Roadmap on nanomedicine.

Since the launch of the Alliance for Nanotechnology in Cancer by the National Cancer Institute in late 2004, several similar initiatives have been promoted all over the globe with the intention of advancing the diagnosis, treatment and prevention of cancer in the wake of nanoscience and nanotechnology. All this has encouraged scientists with diverse backgrounds to team up with one another, learn from each other, and generate new knowledge at the interface between engineering, physics, chemistry and biomedical sciences. Importantly, this new knowledge has been wisely channeled towards the development of novel diagnostic, imaging and therapeutic nanosystems, many of which are currently at different stages of clinical development. This roadmap collects eight brief articles elaborating on the interaction of nanomedicines with human biology; the biomedical and clinical applications of nanomedicines; and the importance of patient stratification in the development of future nanomedicines. The first article reports on the role of geometry and mechanical properties in nanomedicine rational design; the second articulates on the interaction of nanomedicines with cells of the immune system; and the third deals with exploiting endogenous molecules, such as albumin, to carry therapeutic agents. The second group of articles highlights the successful application of nanomedicines in the treatment of cancer with the optimal delivery of nucleic acids, diabetes with the sustained and controlled release of insulin, stroke by using thrombolytic particles, and atherosclerosis with the development of targeted nanoparticles. Finally, the last contribution comments on how nanomedicine and theranostics could play a pivotal role in the development of personalized medicines. As this roadmap cannot cover the massive extent of development of nanomedicine over the past 15 years, only a few major achievements are highlighted as the field progressively matures from the initial hype to the consolidation phase.

Nanostructured lipid carriers accumulate in atherosclerotic plaques of ApoE-/- mice.

Nanostructured lipid carriers (NLC) might represent an interesting approach for the identification and targeting of rupture-prone atherosclerotic plaques. In this study, we evaluated the biodistribution, targeting ability and safety of 64Cu-fonctionalized NLC in atherosclerotic mice. 64Cu-chelating-NLC (51.8±3.1 nm diameter) with low dispersity index (0.066±0.016) were produced by high pressure homogenization at tens-of-grams scale. 24 h after injection of 64Cu-chelated particles in ApoE-/- mice, focal regions of the aorta showed accumulation of particles on autoradiography that colocalized with Oil Red O lipid mapping. Signal intensity was significantly greater in aortas isolated from ApoE-/- mice compared to wild type (WT) control (8.95 [7.58, 10.16]×108 vs 4.59 [3.11, 5.03]×108 QL/mm2, P < 0.05). Moreover, NLC seemed safe in relevant biocompatibility studies. NLC could constitute an interesting platform with high clinical translation potential for targeted delivery and imaging purposes in atherosclerosis.

Pharmaceutical Development and Safety Evaluation of a GMP-Grade Fucoidan for Molecular Diagnosis of Cardiovascular Diseases.

The adhesion molecule P-selectin is present on the cell surface of both activated endothelium and activated platelets. The present study describes the pharmaceutical development, safety evaluation, and preclinical efficacy of a micro-dosed radiotracer. The macromolecular nanoscale assembly consisted of a natural compound made of a sulfated fucose-rich polysaccharides (fucoidan) and a radionuclide (technetium-99m) for the detection of P-selectin expression in cardiovascular diseases. After extraction and fractionation from brown seaweeds, the good manufacturing practice (GMP) production of a low molecular weight (LMW) fucoidan of 7 kDa was achieved and full physicochemical characterization was performed. The regulatory toxicology study in rats of the GMP batch of LMW fucoidan revealed no adverse effects up to 400 µg/kg (×500 higher than the expected human dose) and pseudoallergy was not seen as well. In a myocardial ischemia-reperfusion model in rats, the GMP-grade LMW fucoidan labeled with technetium-99m detected P-selectin upregulation in vivo. The present study supports the potential of using 99mTc-fucoidan as an imaging agent to detect activated endothelium in humans.

Fucoidans in Nanomedicine.

Fucoidans are widespread cost-effective sulfated marine polysaccharides which have raised interest in the scientific community over last decades for their wide spectrum of bioactivities. Unsurprisingly, nanomedicine has grasped these compounds to develop innovative therapeutic and diagnostic nanosystems. The applications of fucoidans in nanomedicine as imaging agents, drug carriers or for their intrinsic properties are reviewed here after a short presentation of the main structural data and biological properties of fucoidans. The origin and the physicochemical specifications of fucoidans are summarized in order to discuss the strategy of fucoidan-containing nanosystems in Human health. Currently, there is a need for reproducible, well characterized fucoidan fractions to ensure significant progress.

