Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NPâeuro0/00>âeuro0/00chitosan DNA NPâeuro0/00=âeuro0/00DNA unloaded chitosan NPâeuro0/00>âeuro0/00control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to investigate the influence of glial cells on the neurotoxicity of two organophosphorus pesticides (OPs), chlorpyrifos and parathion. Mixed-cell aggregate cultures were treated continuously for 10 days between DIV 5 and 15. Parathion induced astrogliosis at concentration at which MAP-2 immunostaining, found here to be more sensitive than neuron-specific enzyme activities, was not affected. In contrast, chlorpyrifos induced a comparatively weak gliotic reaction, and only at concentrations at which neurons were already affected. After similar treatments, increased neurotoxicity of parathion and chlorpyrifos was found in aggregate cultures deprived of glial cells. These results suggest that glial cells provide neuroprotection against OPs toxicity. To address the question of the difference in toxicity between parathion and chlorpyrifos, the toxic effects of their leaving groups, p-nitrophenol and trichloropyridinol, were studied in mixed-cell aggregates. General cytotoxicity was more pronounced for trichloropyridinol and both compounds had similar toxic effects on neuron-specific enzyme activities. In contrast, trichloropyridinol induced a much stronger decrease in glutamine synthetase activity, the enzymatic marker of astrocytes. Trichloropyridinol may exert a toxic effect on astrocytes, compromising their neuroprotective function, thus exacerbating the neurotoxicity of chlorpyrifos. This is in line with the suggestion that glial cells may contribute to OPs neurotoxicity, and with the view that OPs may exert their neurotoxic effects through different mechanisms.

Generation of the primary hair follicle pattern.

Hair follicles are spaced apart from one another at regular intervals through the skin. Although follicles are predominantly epidermal structures, classical tissue recombination experiments indicated that the underlying dermis defines their location during development. Although many molecules involved in hair follicle formation have been identified, the molecular interactions that determine the emergent property of pattern formation have remained elusive. We have used embryonic skin cultures to dissect signaling responses and patterning outcomes as the skin spatially organizes itself. We find that ectodysplasin receptor (Edar)-bone morphogenetic protein (BMP) signaling and transcriptional interactions are central to generation of the primary hair follicle pattern, with restriction of responsiveness, rather than localization of an inducing ligand, being the key driver in this process. The crux of this patterning mechanism is rapid Edar-positive feedback in the epidermis coupled with induction of dermal BMP4/7. The BMPs in turn repress epidermal Edar and hence follicle fate. Edar activation also induces connective tissue growth factor, an inhibitor of BMP signaling, allowing BMP action only at a distance from their site of synthesis. Consistent with this model, transgenic hyperactivation of Edar signaling leads to widespread overproduction of hair follicles. This Edar-BMP activation-inhibition mechanism appears to operate alongside a labile prepattern, suggesting that Edar-mediated stabilization of beta-catenin active foci is a key event in determining definitive follicle locations.

Synergistic effect of calcitriol and interferon-α on hepatitis C virus replication in vitro

Background and aims: V itamin D is an important modulator o fnumerous c ellular processes, including innate and adaptive immunepathways. A recent large-scale genetic validation study performed withinthe framework of the Swiss Hepatitis C Cohort S tudy has demonstratedan association between t he 1α-hydroxylase promoter single nucleotidepolymorphism CYP27B1-1260 rs10877012 and sustained virologicresponse (SVR) after pegylated interferon-α ( PEG-IFN-α) plus ribavirintreatment of c hronic hepatitis C in patients w ith a p oor-response IL28Bgenotype. This suggests an intrinsic role o f vitamin D signaling in theresponse t o treatment of chronic hepatitis C, especially in patients withlimited sensitivity to IFN-α. In the present study, we investigated theeffect of 1,25-(OH)2 v itamin D3 (calcitriol) alone or in combination withIFN-α on the hepatitis C virus (HCV) life cycle in vitro.Methods: H uh-7.5 cells harboring Con1- or JFH-1-derived HCVreplicons or cell culture-derived HCV were exposed to 0.1-100 nMcalcitriol ± 1 -100 IU/ml IFN-α. The effect on HCV RNA replication andviral particle production was investigated by quantitative r eal-time PCR,immunoblot analyses, and infectivity titration analyses. The expression ofinterferon-stimulated genes (ISGs) and of calcitriol target genes wasassessed by quantitative real-time PCR.Results: Calcitriol had no relevant effect on the viability of Huh-7.5 cells.Calcitriol strongly induced and repressed the expression of the calcitrioltarget genes CYP24A1 and CCNC, respectively, confirming that Huh-7.5cells c an respond to c alcitriol signaling. P hysiological doses of calcitrioldid not significantly a ffect HCV RNA replication or i nfectious particleproduction in vitro, and calcitriol alone h ad no significant effect on theexpression of several ISGs. However, calcitriol in combination with IFN-αsubstantially increased the expression of ISGs compared to IFN-α alone.In addition, calcitriol plus IFN-α s ynergistically inhibited HCV RNAreplication.Conclusions: C alcitriol at physiological concentrations and IFN-α a ctsynergistically on the expression of I SGs and HCV RNA replication i nvitro. Experiments exploring the underlying mechanisms are underway.

