Biomedical Nanoparticles: Overview of Their Surface Immune-Compatibility

Diagnostic- and therapeutic release-aimed nanoparticles require the highest degree of biocompatibility. Some physical and chemical characteristics of such nanomaterials are often at odds with this requirement. For instance, metals with specific features used as contrast agents in magnetic resonance imaging need particular coatings to improve their blood solubility and increase their biocompatibility. Other examples come from the development of nanocarriers exploiting the different characteristics of two or more materials, i.e., the ability to encapsulate a certain drug by one core-material and the targeting capability of a different coating surface. Furthermore, all these “human-non-self” modifications necessitate proofs of compatibility with the immune system to avoid inflammatory reactions and resultant adverse effects for the patient. In the present review we discuss the molecular interactions and responses of the immune system to the principal nanoparticle surface modifications used in nanomedicine

Molecularly Imprinted Biodegradable Nanoparticles

Biodegradable polymer nanoparticles are promising carriers for targeted drug delivery in nanomedicine applications. Molecu- lar imprinting is a potential strategy to target polymer nanoparticles through binding of endogenous ligands that may promote recognition and active transport into specific cells and tissues. However, the lock-and-key mechanism of molecular imprinting requires relatively rigid cross-linked structures, unlike those of many biodegradable polymers. To date, no fully biodegradable molecularly imprinted particles have been reported in the literature. This paper reports the synthesis of a novel molecularly- imprinted nanocarrier, based on poly(lactide-co-glycolide) (PLGA) and acrylic acid, that combines biodegradability and molec- ular recognition properties. A novel three-arm biodegradable cross-linker was synthesized by ring-opening polymerization of glycolide and lactide initiated by glycerol. The resulting macromer was functionalized by introduction of end-functions through reaction with acryloyl chloride. Macromer and acrylic acid were used for the synthesis of narrowly-dispersed nanoparticles by radical polymerization in diluted conditions in the presence of biotin as template molecule. The binding capacity of the imprinted nanoparticles towards biotin and biotinylated bovine serum albumin was twentyfold that of non-imprinted nanoparti- cles. Degradation rates and functional performances were assessed in in vitro tests and cell cultures, demonstrating effective biotin-mediated cell internalization

Homozygous loss of autism-risk gene CNTNAP2 results in reduced local and long-range prefrontal functional connectivity

Functional connectivity aberrancies, as measured with resting-state fMRI (rsfMRI), have been consistently observed in the brain of autism spectrum disorders (ASD) patients. However, the genetic and neurobiological underpinnings of these findings remain unclear. Homozygous mutations in Contactin Associated Protein-like 2 (CNTNAP2), a neurexin-related cell-adhesion protein, are strongly linked to autism and epilepsy. Here we used rsfMRI to show that homozygous mice lacking Cntnap2 exhibit reduced long-range and local functional connectivity in prefrontal and midline brain “connectivity hubs”. Long-range rsfMRI connectivity impairments affected heteromodal cortical regions and were prominent between frontoposterior components of the mouse default-mode network (DMN), an effect that was associated with reduced social investigation, a core “autism trait” in mice. Notably, viral tracing revealed reduced frequency of prefrontal-projecting neural clusters in the cingulate cortex of Cntnap2-/- mutants, suggesting a possible contribution of defective mesoscale axonal wiring to the observed functional impairments. Macroscale cortico-cortical white matter organization appeared to be otherwise preserved in these animals. These findings reveal a key contribution of ASD-associated gene CNTNAP2 in modulating macroscale functional connectivity, and suggest that homozygous loss-of-function mutations in this gene may predispose to neurodevelopmental disorders and autism through a selective dysregulation of connectivity in integrative prefrontal areas