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.

Nanomedicine for the molecular diagnosis of cardiovascular pathologies.

Predicting acute clinical events caused by atherosclerotic plaque rupture remains a clinical challenge. Anatomic mapping of the vascular tree provided by standard imaging technologies is not always sufficient for a robust diagnosis. Yet biological mechanisms leading to unstable plaques have been identified and corresponding biomarkers have been described. Nanosystems charged with contrast agents and targeted towards these specific biomarkers have been developed for several types of imaging modalities. The first systems that have reached the clinic are ultrasmall superparamagnetic iron oxides for Magnetic Resonance Imaging. Their potential relies on their passive accumulation by predominant physiological mechanisms in rupture-prone plaques. Active targeting strategies are under development to improve their specificity and set up other types of nanoplatforms. Preclinical results show a huge potential of nanomedicine for cardiovascular diagnosis, as long as the safety of these nanosystems in the body is studied in depth.

Combining magnetic nanoparticles with cell derived microvesicles for drug loading and targeting.

Inspired by microvesicle-mediated intercellular communication, we propose a hybrid vector for magnetic drug delivery. It consists of macrophage-derived microvesicles engineered to enclose different therapeutic agents together with iron oxide nanoparticles. Here, we investigated in vitro how magnetic nanoparticles may influence the vector effectiveness in terms of drug uptake and targeting. Human macrophages were loaded with iron oxide nanoparticles and different therapeutic agents: a chemotherapeutic agent (doxorubicin), tissue-plasminogen activator (t-PA) and two photosensitizers (disulfonated tetraphenyl chlorin-TPCS2a and 5,10,15,20-tetra(m-hydroxyphenyl)chlorin-mTHPC). The hybrid cell microvesicles were magnetically responsive, readily manipulated by magnetic forces and MRI-detectable. Using photosensitizer-loaded vesicles, we showed that the uptake of microvesicles by cancer cells could be kinetically modulated and spatially controlled under magnetic field and that cancer cell death was enhanced by the magnetic targeting. From the clinical editor: In this article, the authors devised a biogenic method using macrophages to produce microvesicles containing both iron oxide and chemotherapeutic agents. They showed that the microvesicles could be manipulated by magnetic force for targeting and subsequent delivery of the drug payload against cancer cells. This smart method could provide a novel way for future fight against cancer.

Purification of a low molecular weight fucoidan for SPECT molecular imaging of myocardial infarction.

Fucoidans constitute a large family of sulfated polysaccharides with several biochemical properties. A commercial fucoidan from brown algae, containing low molecular weight polysaccharidic species constituted of l-fucose, uronic acids and sulfate groups, was simply treated here with calcium acetate solution. This treatment led to a purified fraction with a yield of 45%. The physicochemical characterizations of the purified fucoidan using colorimetric assay, MALLS, dRI, FT-IR, NMR, exhibited molecular weight distributions and chemical profiles similar for both fucoidans whereas the sulfate and l-fucose contents increased by 16% and 71%, respectively. The biodistribution study in rat of both compounds labeled with 99mTc evidenced a predominant renal elimination of the purified fucoidan, but the crude fucoidan was mainly retained in liver and spleen. In rat myocardial ischemia-reperfusion, we then demonstrated the better efficiency of the purified fucoidan. This purified sulfated polysaccharide appears promising for the development of molecular imaging in acute coronary syndrome.

Microbubbles for human diagnosis and therapy.

Microbubbles (MBs) were observed for the first time in vivo as a curious consequence of quick saline injection during ultrasound (US) imaging of the aortic root, more than 50 years ago. From this serendipitous event, MBs are now widely used as contrast enhancers for US imaging. Their intrinsic properties described in this review, allow a multitude of designs, from shell to gas composition but also from grafting targeting agents to drug payload encapsulation. Indeed, the versatile MBs are deeply studied for their dual potential in imaging and therapy. As presented in this paper, new generations of MBs now opens perspectives for targeted molecular imaging along with the development of new US imaging systems. This review also presents an overview of the different therapeutic strategies with US and MBs for cancer, cardiovascular diseases, and inflammation. The overall aim is to overlap those fields in order to find similarities in the MBs application for treatment enhancement associated with US. To conclude, this review explores the new scales of MBs technologies with nanobubbles development, and along concurrent advances in the US imaging field. This review ends by discussing perspectives for the booming future uses of MBs.

