Exploring the role of polymers to overcome ongoing challenges in the field of extracellular vesicles.

Extracellular vesicles (EVs) are naturally occurring nanoparticles released from all eucaryotic and procaryotic cells. While their role was formerly largely underestimated, EVs are now clearly established as key mediators of intercellular communication. Therefore, these vesicles constitute an attractive topic of study for both basic and applied research with great potential, for example, as a new class of biomarkers, as cell-free therapeutics or as drug delivery systems. However, the complexity and biological origin of EVs sometimes complicate their identification and therapeutic use. Thus, this rapidly expanding research field requires new methods and tools for the production, enrichment, detection, and therapeutic application of EVs. In this review, we have sought to explain how polymer materials actively contributed to overcome some of the limitations associated to EVs. Indeed, thanks to their infinite diversity of composition and properties, polymers can act through a variety of strategies and at different stages of EVs development. Overall, we would like to emphasize the importance of multidisciplinary research involving polymers to address persistent limitations in the field of EVs.

Rapid communication: insights into the role of extracellular vesicles during Auger radioimmunotherapy.

PURPOSE: Non-targeted effects, including bystander and systemic effects, play a crucial role during Auger targeted radionuclide therapy. Here, we investigated whether small extracellular vesicles (sEVs) produced by irradiated cells could contribute to the bystander cytotoxic effects in vitro and also to therapeutic efficacy in vivo, after their injection in tumor xenografts. MATERIALS AND METHODS: B16F10 melanoma donor cells were exposed to radiolabeled antibodies (Auger radioimmunotherapy, RIT) for 48 h or to X-rays (donor cells). Then, donor cells were incubated with fresh medium for 2 h to prepare conditioned medium (CM) that was transferred onto recipient cells for bystander effect assessment, or used for sEVs enrichment. Resulting sEVs were incubated in vitro with recipient cells for determining bystander cytotoxicity, or injected in B16F10 melanoma tumors harbored by athymic and C57BL/6 mice. RESULTS: In vitro analysis of bystander cytotoxic effects showed that CM killed about 30-40% of melanoma cells. SEVs isolated from CM contributed to this effect. Moreover, the double-stranded DNA (dsDNA) content was increased in sEVs isolated from CM of exposed cells compared to control (not exposed), but the difference was significant only for the X-ray condition. These results were supported by immunodetection of cytosolic dsDNA in donor cells, a phenomenon that should precede dsDNA enrichment in sEVs. However, sEVs cytotoxicity could not be detected in vivo. Indeed, in athymic and in immunocompetent mice that received four intratumoral injections of sEVs (1/day), tumor growth was not delayed compared with untreated controls. Tumor growth was slightly (not significantly) delayed in immunocompetent mice treated with sEVs from X-ray-exposed cells, and significantly with sEVs purified from CM collected after 48 h of incubation. These results highlight the need to determine the optimal conditions, including radiation absorbed dose and sEVs collection time, to obtain the strongest cytotoxic effects. CONCLUSIONS: This study demonstrates that sEVs could play a role during Auger RIT through bystander effects in vitro. No systemic effects were observed in vivo, under our experimental conditions. However, X-rays experiments showed that sEVs collection time might be influencing the nature of sEVs, a parameter that should also be investigated during Auger RIT.

Novel trehalose-based excipients for stabilizing nebulized anti-SARS-CoV-2 antibody.

