Superparamagnetic Nanoparticles: A Biodistribution Study Using Xenopus laevis Embryos

Various in-vivo biological models have been proposed for studying the interactions of nanomaterials with biological systems. Recently, there has been a significant increase in interest in the use of non-mammalian embryos, such as the frog Xenopus laevis as valid models for research in nanomedicine. In the present work, we demonstrate that X. laevis is a powerful model for the study of the biodistribution of superparamagnetic nanoparticles (SPION), extensively used in biomedical field for cell separation, MRI diagnostics and magnetic drug-targeting. 10 nl of 25 mg/ml of SPIONs (nano-screen MAG/ARA 200 nm, Chemicell) were microinjected. The biodistribution of SPIONs, following cardiac or pronephros injection of anesthetized frog larvae at stage 37, was studied by both in-vivo florescence and by Prussian blue staining of paraffin sections of the larvae after 24, 48, 72 or 96 hours (at 14 °C). The study confirmed that SPIONs diffused from either injection site by blood stream to all larval organs, being still present after 96 hours of injection

Magnetic nanoparticles: a strategy to target the choroidal layer in the posterior segment of the eye

Despite the higher rate of blindness due to population aging, minimally invasive and selective drug delivery to the eye still remains an open challenge, especially in the posterior segment. The retina, the retinal pigment epithelium (RPE) and the choroid are posterior segment cell layers, which may be affected by several diseases. In particular, damages to the choroid are associated with poor prognosis in the most severe pathologies. A drug delivery approach, able to target the choroid, is still missing. Recently, we demonstrated that intravitreally injected magnetic nanoparticles (MNP) are able to rapidly and persistently localise within the RPE in an autonomous manner. In this work we functionalised the MNP surface with the vascular endothelial growth factor, a bioactive molecule capable of transcytosis from the RPE towards more posterior layers. Such functionalisation successfully addressed the MNPs to the choroid, while MNP functionalised with a control polypeptide (poly-L-lysine) showed the same localisation pattern of the naked MNP particles. These data suggest that the combination of MNP with different bioactive molecules could represent a powerful strategy for cell-specific targeting of the eye posterior segment

Kdm7a expression is spatiotemporally regulated in developing Xenopus laevis embryos, and its overexpression influences late retinal development

Background: Post-translational histone modifications are among the most common epigenetic modifications that orchestrate gene expression, playing a pivotal role during embryonic development and in various pathological conditions. Among histone lysine demethylases, KDM7A, also known as KIAA1718 or JHDM1D, catalyzes the demethylation of H3K9me1/2 and H3K27me1/2, leading to transcriptional regulation. Previous data suggest that KDM7A plays a central role in several biological processes, including cell proliferation, commitment, differentiation, apoptosis, and maintenance. However, information on the expression pattern of KDM7A in whole organisms is limited, and its functional role is still unclear. Results: In Xenopus development, kdm7a is expressed early, undergoing spatiotemporal regulation in various organs and tissues, including the central nervous system and the eye. Focusing on retinal development, we found that kdm7a overexpression does not affect the expression of genes critically involved in early neural development and eye-field specification, whereas unbalances the distribution of neural cell subtypes in the mature retina by disfavoring the development of ganglion cells while promoting that of horizontal cells. Conclusions: Kdm7a is dynamically expressed during embryonic development, and its overexpression influences late retinal development, suggesting a potential involvement in the molecular machinery regulating the spatiotemporally ordered generation of retinal neuronal subtypes

Neurotrophin-conjugated nanoparticles prevent retina damage induced by oxidative stress

Glaucoma and other optic neuropathies are characterized by a loss of retinal ganglion cells (RGCs), a cell layer located in the posterior eye segment. Several preclinical studies demonstrate that neurotrophins (NTs) prevent RGC loss. However, NTs are rarely investigated in the clinic due to various issues, such as difficulties in reaching the retina, the very short half-life of NTs, and the need for multiple injections. We demonstrate that NTs can be conjugated to magnetic nanoparticles (MNPs), which act as smart drug carriers. This combines the advantages of the self-localization of the drug in the retina and drug protection from fast degradation. We tested the nerve growth factor and brain-derived neurotrophic factor by comparing the neuroprotection of free versus conjugated proteins in a model of RGC loss induced by oxidative stress. Histological data demonstrated that the conjugated proteins totally prevented RGC loss, in sharp contrast to the equivalent dose of free proteins, which had no effect. The overall data suggest that the nanoscale MNP-protein hybrid is an excellent tool in implementing ocular drug delivery strategies for neuroprotection and therapy

Nano-topography:Quicksand for cell cycle progression?