Brain-derived neurotrophic factor-like immunoreactivity is localized mainly in small sensory neurons of rat dorsal root ganglia.

Brain-derived neurotrophic factor (BDNF) is a protein capable of supporting the survival and fiber outgrowth of peripheral sensory neurons. It has been argued that histological detection of BDNF has proven difficult because of its low molecular weight and relatively low expression. In the present study we report that rapid removal of dorsal root ganglia (DRG) from the rat, followed by rapid freezing and appropriate fixation with cold acetone, preserves BDNF in situ without altering protein antigenicity. Under these conditions, specific BDNF-like immunoreactivity was detected in DRG both in vivo and in vitro. During DRG development in vivo, BDNF-like immunoreactivity (BDNF-LI) was observed only in a subset of sensory neurons. BDNF-LI was confined to small neurons, after neurons became morphologically distinct on the basis of size. BDNF-L immunoprecipitate was detected only in neuronal cells, and not in satellite or Schwann cells. While in vivo BDNF localization was restricted to small neurons, practically all neurons in DRG cell culture displayed BDNF-LI. Small or large primary afferent neurons exhibited a faint but clear BDNF-LI during the whole life span of cultures. Again, non-neuronal cells were devoid of BDNF-LI. In conclusion, in DRG in vivo, specific BDNF-LI was confined to small B sensory neurons. In contrast, all DRG sensory neurons displayed BDNF-LI in vitro. The finding that BDNF expressed in all DRG neurons in vitro but not in vivo suggests that BDNF expression may be modulated by environmental factors.

Fine-Tuning Translation Kinetics Selection as the Driving Force of Codon Usage Bias in the Hepatitis A Virus Capsid

Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness.

Hepatitis E virus: a zoonosis adapting to humans.

Hepatitis E virus (HEV) infection is gaining global attention, not only because of the increasing burden of the disease in low endemicity countries, in terms of morbidity and mortality rates, but also due to recent advances in the molecular virology and epidemiology of this emerging pathogen. HEV infection spread can be described as the evolution of a zoonosis towards an established human infection. As known from other viruses, such as the human immunodeficiency virus or the influenza viruses, crossing the species barriers from animals to humans is a recurrent phenomenon. Albeit slow at the beginning, once the virus has adapted to humans, the person-to-person spread can proceed very quickly. Although an optimal cell culture system for HEV is not yet available, outstanding progress has been made with the in vitro expression of HEV-like particles. These new tools have fostered new research to understand the molecular, structural and immunological aspects of human HEV infection. Although some promising data from Phase II vaccine trials are available, recent discoveries will certainly open new avenues for HEV-specific prophylaxis and therapy.

NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesity and antidiabetic activity.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) plays a key role in adipocyte differentiation and insulin sensitivity. Its synthetic ligands, the thiazolidinediones (TZD), are used as insulin sensitizers in the treatment of type 2 diabetes. These compounds induce both adipocyte differentiation in cell culture models and promote weight gain in rodents and humans. Here, we report on the identification of a new synthetic PPARgamma antagonist, the phosphonophosphate SR-202, which inhibits both TZD-stimulated recruitment of the coactivator steroid receptor coactivator-1 and TZD-induced transcriptional activity of the receptor. In cell culture, SR-202 efficiently antagonizes hormone- and TZD-induced adipocyte differentiation. In vivo, decreasing PPARgamma activity, either by treatment with SR-202 or by invalidation of one allele of the PPARgamma gene, leads to a reduction of both high fat diet-induced adipocyte hypertrophy and insulin resistance. These effects are accompanied by a smaller size of the adipocytes and a reduction of TNFalpha and leptin secretion. Treatment with SR-202 also dramatically improves insulin sensitivity in the diabetic ob/ob mice. Thus, although we cannot exclude that its actions involve additional signaling mechanisms, SR-202 represents a new selective PPARgamma antagonist that is effective both in vitro and in vivo. Because it yields both antiobesity and antidiabetic effects, SR-202 may be a lead for new compounds to be used in the treatment of obesity and type 2 diabetes.

Drug-eluting beads loaded with antiangiogenic agents for chemoembolization: in vitro sunitinib loading and release and in vivo pharmacokinetics in an animal model.

PURPOSE: The combination of embolic beads with a multitargeted tyrosine kinase inhibitor that inhibits tumor vessel growth is suggested as an alternative and improvement to the current standard doxorubicin-eluting beads for use in transarterial chemoembolization. This study demonstrates the in vitro loading and release kinetics of sunitinib using commercially available embolization microspheres and evaluates the in vitro biologic efficacy on cell cultures and the resulting in vivo pharmacokinetics profiles in an animal model. MATERIALS AND METHODS: DC Bead microspheres, 70-150 µm and 100-300 µm (Biocompatibles Ltd., Farnham, United Kingdom), were loaded by immersion in sunitinib solution. Drug release was measured in saline in a USP-approved flow-through apparatus and quantified by spectrophotometry. Activity after release was confirmed in cell culture. For pharmacokinetics and in vivo toxicity evaluation, New Zealand white rabbits received sunitinib either by intraarterial injection of 100-300 µm sized beads or per os. Plasma and liver tissue drug concentrations were assessed by liquid chromatography-tandem mass spectroscopy. RESULTS: Sunitinib loading on beads was close to complete and homogeneous. A total release of 80% in saline was measured, with similar fast-release profiles for both sphere sizes. After embolization, drug plasma levels remained below the therapeutic threshold (< 50 ng/mL), but high concentrations at 6 hours (14.9 µg/g) and 24 hours (3.4 µg/g) were found in the liver tissue. CONCLUSIONS: DC Bead microspheres of two sizes were efficiently loaded with sunitinib and displayed a fast and almost complete release in saline. High liver drug concentrations and low systemic levels indicated the potential of sunitinib-eluting beads for use in embolization.

Sequence analysis and expression of the attachment and fusion proteins of canine distemper virus wild-type strain A75/17.

The biological properties of wild-type A75/17 and cell culture-adapted Onderstepoort canine distemper virus differ markedly. To learn more about the molecular basis for these differences, we have isolated and sequenced the protein-coding regions of the attachment and fusion proteins of wild-type canine distemper virus strain A75/17. In the attachment protein, a total of 57 amino acid differences were observed between the Onderstepoort strain and strain A75/17, and these were distributed evenly over the entire protein. Interestingly, the attachment protein of strain A75/17 contained an extension of three amino acids at the C terminus. Expression studies showed that the attachment protein of strain A75/17 had a higher apparent molecular mass than the attachment protein of the Onderstepoort strain, in both the presence and absence of tunicamycin. In the fusion protein, 60 amino acid differences were observed between the two strains, of which 44 were clustered in the much smaller F2 portion of the molecule. Significantly, the AUG that has been proposed as a translation initiation codon in the Onderstepoort strain is an AUA codon in strain A75/17. Detailed mutation analyses showed that both the first and second AUGs of strain A75/17 are the major translation initiation sites of the fusion protein. Similar analyses demonstrated that, also in the Onderstepoort strain, the first two AUGs are the translation initiation codons which contribute most to the generation of precursor molecules yielding the mature form of the fusion protein.

