Erythro-Magneto-HA-Virosome: A Bio-Inspired Drug Delivery System for Active Targeting of Drugs in the Lungs.

Over the past few decades, finding more efficient and selective administration routes has gained significant attention due to its crucial role in the bioavailability, absorption rate and pharmacokinetics of therapeutic substances. The pulmonary delivery of drugs has become an attractive target of scientific and biomedical interest in the health care research area, as the lung, thanks to its high permeability and large absorptive surface area and good blood supply, is capable of absorbing pharmaceuticals either for local deposition or for systemic delivery. Nevertheless, the pulmonary drug delivery is relatively complex, and strategies to mitigate the effects of mechanical, chemical and immunological barriers are required. Herein, engineered erythrocytes, the Erythro-Magneto-Hemagglutinin (HA)-virosomes (EMHVs), are used as a novel strategy for efficiently delivering drugs to the lungs. EMHV bio-based carriers exploit the physical properties of magnetic nanoparticles to achieve effective targeting after their intravenous injection thanks to an external magnetic field. In addition, the presence of hemagglutinin fusion proteins on EMHVs' membrane allows the DDS to anchor and fuse with the target tissue and locally release the therapeutic compound. Our results on the biomechanical and biophysical properties of EMHVs, such as the membrane robustness and deformability and the high magnetic susceptibility, as well as their in vivo biodistribution, highlight that this bio-inspired DDS is a promising platform for the controlled and lung-targeting delivery of drugs, and represents a valuable alternative to inhalation therapy to fulfill unmet clinical needs.

miR-182-5p is an evolutionarily conserved Tbx5 effector that impacts cardiac development and electrical activity in zebrafish.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

Pectin-coated boron nitride nanotubes: In vitro cyto-/immune-compatibility on RAW 264.7 macrophages.

BACKGROUND: Boron nitride nanotubes (BNNTs) represent a new opportunity for drug delivery and clinical therapy. The present work has the objective to investigate pectin-coated BNNTs (P-BNNTs) for their biocompatibility on macrophage cultures, since these cells are among the first components of the immune system to interact with administered nanoparticles. METHODS: As first step, the potential toxicity of P-BNNTs is verified in terms of proliferation, oxidative stress induction and apoptosis/necrosis phenomena. Thereafter, the modulation of immune cell response following P-BNNT exposure is evaluated at gene and protein level, in particular focusing on cytokine release. Finally, P-BNNT internalization is assessed through transmission electron microscopy and confocal microscopy. RESULTS: The results proved that P-BNNTs are not toxic for macrophages up to 50 µg/ml after 24 h of incubation. The cytokine expression is not affected by P-BNNT administration both at gene and protein level. Moreover, P-BNNTs are internalized by macrophages without impairments of the cell structures. CONCLUSIONS: Collected data suggest that P-BNNTs cause neither adverse effects nor inflammation processes in macrophages. GENERAL SIGNIFICANCE: These findings represent the first and fundamental step in immune compatibility evaluation of BNNTs, mandatory before any further pre-clinical testing.

AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells.

Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.

Measuring the elastic properties of living cells through the analysis of current-displacement curves in scanning ion conductance microscopy.

Knowledge of mechanical properties of living cells is essential to understand their physiological and pathological conditions. To measure local cellular elasticity, scanning probe techniques have been increasingly employed. In particular, non-contact scanning ion conductance microscopy (SICM) has been used for this purpose; thanks to the application of a hydrostatic pressure via the SICM pipette. However, the measurement of sample deformations induced by weak pressures at a short distance has not yet been carried out. A direct quantification of the applied pressure has not been also achieved up to now. These two issues are highly relevant, especially when one addresses the investigation of thin cell regions. In this paper, we present an approach to solve these problems based on the use of a setup integrating SICM, atomic force microscopy, and optical microscopy. In particular, we describe how we can directly image the pipette aperture in situ. Additionally, we can measure the force induced by a constant hydrostatic pressure applied via the pipette over the entire probe-sample distance range from a remote point to contact. Then, we demonstrate that the sample deformation induced by an external pressure applied to the pipette can be indirectly and reliably evaluated from the analysis of the current-displacement curves. This method allows us to measure the linear relationship between indentation and applied pressure on uniformly deformable elastomers of known Young's modulus. Finally, we apply the method to murine fibroblasts and we show that it is sensitive to local and temporally induced variations of the cell surface elasticity.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells.