The 3-D spatial and mechanical features of nano-topography can create alternative environments, which influence cellular response. In this paper, murine fibroblast cells were grown on surfaces characterized by protruding nanotubes. Cells cultured on such nano-structured surface exhibit stronger cellular adhesion compared to control groups, but despite the fact that stronger adhesion is generally believed to promote cell cycle progression, the time cells spend in G1 phase is doubled. This apparent contradiction is solved by confocal microscopy analysis, which shows that the nano-topography inhibits actin stress fiber formation. In turn, this impairs RhoA activation, which is required to suppress the inhibition of cell cycle progression imposed by p21/p27. This finding suggests that the generation of stress fibers, required to impose the homeostatic intracellular tension, rather than cell adhesion/spreading is the limiting factor for cell cycle progression. Indeed, nano-topography could represent a unique tool to inhibit proliferation in adherent well-spread cells

Sheets of vertically aligned BaTiO₃ nanotubes reduce cell proliferation but not viability of NIH-3T3 cells

All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO3, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO3 nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants

Unravelling the mechanisms that determine the uptake and metabolism of magnetic single and multicore nanoparticles in a Xenopus laevis model.

Multicore superparamagnetic nanoparticles have been proposed as ideal tools for some biomedical applications because of their high magnetic moment per particle, high specific surface area and long term colloidal stability. Through controlled aggregation and packing of magnetic cores it is possible to obtain not only single-core but also multicore and hollow spheres with internal voids. In this work, we compare toxicological properties of single and multicore nanoparticles. Both types of particles showed moderate in vitro toxicity (MTT assay) tested in Hep G2 (human hepatocellular carcinoma) and Caco-2 (human colorectal adenocarcinoma) cells. The influence of surface chemistry in their biological behavior was also studied after functionalization with O,O′-bis(2-aminoethyl) PEG (2000 Da). For the first time, these nanoparticles were evaluated in a Xenopus laevis model studying their whole organism toxicity and their impact upon iron metabolism. The degree of activation of the metabolic pathway depends on the size and surface charge of the nanoparticles which determine their uptake. The results also highlight the potential of Xenopus laevis model bridging the gap between in vitro cell-based assays and rodent models for toxicity assessment to develop effective nanoparticles for biomedical applications

Caratterizzazione di geni regolati dal fattore di trascrizione Xrx1: implicazioni nello sviluppo dell'occhio in Xenopus laevis

Durante lo sviluppo dell’occhio nei Vertebrati sono necessari una serie di eventi altamente coordinati, tra cui la specificazione della piastra neurale anteriore, l’evaginazione laterale delle vescicole ottiche dal prosencefalo e infine il differenziamento del cristallino e dei vari strati della retina. Tra i geni essenziali per il corretto sviluppo di questo organo un ruolo critico e’ svolto dal fattore di trascrizione di tipo paired-like Rx1. In Xenopus laevis, Xrx1 è espresso a partire dallo stadio di neurula nella piastra neurale anteriore. Successivamente, la sua espressione si localizza nelle vescicole e nelle coppe ottiche, nel diencefalo ventrale e nella ghiandola pineale. Una volta terminato il differenziamento della retina, la sua espressione nell’occhio si restringe alla zona marginale ciliare, dove è implicato nel mantenimento della staminalità dei progenitori che permettono la rigenerazione dei neuroni retinici. Nell’ambito di un progetto volto a identificare i geni regolati da questo fattore di trascrizione, sono stati effettuati esperimenti utilizzando i “microarray” commerciali “GeneChip Xenopus laevis genome array”. Grazie a queste tecniche è stato possibile evidenziare i trascritti che cambiano livello di espressione in seguito alla sovraespressione o all’inattivazione funzionale di Xrx1 allo stadio di neurula precoce, e che quindi hanno maggiore probabilità di essere effettivamente controllati da Xrx1 durante lo sviluppo. Tra questi sono stati selezionati trascritti dotati di un comportamento coerente con il piu’ semplice modello ipotizzabile, quindi quelli che rispettivamente aumentano nella sovraespressione e diminuiscono nella inattivazione funzionale, o viceversa. Lo scopo di questa tesi è di caratterizzare il profilo di espressione dei trascritti selezionati, fornendo così anche una prima conferma dei dati provenienti dagli esperimenti di microarray e, inoltre, di identificare i trascritti analizzati per evidenziare eventuali relazioni funzionali con Xrx1. Sono stati analizzati undici dei trascritti selezionati. Il profilo di espressione di questi geni è stato studiato mediante ibridazione in situ su embrioni interi a diversi stadi di sviluppo, da nerula precoce a larva natante. Inoltre sono state effettuate ibridazioni in situ su sezioni di retina differenziata a stadio di girino per verificarne la localizzazione, in particolare a livello della CMZ. Dalle analisi su embrioni interi e sezioni di retine è risultato che sette trascritti sono espressi ad alti livelli nell’occhio e nel sistema nervoso centrale anteriore: di questi, quattro sono specificamente localizzati o arricchiti nella zona marginale ciliare. Infine uno dei trascritti selezionati, L11, è localizzato nell’endoderma a stadio di neurula, mentre non risulta trascritto a stadi successivi. Per indagare più approfonditamente sulla sua eventuale interazione con Xrx1 sono stati effettuati esperimenti di guadagno di funzione sovraesprimendo Xrx1 e analizzando l’espressione di L11 a stadio di neurula. Inoltre sono state effettuate analisi bioinformatiche di omologia di sequenza e successivamente ricerche in letteratura per tentare di identificare, anche solo parzialmente, i trascritti sconosciuti o inferirne una funzione. I risultati confermano che, ad eccezione di due trascritti, tutti gli altri hanno domini di espressione parzialmente sovrapposti o riconducibili a quello di Xrx1, e sono quindi dotati della giusta localizzazione per essere effettivamente regolati da Xrx1 durante lo sviluppo. Per mezzo delle indagini bioinformatiche sono stati individuati alcuni trascritti particolarmente interessanti per la funzione svolta in relazione alle attività di Xrx1, ad esempio nel mantenimento della staminalità