COVID-19 is caused by the infection of the lungs by SARS-CoV-2. Monoclonal antibodies, such as sotrovimab, showed great efficiency in neutralizing the virus before its internalization by lung epithelial cells. However, parenteral routes are still the preferred route of administration, even for local infections, which requires injection of high doses of antibody to reach efficacious concentrations in the lungs. Lung administration of antibodies would be more relevant requiring lower doses, thus reducing the costs and the side effects. But aerosolization of therapeutic proteins is very challenging, as the different processes available are harsh and trigger protein aggregation and conformational changes. This decreases the efficiency of the treatment, and can increase its immunogenicity. To address those issues, we developed a series of new excipients composed of a trehalose core, a succinyl side chain and a hydrophobic carbon chain (from 8 to 16 carbons). Succinylation increased the solubility of the excipients, allowing their use at relevant concentrations for protein stabilization. In particular, the excipient with 16 carbons (C16TreSuc) used at 5.6 mM was able to preserve colloidal stability and antigen-binding ability of sotrovimab during the nebulization process. It could also be used as a cryoprotectant, allowing storage of sotrovimab in a lyophilized form during weeks. Finally, we demonstrated that C16TreSuc could be used as an excipient to stabilize antibodies for the treatment against COVID-19, by in vitro and in vivo assays. The presence of C16TreSuc during nebulization preserved the neutralization capacity of sotrovimab against SARS-CoV-2 in vitro; an increase of its efficacy was even observed, compared to the non-nebulized control. The in vivo study also showed the wide distribution of sotrovimab in mice lungs, after nebulization with 5.6 mM of excipient. This work brings a solution to stabilize therapeutic proteins during storage and nebulization, making pulmonary immunotherapy possible in the treatment of COVID-19 and other lung diseases.

Size-tunable lipid vectors for controlled local delivery of siRNA from gene activated matrix.

Tissue engineering aims to restore or replace different types of biological tissues through the association of cells, biologic factors and biomaterials. Currently, stem cells arise as a major cell source for many therapeutic indications, and their association with 3D scaffolds allow increasing regenerative medicine efficiency. In this context, the use of RNA interference to enhance or control stem cell differentiation into the desired phenotype appears as a promising strategy. However, achieving high transfection efficiency of cells in a 3D structure requires the use of a vector allowing for the spatiotemporally controlled release of the genetic material from these scaffolds. In this study, we report a new siRNA nanovector, called solvent exchange lipoplexe formulation (SELF), which has a tunable size, is stable over time in cell culture conditions and possess a high efficiency to transfect primary human mesenchymal stromal cells (hMSC). We associated SELFs with porous 3D collagen microspheres and demonstrated that the loading capacity and release kinetics were different depending on the size of the associated SELF. Interestingly, these different release profiles resulted in differences in the transfection kinetics of hMSCs. This original and unique type of gene activated matrix, with adaptable release kinetics, could be of interest for long-term and/or sequential transfection profiles of stem cells in 3D culture. STATEMENT OF SIGNIFICANCE: This work combines the use of human mesenchymal stromal cell (hMSC) and gene therapy for tissue engineering. Here, a gene-activated matrix was elaborated with collagen microspheres supporting hMSCs and acting as a reservoir for transfection vectors. This injectable GAM allows for the local and sustained delivery of nucleic acids, hence long-lasting transfection of the supported cells. With the original synthesis protocol presented herein, the size of the nanocarriers can be easily adapted, resulting in different siRNA release profiles from the microspheres. Most interestingly, different siRNA release profiles gave rise to different cell transfection profiles as assessed by the downregulation of a target gene. This highlights the versatility of the system and its suitability for various pathophysiological needs in regenerative medicine.

Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France".

Extracellular vesicles (EV) are emergent therapeutic effectors that have reached clinical trial investigation. To translate EV-based therapeutic to clinic, the challenge is to demonstrate quality, safety, and efficacy, as required for any medicinal product. EV research translation into medicinal products is an exciting and challenging perspective. Recent papers, provide important guidance on regulatory aspects of pharmaceutical development, defining EVs for therapeutic applications and critical considerations for the development of potency tests. In addition, the ISEV Task Force on Regulatory Affairs and Clinical Use of EV-based Therapeutics as well as the Exosomes Committee from the ISCT are expected to contribute in an active way to the development of EV-based medicinal products by providing update on the scientific progress in EVs field, information to patients and expert resource network for regulatory bodies. The contribution of our work group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France", created in 2020, can be positioned in complement to all these important initiatives. Based on complementary scientific, technical, and medical expertise, we provide EV-specific recommendations for manufacturing, quality control, analytics, non-clinical development, and clinical trials, according to current European legislation. We especially focus on early phase clinical trials concerning immediate needs in the field. The main contents of the investigational medicinal product dossier, marketing authorization applications, and critical guideline information are outlined for the transition from research to clinical development and ultimate market authorization.