REVIEW: "ISCHEMIC STROKE: From Fibrinolysis to Functional Recovery" Nanomedicine: Emerging Approaches to Treat Ischemic Stroke.

Stroke is responsible for 11% of all deaths worldwide, the majority of which are caused by ischemic strokes, thus making the need to urgently find safe and effective therapies. Today, these can be cured either by mechanical thrombectomy when the thrombus is accessible, or by intravenous injection of fibrinolytics. However, the latter present several limitations, such as potential severe side effects, few eligible patients and low rate of partial and full recovery. To design safer and more effective treatments, nanomedicine appeared in this medical field a few decades ago. This review will explain why nanoparticle-based therapies and imaging techniques are relevant for ischemic stroke management. Then, it will present the different nanoparticle types that have been recently developed to treat this pathology. It will also study the various targeting strategies used to bring nanoparticles to the stroke site, thereby limiting side effects and improving the therapeutic efficacy. Finally, this review will present the few clinical studies testing nanomedicine on stroke and discuss potential causes for their scarcity.

rtPA-loaded fucoidan polymer microbubbles for the targeted treatment of stroke.

Systemic injection of thrombolytic drugs is the gold standard treatment for non-invasive blood clot resolution. The most serious risks associated with the intravenous injection of tissue plasminogen activator-like proteins are the bleeding complication and the dose related neurotoxicity. Indeed, the drug has to be injected in high concentrations due to its short half-life, the presence of its natural blood inhibitor (PAI-1) and the fast hepatic clearance (0.9 mg/kg in humans, 10 mg/kg in mouse models). Overall, there is a serious need for a dose-reduced targeted treatment to overcome these issues. We present in this article a new acoustic cavitation-based method for polymer MBs synthesis, three times faster than current hydrodynamic-cavitation method. The generated MBs are ultrasound responsive, stable and biocompatible. Their functionalization enabled the efficient and targeted treatment of stroke, without side effects. The stabilizing shell of the MBs is composed of Poly-Isobutyl Cyanoacrylate (PIBCA), copolymerized with fucoidan. Widely studied for its targeting properties, fucoidan exhibit a nanomolar affinity for activated endothelium and activated platelets (P-selectins). Secondly, the thrombolytic agent (rtPA) was loaded onto microbubbles (MBs) with a simple adsorption protocol. Hence, the present study validated the in vivo efficiency of rtPA-loaded Fuco MBs to be over 50 % more efficient than regular free rtPA injection for stroke resolution. In addition, the relative injected rtPA grafted onto targeting MBs was 1/10th of the standard effective dose (1 mg/kg in mouse). As a result, no hemorrhagic event, BBB leakage nor unexpected tissue distribution were observed.

Enabling MedTech Translation in Academia: Redefining Value Proposition with Updated Regulations.

Academic institutions are becoming more focused on translating new technologies for clinical applications. A transition from "bench to bedside" is often described to take basic research concepts and methods to develop a therapeutic or diagnostic solution with proven evidence of efficacy at the clinical level while also fulfilling regulatory requirements. The regulatory environment is evolving in Europe with transition and grace periods for the full enforcement of the Medical Device Regulation 2017/745 (MDR), replacing the Medical Device Directive 93/42/EEC (MDD). These new guidelines increase demands for scientific, technical, and clinical data with reduced capacity in regulatory bodies creating uncertainty in future product certification. Academic translational activities will be uniquely affected by this new legislation. The barriers and threats to successful translation in academia can be overcome by strong clinical partnerships, close-industrial collaborations, and entrepreneurial programs, enabling continued product development to overcome regulatory hurdles, reassuring their foothold of medical device development.

P-selectin targeting polysaccharide-based nanogels for miRNA delivery.

Nanogels were prepared in aqueous media without the use of any organic solvent via a simple polyelectrolyte complexation method between aminated pullulan and fucoidan followed by covalent crosslinking with genipin. Homogeneously distributed genipin crosslinked nanogels (G-PECs) were obtained with a mean hydrodynamic diameter of ~155 nm and zeta potential of 0.86 ± 4.35 mV. Their capacity to bind to human activated platelets was evaluated in vitro, as well as their cytocompatibility within human endothelial cells after 1 day of incubation up to 1000 µg/mL of G-PECs (94.56 ± 7.82% of viable cells). Additional hemolysis tests support the biocompatible character of the developed nanosystems (hemolysis rate of 2.09 ± 0.06% for 1000 µg/mL of G-PECs). Under acid conditions, the surface charge of G-PECs was tuned to around ~10 mV allowing miRNA incorporation via electrostatic interactions. G-PECs were able to promote miRNA delivery inside cells, as demonstrated by fluorescence microscopy images of labelled miRNA. With further studies to demonstrate the biological activity of delivered miRNA, these nanogels could be an interesting platform for miRNA-based therapeutics in atherothrombotic-related diseases thanks to the possibility to target over-expressed P-selectin.