DDT is a highly lipophilic molecule known to deplete membrane rafts of their phosphoglycolipid and cholesterol contents. However, we have recently shown that DDT can also alter the thyroid homeostasis by inhibiting TSH receptor (TSHr) internalization. The present study was undertaken to verify whether DDT goitrogenic effects are due to the insecticide acting directly on TSHr or via alteration of the membrane rafts hosting the receptor itself. Our results demonstrate that, in CHO-TSHr transfected cells, TSHr is activated in the presence of TSH, while it is inhibited following DDT exposure. DDT can also reduce the endocytic vesicular traffic, alter the extension of multi-branched microvilli along their plasma membranes and induce TSHr shedding in vesicular forms. To verify whether TSHr displacement might depend on DDT altering the raft constitution of CHO-TSHr cell membranes the extent of TSHr and lipid raft co-localization was examined by confocal microscopy. Evidence shows that receptor/raft co-localization increased significantly upon exposure to TSH, while receptors and lipid rafts become dislodged on opposite cell poles in DDT-exposed CHO-TSHr cells. As a control, under similar culturing conditions, diphenylethylene, which is known to be a lipophilic substance that is structurally related to DDT, did not affect the extent of TSHr and lipid raft co-localization in CHO-TSHr cells treated with TSH. These findings corroborate and extend our view that, in CHO cells, the DDT disrupting action on TSHr is primarily due to the insecticide acting on membranes to deplete their raft cholesterol content, and that the resulting inhibition on TSHr internalization is due to receptor dislodgement from altered raft microdomains of the plasma membrane.

Exposure to the natural alkaloid Berberine affects cardiovascular system morphogenesis and functionality during zebrafish development.

The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation.

Nonlinear indentation of single human erythrocytes under application of a localized mechanical force.

Despite the accepted notion that erythrocytes are uniquely deformable cells, the apparent Young's modulus values reported in the literature do not differ so much from those of other cells. We devised to measure the local deformability of living immobilized human erythrocytes at a low force, in contact-free mode, using an application of Scanning Ion Conductance Microscopy (SICM) previously developed in our laboratory. Reversible indentations were induced by forces of up to few hundreds pN. The indentation did not grow linearly with the force. The apparent Young's modulus varied from 0.2 to 1.5 kPa applying forces from 20 to 500 pN on a cell surface area of about 0.2 µm2, exhibiting a progressive stiffening at increasing force. Control measurements showed that A549 cells exhibit a constant value of the apparent Young's modulus (about 2 kPa) for forces up to about 800 pN. These findings show that SICM is a suitable tool to investigate cell mechanical properties, when forces in the range of tens of pN are required, in the absence of mechanical contact between probe and sample. The nonlinear deformation of the erythrocyte has to be taken into account in modeling the complex regulation mechanism of the microvascular beds.

Genistein as a Potential Anticancer Agent Against Head and Neck Squamous Cell Carcinoma.

The use of nutraceuticals as protection drugs against chronic diseases gained a vast success. Many studies found that nutraceuticals may reduce the tumorigenic actions of carcinogens, inhibiting the adhesion and proliferation of tumor cells. Genistein is a natural isoflavone preventing osteoporosis, menopause problems and heart diseases. It is also known in China and Japan for its anticancer properties. The available treatment protocols for Head and neck squamous cell cancer (HNSCC) have led to poor results and new therapies are necessary. In this paper, we will review anticancer therapeutic potential of genistein and in vitro and in vivo studies that suggest its potential role in the treatments of HNSCC.

Polymeric nanoparticles promote endocytosis of a survivin molecular beacon: Localization and fate of nanoparticles and beacon in human A549 cells.

Polymethylmethacrylate core-shell fluorescent nanoparticles promote, in human lung A549 cancer cells, the internalization of a molecular beacon (MB) specific for survivin mRNA, an anti-apoptotic protein overexpressed in cancer cells. AIMS: To design an effective drug delivery system, the knowledge of the uptake mechanism and of the nanoparticles (NPs) and MB fate is required. MATERIALS AND METHODS AND KEY FINDINGS: Experiments with dextran as marker for endocytosis showed that in the presence of NPs the number of endocytic vesicles per cell doubled and their mean size significantly (p < 0.001) increased with respect to controls in absence of NPs, indicating an involvement of NPs in the endocytotic process. By using LysoTracker™ Deep Red, as marker of lysosomes, we found that nanoparticles co-localize with lysosomes. Moreover, a cellular release of nanoparticles detected in the culture medium, suggested a role of lysosomal exocytosis in nanoparticle elimination. The MB fluorescence in proximity of the labeled Endoplasmic Reticulum was indicative that the opening of the MB occurs in proximity of its target mRNA. SIGNIFICANCE: The results show the involvement of endocytotic pathway in the uptake of NPs, which are an appropriate delivery system capable of being eliminated by cells. Furthermore the data confirm that the MB can be considered an effective tool for the intracellular sensing.

Regulation of the mechanosensitive cation channels by ATP and cAMP in leech neurons.