Interest of extracellular vesicles in regards to lipid nanoparticle based systems for intracellular protein delivery.

Compared to chemicals that continue to dominate the overall pharmaceutical market, protein therapeutics offer the advantages of higher specificity, greater activity, and reduced toxicity. While nearly all existing therapeutic proteins were developed against soluble or extracellular targets, the ability for proteins to enter cells and target intracellular compartments can significantly broaden their utility for a myriad of exiting targets. Given their physical, chemical, biological instability that could induce adverse effects, and their limited ability to cross cell membranes, delivery systems are required to fully reveal their biological potential. In this context, as natural protein nanocarriers, extracellular vesicles (EVs) hold great promise. Nevertheless, if not present naturally, bringing an interest protein into EV is not an easy task. In this review, we will explore methods used to load extrinsic protein into EVs and compare these natural vectors to their close synthetic counterparts, liposomes/lipid nanoparticles, to induce intracellular protein delivery.

Melanotransferrin is efficiently sorted on the surface of exosomes secreted by melanoma cells.

Cutaneous melanoma is the most lethal type of skin cancer. Early detection is crucial to improve the outcome of melanoma patients. The identification of noninvasive prognostic biomarkers for the follow-up of melanoma patients is still in demand for clinical use. We show here that exosomal melanotransferrin fulfills the biomarker characteristics required to meet this demand. Melanotransferrin is typically overexpressed in melanoma cells compared to other cell types - including cancer cells - and is efficiently sorted and secreted with nanovesicles, or so-called exosomes, due to its membrane-anchoring by a glycosylphosphatidylinositol. Melanotransferrin is exposed on the surface of exosomes and is accessible for antibody recognition. An ELISA was set up to quantify melanotransferrin after immobilization of nanovesicles through the exosomal constituent tetraspanins CD63. Melanotransferrin was detected using a low number of exosomes purified from melanoma cell line cultures, and melanotransferrin detection was abolished by phosphatidylinositol-specific phospholipase C treatment. This exosomal melanotransferrin ELISA was able to discriminate an equal number of assayed exosomes purified from two different melanoma cell lines (A-375 vs. SK-MEL-28). Moreover, plasma samples from patients with melanoma and noncancer disease were assayed using this ELISA and elevated levels of exosomal melanotransferrin were seen in the plasma of patients with melanoma. We propose that exosomal melanotransferrin should be assessed as a potential melanoma biomarker.

PySHS: Python Open Source Software for Second Harmonic Scattering.

The PySHS package is a new python open source software tool which simulates the second harmonic scattering (SHS) of different kinds of colloidal nano-objects in various experimental configurations. This package is able to compute polarizations resolved at a fixed scattered angle or angular distribution for different polarization configurations. This article presents the model implemented in the PySHS software and gives some computational examples. A comparison between computational results and experimental data concerning molecular dye intercalated inside liposomes membrane is presented to illustrate the possibilities with PySHS.

Degradable double hydrophilic block copolymers and tripartite polyionic complex micelles thereof for small interfering ribonucleic acids (siRNA) delivery.