Fucoidan-functionalized polysaccharide submicroparticles loaded with alteplase for efficient targeted thrombolytic therapy.

Intravenous administration of fibrinolytic drugs is the standard treatment of acute thrombotic diseases. However, current fibrinolytics exhibit limited clinical efficacy because of their short plasma half-lives and might trigger hemorrhagic transformations. Therefore, it is mandatory to develop innovative nanomedicine-based solutions for more efficient and safer thrombolysis with biocompatible and biodegradable thrombus-targeted nanocarrier. Herein, fucoidan-functionalized hydrogel polysaccharide submicroparticles with high biocompatibility are elaborated by the inverse miniemulsion/crosslinking method. They are loaded with the gold standard fibrinolytic - alteplase - to direct site-specific fibrinolysis due to nanomolar interactions between fucoidan and P-selectin overexpressed on activated platelets and endothelial cells in the thrombus area. The thrombus targeting properties of these particles are validated in a microfluidic assay containing recombinant P-selectin and activated platelets under arterial and venous blood shear rates as well as in vivo. The experiments on the murine model of acute thromboembolic ischemic stroke support this product's therapeutic efficacy, revealing a faster recanalization rate in the middle cerebral artery than with free alteplase, which reduces post-ischemic cerebral infarct lesions and blood-brain barrier permeability. Altogether, this proof-of-concept study demonstrates the potential of a biomaterial-based targeted nanomedicine for the precise treatment of acute thrombotic events, such as ischemic stroke.

Nanomedicine progress in thrombolytic therapy.

Thrombotic occlusions of blood vessels are responsible for life-threatening cardiovascular disorders such as myocardial infarction, ischemic stroke, and venous thromboembolism. Current thrombolytic therapy, the injection of Plasminogen Activators (PA), is yet limited by a narrow therapeutic window, rapid drug elimination, and risks of hemorrhagic complications. Nanomedicine-based vectorization of PA protects the drug from the enzymatic degradation, improves the therapeutic outcomes, and diminishes adverse effects in preclinical models. Herein, we review the pathophysiology of arterial and venous thrombosis and summarize clinically approved PA for the treatment of acute thrombotic diseases. We examine current challenges and perspectives in the recent key research on various (lipid, polymeric, inorganic, biological) targeted nanocarriers intended for the site-specific delivery of PA. Microbubbles and ultrasound-assisted sonothrombolysis that demonstrate thrombolysis enhancement in clinical trials are further discussed. Moreover, this review features strategies for the rational design of nanocarriers for targeted thrombolysis and effective PA encapsulation in view of interactions between nanomaterials and biological systems. Overall, nanomedicine represents a valued approach for the precise treatment of acute thrombotic pathologies.

Synthesis of cationic quaternized pullulan derivatives for miRNA delivery.

Pullulan is a natural polysaccharide of potential interest for biomedical applications due to its non-toxic, non-immunogenic and biodegradable properties. The aim of this work was to synthesize cationic pullulan derivatives able to form complexes with microRNAs (miRNAs) driven by electrostatic interaction (polyplexes). Quaternized ammonium groups were linked to pullulan backbone by adding the reactive glycidyltrimethylammonium chloride (GTMAC). The presence of these cationic groups within the pullulan was confirmed by elemental analysis, Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The alkylated pullulan was able to interact with miRNA and form stable polyplexes that were characterized regarding size, zeta potential and morphology. The presence of miRNA was confirmed by agarose gel electrophoresis and UV spectrophotometry. In vitro tests on human umbilical vein endothelial cells did not show any cytotoxicity after 1 day of incubation with nanosized polyplexes up to 200 µg/mL. QA-pullulan was able to promote miRNA delivery inside cells as demonstrated by fluorescence microscopy images of labelled miRNA. In conclusion, the formation of polyplexes using cationic derivatives of pullulan with miRNA provided an easy and versatile method for polysaccharide nanoparticle production in aqueous media and could be a new promising platform for gene delivery.

Gd(DOTA)-grafted submicronic polysaccharide-based particles functionalized with fucoidan as potential MR contrast agent able to target human activated platelets.