Deletion of autism risk gene Shank3 disrupts prefrontal connectivity

Mutations in the synaptic scaffolding protein Shank3 are a major cause of autism, and are associated with prominent intellectual and language deficits. However, the neural mechanisms whereby SHANK3 deficiency affects higher order socio-communicative functions remain unclear. Using high-resolution functional and structural MRI in adult male mice, here we show that loss of Shank3 (Shank3B-/-) results in disrupted local and long-range prefrontal and fronto-striatal functional connectivity. We document that prefrontal hypo-connectivity is associated with reduced short-range cortical projections density, and reduced gray matter volume. Finally, we show that prefrontal disconnectivity is predictive of social communication deficits, as assessed with ultrasound vocalization recordings. Collectively, our results reveal a critical role of SHANK3 in the development of prefrontal anatomy and function, and suggest that SHANK3 deficiency may predispose to intellectual disability and socio-communicative impairments via dysregulation of higher-order cortical connectivity

mTOR-related synaptic pathology causes autism spectrum disorder-associated functional hyperconnectivity.

Postmortem studies have revealed increased density of excitatory synapses in the brains of individuals with autism spectrum disorder (ASD), with a putative link to aberrant mTOR-dependent synaptic pruning. ASD is also characterized by atypical macroscale functional connectivity as measured with resting-state fMRI (rsfMRI). These observations raise the question of whether excess of synapses causes aberrant functional connectivity in ASD. Using rsfMRI, electrophysiology and in silico modelling in Tsc2 haploinsufficient mice, we show that mTOR-dependent increased spine density is associated with ASD -like stereotypies and cortico-striatal hyperconnectivity. These deficits are completely rescued by pharmacological inhibition of mTOR. Notably, we further demonstrate that children with idiopathic ASD exhibit analogous cortical-striatal hyperconnectivity, and document that this connectivity fingerprint is enriched for ASD-dysregulated genes interacting with mTOR or Tsc2. Finally, we show that the identified transcriptomic signature is predominantly expressed in a subset of children with autism, thereby defining a segregable autism subtype. Our findings causally link mTOR-related synaptic pathology to large-scale network aberrations, revealing a unifying multi-scale framework that mechanistically reconciles developmental synaptopathy and functional hyperconnectivity in autism

Mapping the structural and functional organization of the serotonergic system connectome

Serotonin-synthetizing neurons, which are confined in the raphe nuclei of the rhombencephalon, provide a profuse innervation network throughout the central nervous system and are involved in the modulation of a plethora of brain functions. In the last few years, the development of novel genetic tools and high-throughput techniques has added unforeseen degrees of heterogeneity among serotonergic neurons in terms of neurodevelopmental origins, gene programs and electrophysiological properties. Recently, a map of the complex topographical organization of the serotonergic fibers has been drawn using intersectional fate mapping strategy, as well as retrograde or anterograde tracing. However, no comprehensive retrograde study has been performed yet, and anterograde approaches still set a series of limitation. Here, combining the recombinant G-deleted rabies virus system with Tph2GFP knock-in mice, in which serotonergic neurons were clearly labeled by the expression of GFP, I present a map of the topographic organization of 5-HT ascending projection to 19 rostral brain regions, known to be regulated by 5-HT. This experimental approach revealed that each brain district hereby investigated is innervated by a relatively small and region-specific number of serotonergic neurons. Thanks to Cre-independent and -dependent monosynaptic tracing, coupling pseudotyped recombinant rabies virus with a helper adeno-associated virus, it was possible to deduce that such narrow subpopulation of 5-HT neurons is characterized by a wiring transmission mode. Moreover, the balance between wiring and volume transmission appears to be region-specific, and may underlie specific regulation mechanism on neuronal functions. Altogether, my results can shed new lights on the communication properties of the serotonergic system, highlighting a further level of serotonergic control of brain function, possibly helping to better understand its role in health and disease

A rabies virus based approach to map serotonergic neurons innervating different brain structures