Polymer vectors for gene therapy have been largely investigated as an alternative to viral vectors. In particular, double hydrophilic block copolymers (DHBCs) have shown potential in this domain, but to date studies mainly focus on non-degradable copolymers, which may be a restriction for further development. To overcome this limitation, we synthesized a DHBC (PEG43-b-PCL12(COOH)6.5) composed of a poly(ethylene glycol) (PEG) non-ionic and bioeliminable block and a degradable carboxylic acid-functionalized poly(ε-caprolactone) (PCL) block. The potential of this DHBC as an original vector for small interfering ribonucleic acids (siRNA) to formulate tripartite polyionic complex (PIC) micelles with poly(lysine) (PLL) was evaluated. We first studied the impact of the charge ratio (R) on the size and the zeta potential of the resulting micelles. With a charge ratio R = 1, one formulation with optimized physico-chemical properties showed the ability to complex 75% of siRNA. We showed a stability of the micelles at pH 7.4 and a disruption at pH 5, which allowed a pH-triggered siRNA release and proved the pH-stimuli responsive character of the tripartite micelles. In addition, the tripartite PIC micelles were shown to be non-cytotoxic below 40 µg/mL. The potential of these siRNA vectors was further evaluated in vitro: it was found that the tripartite PIC micelles allowed siRNA internalization to be 3 times higher than PLL polyplexes in murine mesenchymal stem cells, and were able to transfect human breast cancer cells. Overall, this set of data pre-validates the use of degradable DHBC as non-viral vectors for the encapsulation and the controlled release of siRNA, which may therefore constitute a sound alternative to non-degradable and/or cytotoxic polycationic vectors.

Post-production modifications of murine mesenchymal stem cell (mMSC) derived extracellular vesicles (EVs) and impact on their cellular interaction.

In regards to their key role in intercellular communication, extracellular vesicles (EVs) have a strong potential as bio-inspired drug delivery systems (DDS). With the aim of circumventing some of their well-known issues (production yield, drug loading yield, pharmacokinetics), we specifically focused on switching the biological vision of these entities to a more physico-chemical one, and to consider and fine-tune EVs as synthetic vectors. To allow a rational use, we first performed a full physico-chemical (size, concentration, surface charge, cryoTEM), biochemical (western blot, proteomics, lipidomics, transcriptomics) and biological (cell internalisation) characterisation of murine mesenchymal stem cell (mMSC)-derived EVs. A stability study based on evaluating the colloidal behaviour of obtained vesicles was performed in order to identify optimal storage conditions. We evidenced the interest of using EVs instead of liposomes, in regards to target cell internalisation efficiency. EVs were shown to be internalised through a caveolae and cholesterol-dependent pathway, following a different endocytic route than liposomes. Then, we characterised the effect of physical methods scarcely investigated with EVs (extrusion through 50 nm membranes, freeze-drying, sonication) on EV size, concentration, structure and cell internalisation properties. Our extensive characterisation of the effect of these physical processes highlights their promise as loading methods to make EVs efficient delivery vehicles.

Near-Infrared Optical Imaging of Nucleic Acid Nanocarriers In Vivo.

Noninvasive, real-time optical imaging methods are well suited to follow the in vivo distribution of nucleic acid nanocarriers, their dissociation and the resulting gene expression or inhibition. Indeed, most small animal imaging devices are performing bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid, and cost-effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks.Here we help the reader in choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for FRET assays, imaging of reporter genes as well as nanocarriers for in vivo gene and siRNA delivery. In addition, we present the labeling methods of these macromolecules, and of their payload and the protocols to detect them using bioluminescence and NIR fluorescence imaging in mice.

Fluorescent Biosensor for Detection of the R248Q Aggregation-Prone Mutant of p53.

The p53 tumour suppressor and guardian of the genome undergoes missense mutations that lead to functional inactivation in 50 % of human cancers. These mutations occur mostly in the DNA-binding domain of the protein, and several of these result in conformational changes that lead to amyloid-like protein aggregation. Herein, we describe a fluorescent biosensor that reports on the R248Q mutant of p53 in vitro and in living cells, engineered through conjugation of an environmentally sensitive probe onto a peptide derived from the primary aggregation segment of p53. This biosensor was characterised both in vitro and by means of fluorescence microscopy following facilitated delivery into cultured cells. It is shown that this biosensor preferentially reports on the p53 R248Q mutant in the PC9 lung cancer cell line compared with other lung cancer cell lines harbouring either wild-type or no p53.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair.