Non-mammalian vertebrate embryos as models in nanomedicine

Various in vivo biological models have been proposed for studying the interactions of nano-materials in biological systems. Unfortunately, the widely used small mammalian animal models (rodents) are costly and labor intensive and generate ethical issues and antagonism from the anti-vivisectionist movement. Recently, there has been increasing interest in the scientific community in the interactions between nano-materials and non-mammalian developmental organisms, which are now being recognized as valid models for the study of human disease. This review examines and discusses the biomedical applications and the interaction of nano-materials with embryonic systems, focusing on non-mammalian vertebrate models, such as chicken, zebrafish and Xenopus. From the Clinical Editor: Animal models are critical components of preclinical biomedical research. This review discusses the feasibility and potential applications of non-mammalian vertebral animals, such as zebrafish, xenopus, and chicken as animal models in nanomedicine research. © 2014 Elsevier Inc

Targeting drugs to specific cell layers of the posterior eye segment: implementation of “smart” strategies of ocular neuroprotection in zebrafish model

Despite the higher rate of blindness due to population aging, minimally invasive and selective drug delivery to the eye still remains an open challenge, especially in the posterior segment. The posterior eye segment is composed by several cell layers, each one specialized in different functions. It is affected by several diseases, which account for the majority of blindness worldwide. One of the most effective routes to reach the eye posterior segment is represented by intravitreal (IVT) injections. However, current procedures have severe collateral effects due to the need of repetitive injections over the time, the unfavorable drug kinetics (the initial dose is very high and quickly drops to zero due to physiological washing), the lack of specificity for the target (all cell layers are exposed to the drug), etc. In this work we used a drug carrier, with the intent to release the drug in a specific region or tissue and to prolong its half-life. Specifically, we validated a carrier based on magnetic nanoparticles (MNPs). Our studies suggest that zebrafish embryos are an excellent animal model for proof of concept studies. In zebrafish, the IVT injection has been already used for the delivery of drugs to posterior segment, e.g., antioxidants [1] and growth factors [2]. We found that magnetic nanoparticle, after intraocular injection, are able to rapidly and persistently localize within the retinal pigment epithelium (RPE) in an autonomously manner [3]. Most importantly, our results show that surface functionalization could change the fate of the particles, driving their localization in different cell layers of the posterior eye chamber, such as the choroid or the retina [4]. We used MNPs to deliver neurotrophic factors, which have a very short half-life in vivo, usually making ineffective their clinical use. We developed a model of damage to the posterior eye chamber in zebrafish embryos (0-96 hours post fertilization). We found that the injection of MNPs functionalized with nerve growth factor (NGF) or brain derived nerve factor (BDNF) strongly prevents injuries to the posterior eye chamber and sustain the regeneration process compared to that obtained using the free factors. We postulate that the increase in stability and the localization of growth factors mediated by MNPs are responsible for the enhanced neuroprotective effects of NGF and BDNF in the MNP group compared with the sham group. Our data suggest that MNP could represent a powerful strategy for the design of novel minimally invasive carriers for cell-specific ocular delivery and neuroprotection