Identification of GPx8 as a novel cellular substrate of the hepatitis C virus NS3-4A protease

Background: The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity (MAVS and TRIF) as well as a phosphatase involved in growth factor signaling (TC-PTP). The aim of this study was to identify novel cellular substrates of the NS3-4A protease and to investigate their role in the life cycle and pathogenesis of HCV. Methods: Cell lines inducibly expressing the NS3-4A protease were analyzed in basal as well as interferon- α -stimulated states by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. Candidates fulfilling strin- gent criteria for potential substrates or products of the NS3-4A protease were further investigated in different experimental sys- tems as well as in liver biopsies from patients with chronic hep- atitis C. Results: SILAC coupled with protein separation and mass spectrometry yielded > 5000 proteins of which 21 can- didates were selected for further analyses. These allowed us to identify GPx8, a membrane-associated peroxidase involved in disulfide bond formation in the endoplasmic reticulum, as a novel cellular substrate of the HCV NS3-4A protease. Cleavage occurs at cysteine in position 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic hepatitis C. Further functional studies, involving overexpression and RNA silencing, revealed that GPx8 is a proviral factor involved in viral particle production but not in HCV entry or RNA replica- tion. Conclusions: GPx8 is a proviral host factor cleaved by the HCV NS3-4A protease. Studies investigating the consequences of cleavage for GPx8 function are underway. The identification of novel cellular substrates of the HCV NS3-4A protease should yield new insights into the HCV life cycle and the pathogenesis of hepatitis C and may reveal novel angles for therapeutic inter- vention.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

Angiotensin-II mediates norepinephrine and neuropeptide-Y secretion in a human pheochromocytoma.

The potential role of angiotensin-II in mediating catecholamine and neuropeptide-Y release in a human pheochromocytoma has been investigated. Angiotensin-II type I receptors are transcribed and translated into functional proteins in a surgically removed pheochromocytoma. Primary cell culture of the tumor has been studied in a perfused system. Angiotensin-II increased the release of norepinephrine and neuropeptide-Y by the pheochromocytes. Activation of the angiotensin-II type I receptors by angiotensin-II was associated with a rise in cytosolic free calcium. The renin-angiotensin system may, therefore, contribute to the secretion of catecholamines and NPY occurring in patients with pheochromocytoma and when stimulated trigger hypertensive crisis.