PLGA-based microcarriers induce mesenchymal stem cell chondrogenesis and stimulate cartilage repair in osteoarthritis.

In the present study, we aimed at evaluating the ability of novel PLGA-P188-PLGA-based microspheres to induce the differentiation of mesenchymal stem/stromal cells (MSC) into chondrocytes. To this aim, we tested microspheres releasing TGFß3 (PAM-T) in vitro and in situ, in a pathological osteoarthritic (OA) environment. We first evaluated the chondrogenic differentiation of human MSCs seeded onto PAM-T in vitro and confirmed the up-regulation of chondrogenic markers while the secretome of the cells was not changed by the 3D environment. We then injected human MSC seeded onto PAM-T in the knee joints of mice with collagenase-induced OA. After 6 weeks, histological analysis revealed that formation of a cartilage-like tissue occurred at the vicinity of PAM-T that was not observed when MSCs were seeded onto PAM. We also noticed that the endogenous articular cartilage was less degraded. The extent of cartilage protection was further analysed by confocal laser microscopy. When MSCs seeded onto PAM-T were injected early after OA induction, protection of cartilage against degradation was evidenced and this effect was associated to a higher survival of MSCs in presence of TGFß3. This study points to the interest of using MSCs seeded onto PAM for cartilage repair and stimulation of endogenous cartilage regeneration.

Scaffolds for Controlled Release of Cartilage Growth Factors.

In recent years, cell-based therapies using adult stem cells have attracted considerable interest in regenerative medicine. A tissue-engineered construct for cartilage repair should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable and biocompatible poly (D,L-lactide-co-glycolide acid) (PLGA), are a unique system which combines these properties in an adaptable and simple microdevice. This device relies on nanoprecipitation of proteins encapsulated in polymeric microspheres with a solid in oil in water emulsion-solvent evaporation process, and their subsequent coating with extracellular matrix protein molecules. Here, we describe their preparation process, and some of their characterization methods for an application in cartilage tissue engineering.

New PLGA-P188-PLGA matrix enhances TGF-ß3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells.

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest in various clinical applications because it allows cell implantation through minimally invasive surgical procedures. In case of cartilage repair, a tissue engineered construct should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable poly(d,l-lactide-co-glycolide acid) (PLGA), are a unique system, which combines these properties in an adaptable and simple microdevice. However, a limitation of such scaffold is low and incomplete protein release that occurs using the hydrophobic PLGA based microspheres. To circumvent this problem, we developed a novel formulation of polymeric PAMs containing a P188 poloxamer, which protects the protein from denaturation and may positively affect chondrogenesis. This poloxamer was added as a free additive for protein complexation and as a component of the scaffold covalently linked to PLGA. This procedure allows getting a more hydrophilic scaffold but also retaining the protective polymer inside the microcarriers during their degradation. The novel PLGA-P188-PLGA PAMs presenting a fibronectin-covered surface allowed enhanced MSC survival and proliferation. When engineered with TGFß3, they allowed the sustained release of 70% of the incorporated TGF-ß3 over time. Importantly, they exerted superior chondrogenic differentiation potential compared to previous FN-PAM-PLGA-TGF-ß3, as shown by an increased expression of specific cartilage markers such as cartilage type II, aggrecan and COMP. Therefore, this microdevice represents an efficient easy-to-handle and injectable tool for cartilage repair.

DNA nanocarriers for systemic administration: characterization and in vivo bioimaging in healthy mice.

We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI), revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.Molecular Therapy - Nucleic Acids (2013) 2, e64; doi:10.1038/mtna.2012.56; published online 8 January 2013.