Serotonergic neurons are part of one of the most widely distributed neural systems in the mammalian brain (Lauder and Bloom, 1974). Serotonergic neurons form the raphe nuclei in the brain stem, and are organized in distinct nuclei (B1-9) that project to the whole central nervous system. Consistently with such a broad innervation, serotonin is involved in a wide range of physiological processes including the control of appetite, sleep, memory, mood, stress and sexual behavior (Veenstra-Vanderweele et al, 2000). Several studies using anatomical tracing methods and anterograde viral tracers have led to the hypotheses of a topographic organization of the serotonergic system, with different projections from the caudal, median/central and dorsal raphe neurons to specific target districts in the rostral brain (Muzerelle et al. 2014). Experimental evidence suggests that clusters of serotonergic neurons within the raphe nuclei may have distinct functional properties, but the complex organization of serotonergic neuron projections remains poorly understood. The aim of the present study is to map at a finer scale the organization of serotonin neurons projecting to different target areas, thus contributing to understanding the functional role of specific serotonergic neuronal subpopulations. To this end, we used a Tph2::GFP knock-in mouse model, in which serotonergic neurons are clearly labeled by the expression of GFP (Migliarini et al, 2013), and the retrograde recombinant rabies viral tracer to map the serotonergic neurons innervating different brain structures. Moreover, we developed a conditional GFP expressing mouse model, in which the reporter is maintained under the transcriptional control of the Tph2 gene and activated upon an flp mediated somatic recombination, to map the organization of serotonergic neuron subgroups sharing common targets in the brain

Wiring transmission in the serotonergic system

Serotonergic neurons are part of one of the most widely distributed systems of the mammalian brain. Indeed, serotonin is involved in a wide range of physiological processes, including the control of appetite, sleep, memory, mood, stress and sexual behavior. The raphe nuclei (B1-9) of the brain stem are the origin of serotonergic projections to the whole central nervous system. In the last years, several studies have unraveled the heterogeneity of serotonergic neurons, in terms of developmental programs, molecular and electrophysiological properties. Recently, a map of the complex topographical organization of the serotonergic fibers has been drawn using intersectional fate mapping strategy, as well as retrograde or anterograde tracing (Bang et al, 2012; Fernandez et al. 2015; Muzerelle et al, 2014). Serotonergic neurons have un-myelinated fiber varicosities, where the transmitter is synthesized, stored and released in a “volume transmission” (VT) mode (Agnati et al, 1995). To a lesser extent, serotonergic fibers also present synapse-like specializations where synaptic contacts are established by 5-HT terminals with specific neuronal targets acting in a conventional “wiring transmission” (WT) mode. However, experimental strategies used to map serotonergic projections so far where not selective for VT versus WT, and the organization of WT is still the object of investigation. Taking advantage of the properties of the rabies virus, whose envelope can drive the infection of neurons exclusively through their presynaptic terminals, we have selectively mapped the serotonergic WT system originating in the raphe nuclei. We injected recombinant G-deleted rabies virus in several brain regions of Tph2::GFP knock-in mice, in which serotonergic neurons were clearly labeled by the expression of GFP (Migliarini et al, 2013). We also used monosynaptic tracing, coupling pseudotyped recombinant rabies virus with a helper adeno-associated virus (Wall et al, 2010). This experimental approach revealed that each brain district hereby investigated receives WT from a relatively small and region-specific number of serotonergic neurons, thus making it possible to establish a correlation map between specific serotonergic neurons in the raphe nuclei and distinct brain areas. Altogether, this study sheds new light on communication properties of serotonergic system, and may help understand the selective role of serotonergic WT in health and disease

A poly(ether-ester) copolymer for the preparation of nanocarriers with improved degradation and drug delivery kinetics

Abstract This paper reports the synthesis and the physicochemical, functional and biological characterisations of nanocarriers made of a novel di-block biodegradable poly(ether-ester) copolymer. This material presents tunable, fast biodegradation rates, but its products are less acidic than those of other biosorbable polymers like PLGA, thus presenting a better biocompatibility profile and the possibility to carry pH-sensitive payloads. A method for the production of monodisperse and spherical nanoparticles is proposed; drug delivery kinetics and blood protein adsorption were measured to evaluate the functional properties of these nanoparticles as drug carriers. The copolymer was labelled with a fluorescent dye for internalisation tests, and rhodamine B was used as a model cargo to study transport and release inside cultured cells. Biological tests demonstrated good cytocompatibility, significant cell internalisation and the possibility to vehiculate non-cell penetrating moieties into endothelial cells. Taken together, these results support the potential use of this nanoparticulate system for systemic administration of drugs