Interactions between cells and functionalized nanoparticles

In this present thesis Superparamagnetic Iron Oxide Nanoparticles (SPIONs) with 9 nm in diameter were selected as nanocarriers in order to study their potential application as drug delivery systems. Therefore the aim of the study was to demonstrate the proof of concept by establishing an efficient system of drug delivery, which would be a valuable tool in biomedical applications, such as the treatement of cancer, by reducing the side effects due to administration of a high concentration of therapeutic agents. As demonstrated in a previous study, the uptake of SPIONs by tumoral human cells was enhanced by the presence of amino groups on their surface. The stabilization of SPIONs were then performed and optimized by the coating of poly(vinylalcohol) and poly(vinylalcohol/vinylamine). Such nanoparticles were known as aminoPVA-SPIONs. The toxicity and the inflammatory reaction of aminoPVA-SPIONs were evaluated in order to establish their potentiel use in the human body. The results demonstrated that the human cells were able to invaginate aminoPVA-SPIONS without revealing any toxicity and inflammatory reaction. The analysis by transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo-TEM, confocal microscopy and histological staining (i.e. Prussian Blue) showed that the iron oxide core of SPIONs were located in the cytoplasm of cells and concentrated in vesicles. The evaluation of the mechanism of uptake of aminoPVA-SPIONs revealed that their uptake by monolayer cell culture was performed via an active mechanism, which was achieved by a clathrin-mediated endocytosis. Consequently, it was suggested that aminoPVA-SPIONs were good candidates as nanocarriers in drug delivery systems, which were able to reach the cytoplasm of cells. Their incubation with three-dimensional models mimicing tissues, such as differentiated rat brain cell-derived aggregates and spheroids, revealed that aminoPVA-SPIONs were able to invade into deep cell layers according to the stage of growth of these models. In the view of these promising results, drug-SPIONs were prepared by the functionalization of aminoPVA-SPIONs via a biological labile chemical bond by one of these three antineoplastic agents, which are widely used in clinical practice: 5-fluorourdine (Fur) (an antimetabolite), or camptothecin (CPT) (a topoisomerase inhibitor) or doxorubicin (DOX) (an anthracycline which interfere with DNA). The results shown that drug-SPIONs were internalized by human melanoma cells, as it was expected due the previous results with aminoPVA-SPIONs, and in addition they were active as anticancer agents, suggesting the efficient release of the drug from the drug-SPIONs. The results with CPT-SPIONs were the most promising, whereas DOX- SPIONs did not demonstrate a prononced activity of DOX. In conclusion, the results demonstrated that functionalized iron oxide nanoparticles are a promising tool in order to deliver therapeutic agents. - Dans le cadre de ce travail de thèse, les nanoparticules superparamagnétiques d'oxyde de fer (SPIONs) ayant un diamètre de 9 nm ont été choisies, afin d'étudier leur éventuelle utilisation dans un système de délivrance d'agents thérapeutiques. Ainsi le but de la thèse est de démontrer la faisabilité de fabriquer un système efficace de délivrance d'agents thérapeutiques, qui serait un outil intéressant dans le cadre d'une utilisation biomédicale, par exemple lors du traitement du cancer, qui pourrait réduire les effets secondaires provoqués par le dosage trop élevé de médicaments. Comme il a été démontré dans une précédente étude, l'invagination des SPIONs par des cellules humaines cancéreuses est améliorée par la présence de groupes fonctionnels amino à leur surface. La stabilisation des SPIONs est ainsi effectuée et optimisée par l'enrobage de poly(vinylalcool) et de (poly(vinylalcool/vinylamine), qui sont connues sous le nom de aminoPVA-SPIONs. La toxicité et la réaction inflammatoire des aminoPVA-SPIONs ont été évaluées dans le but de déterminer leur potentielle utilisation dans le corps humain. Les résultats démontrèrent que les cellules humaines sont capables d'invaginer les aminoPVAS-SPIONs sans induire une réaction toxique ou inflammatoire. L'analyse par la microscopie électronique en transmission électronique (TEM), la microscopie électronique à balayage (SEM), le cryo-microscopie électronique (SEM), la microscopie confocale et la coloration histologique (par ex, le bleu de Prusse) a montré que l'oxyde de fer des SPIONs est localisé dans le cytoplasme des cellules et est concentré dans des vesicules. L'évaluation du méchanisme d'invagination des aminoPVA-SPIONs ont révélé que leur invagination par des monocultures de cellules est effectué par un méchanisme actif, contrôlé par une endocytose induite par les clathrins. Par conséquent, les aminoPVA-SPIONs sont de bons candidats en tant que transporteurs (nanocamers) dans un système de délivrance d'agents thérapeuthique, capable d'atteindre le cytoplasme des cellules. Leur incubation avec des modèles tridimenstionnels imitant les tissues, tels que les aggrégats de cellules de cerveau différenciées et les sphéroïdes, a montré que les aminoPVA-SPIONs sont capable de pénétrer dans les couches profondes des modèles, selon l'état d'avancement de leur croissance. En vue de ces résultats prometteurs, les drug-SPIONs ont été préparés en fonctionalisant les aminoPVA-SPIONs par le biai d'une liaison chimique labile par un des trois agents thérapeutiques, déjà utilisé en pratique : 5-fluorourdine (Fur) (un antimétabolite), or camptothecin (CPT) (un inhibiteur de la topoisomerase) or doxorubicin (DOX) (un anthracycline qui interfère avec le DNA). Les résultats ont montré que les drug-SPIONs sont capable d'être internalisés par les mélanomes, comme il a été attendu d'après les résultats obtenus précédemment avec les aminoPVA-SPIONs, et de plus, les drug-SPIONs sont actifs, ce qui suggère un relargage efficace de l'agent thérapeutique du drug-SPIONs. Les résultats obtenus avec les CPT-SPIONs sont les plus prometteurs, tandis que ceux avec les DOX-SPIONs, ce n'est pas le cas, dont l'activité thérapeutique de DOX n'a pas été aussi efficace. En conclusion, les résultats ont pu démontrer que les nanoparticules d'oxyde de fer fonctionnalisées sont un outil prometteur dans la délivrance d'agents thérapeutiques.