Early detection of thrombotic events remains a big medical challenge. Dextran-based submicronic particles bearing Gd(DOTA) groups and functionalized with fucoidan have been produced via a simple and green water-in-oil emulsification/co-crosslinking process. Their capacity to bind to human activated platelets was evidenced in vitro as well as their cytocompatibility with human endothelial cells. The presence of Gd(DOTA) moieties was confirmed by elemental analysis and total reflection X-ray fluorescence (TRXF) spectrometry. Detailed characterization of particles was performed in terms of size distribution, morphology, and relaxation rates. In particular, longitudinal and transversal proton relaxivities were respectively 1.7 and 5.0 times higher than those of DOTAREM. This study highlights their potential as an MRI diagnostic platform for atherothrombosis.

Octamers and nanoparticles as hemoglobin based blood substitutes.

Progress in developing a blood substitute is aided by new biotechnologies and a better understanding of the circulatory system. For Hb based solutions, there is still a debate over the best set of fundamental parameters concerning the oxygen affinity which is correlated with the oxidation rate, the cooperativity, the transporter size, and of course the final source of material. Genetic engineering methods have helped discover novel globins, but not yet the quantity necessary for the high demand of blood transfusions. The expanding database of globin properties has indicated that certain individual parameters are coupled, such as the oxygen affinity and the oxidation rate, indicating that one must accept a compromise of the best parameters. After a general introduction of these basic criteria, we will focus on two strategies concerning the size of the oxygen transporter: Hb octamers, and Hb integrated within a nanoparticle.

Functionalized polymer microbubbles as new molecular ultrasound contrast agent to target P-selectin in thrombus.

Thrombotic diseases rarely cause symptoms until advanced stage and sudden death. Thus, early detection of thrombus by a widely spread imaging modality can improve the prognosis and reduce mortality. Here, polymer microbubbles (MBs) made of degradable poly(IsoButylCyanoAcrylate) and functionalized with fucoidan (Fucoidan-MBs) were designed as a new targeted ultrasound contrast agent to image venous thrombus. The physicochemical characterizations demonstrate that the MBs with fucoidan surface exhibit a size of 2-6 µm and stability in suspension at 4 °C up to 2 months. MBs exhibit high echogenicity and could be completely burst under high destructive pulse. Flow chamber experiments on activated human platelets show a higher affinity of Fucoidan-MBs than control anionic MBs (CM-Dextran-MBs) under shear stress conditions. In vivo analysis by ultrasound and histological results demonstrate that Fucoidan-MBs are localized in rat venous thrombotic wall, whereas few CM-Dextran-MBs are present. In addition, the binding of Fucoidan-MBs in healthy vein is not observed. Collectively, Fucoidan-MBs appear as a promising functionalized carrier for ultrasound molecular imaging in thrombotic diseases.

Core-Shell Polymer-Based Nanoparticles Deliver miR-155-5p to Endothelial Cells.

Heart failure occurs in over 30% of the worldwide population and most commonly originates from cardiovascular diseases such as myocardial infarction. microRNAs (miRNAs) target and silence specific mRNAs, thereby regulating gene expression. Because the endogenous miR-155-5p has been ascribed to vasculoprotection, loading it onto positively charged, core-shell poly(isobutylcyanoacrylate) (PIBCA)-polysaccharide nanoparticles (NPs) was attempted. NPs showed a decrease (p < 0.0001) in surface electrical charge (ζ potential), with negligible changes in size or shape when loaded with the anionic miR-155-5p. Presence of miR-155-5p in loaded NPs was further quantified. Cytocompatibility up to 100 µg/mL of NPs for 2 days with human coronary artery endothelial cells (hCAECs) was documented. NPs were able to enter hCAECs and were localized in the endoplasmic reticulum (ER). Expression of miR-155-5p was increased within the cells by 75-fold after 4 hours of incubation (p < 0.05) and was still noticeable at day 2. Differences between loaded NP-cultured cells and free miRNA, at days 1 (p < 0.05) and 2 (p < 0.001) suggest the ability of prolonged load release in physiological conditions. Expression of miR-155-5p downstream target BACH1 was decreased in the cells by 4-fold after 1 day of incubation (p < 0.05). This study is a first proof of concept that miR-155-5p can be loaded onto NPs and remain intact and biologically active in endothelial cells (ECs). These nanosystems could potentially increase an endogenous cytoprotective response and decrease damage within infarcted hearts.