Single-channel recordings were used to study the modulation of stretch-activated channels (SACs) by intracellular adenosine nucleotides in identified leech neurons. These channels exhibited two activity modes, spike-like (SL) and multiconductance (MC), displaying different polymodal activation. In the absence of mechanical stimulation, internal perfusion of excised patches with ATP induced robust and reversible activation of the MC but not of the SL mode. The ATP effect on channel activity was dose-dependent within a range of 1 microM-1 mM and was induced at different values of intracellular pH and Ca2+. The non-hydrolyzable ATP analog AMP-PNP, ATP without Mg2+ or ADP also effectively enhanced MC activity. Adenosine mimicked the effect of its nucleotides. At negative membrane potentials, both ATP and adenosine activated the channel. Moreover, ATP but not adenosine induced a flickering block. Addition of cAMP during maximal ATP activation completely and reversibly inhibited the channel, with activation and deactivation times of minutes. However, cAMP alone only induced a weak and rapid channel activation, without inhibitory effects. The expression of these channels in the growth cones of leech neurons, their permeability to Ca2+ and their sensitivity to intracellular cAMP are consistent with a role in the Ca2+ oscillations associated with cell growth.

Mechanosensitive channels in neurite outgrowth.

This chapter focuses on the convergence of two areas of investigation in the past: the first is the study of the molecular basis for Ca(2+)-dependent axon pathfinding, and the second is the molecular and the functional characterization of mechanosensitive Ca(2+)-permeant cation channels (MscCa). The convergence of these two fields has reached a pivotal point when some ion channels belonging to the transient receptor potential (TRP) superfamily of proteins play essential roles in the growth cone guidance, and, independently, some of these channels are found to form MscCa of vertebrate cells. Various lines of evidence taken together make likely the idea that MscCa can substantially contribute to the spatial and temporal shaping of Ca(2+) responses in growing neurites. These findings are described and the possible contributions of MscCa to the neurite outgrowth are also discussed in the chapter.

In vitro study on anti-cancer properties of genistein in tongue cancer.

PURPOSE: Tongue cancer is an extremely aggressive disease and is characterized by a poor prognosis. It is a complex disease to treat and current therapies have produced mediocre results with many side effects. Some facts suggest that natural essences can support traditional cancer therapy by carrying out a synergistic function with chemotherapy. Therefore, we evaluated the antitumor effects of genistein on tongue carcinoma cells. METHODS: Genistein 20, 50 and 100 µM were used for 24, 48 and 72 hours on 3 tongue carcinoma cell lines. xCELLigence system was used to evaluate the effects on cell adhesion, proliferation and to calculate IC50 values. Both MTT assay and Trypan blue assay were used to evaluate alterations in cell viability, scratch assay for cell migration and Western blot analysis for expression of some proteins. RESULTS: Cell adhesion was inhibited especially between 20 and 50 µM of genistein treatment. Proliferation was reduced by 50% for treatments with 20 µM at 24 hours, with 20 or 50 µM at 48 and 50 µM at 72 hours (P<0.0001). Viability tests confirmed a proportional reduction in concentration of genistein and duration of treatments. Even cell migration was reduced significantly (P<0.001). Genistein down-regulates vitronectin, OCT4 and survivin. CONCLUSION: This in vitro study clarifies the anti-tumor effect of genistein on tongue carcinoma. In vivo studies are needed to confirm these data and develop a suitable delivery system that is capable of acting directly on tumor.

Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells.

One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm2), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm2). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation.

A Tph2GFP Reporter Stem Cell Line To Model in Vitro and in Vivo Serotonergic Neuron Development and Function.

Modeling biological systems in vitro has contributed to clarification of complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated toward specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2GFP, in which the vital reporter GFP replaces the tryptophan hydroxylase 2 (Tph2) gene. Tph2GFP mES cells selectively express GFP upon in vitro differentiation toward the serotonergic fate, they synthesize serotonin, possess excitable membranes, and show the typical morphological, morphometrical, and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP, we highlighted by time-lapse video microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that predifferentiated Tph2GFP cells are able to terminally differentiate, integrate, and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2GFP mES cell line as a useful tool allowing accurate developmental and dynamic studies and representing a reliable platform for the study of serotonergic neurons in health and disease.

Characterization of tip size and geometry of the pipettes used in scanning ion conductance microscopy.

Scanning ion-conductance microscopy (SICM) belongs to the family of scanning-probe microscopies. The spatial resolution of these techniques is limited by the size of the probe. In SICM the probe is a pipette, obtained by heating and pulling a glass capillary tubing. The size of the pipette tip is therefore an important parameter in SICM experiments. However, the characterization of the tip is not a consolidated routine in SICM experimental practice. In addition, potential and limitations of the different methods available for this characterization may not be known to all users. We present an overview of different methods for characterizing size and geometry of the pipette tip, with the aim of collecting and facilitating the use of several pieces of information appeared in the literature in a wide interval of time under different disciplines. In fact, several methods that have been developed for pipettes used in cell physiology can be also fruitfully employed in the characterization of the SICM probes. The overview includes imaging techniques, such as scanning electron microscopy and atomic Force microscopy, and indirect methods, which measure some physical parameter related to the size of the pipette. Examples of these parameters are the electrical resistance of the pipette filled with a saline solution and the surface tension at the pipette tip. We discuss advantages and drawbacks of the methods, which may be helpful in answering a wide range of experimental questions.

Zolfino landrace (Phaseolus vulgaris L.) from Pratomagno: general and specific features of a functional food.

BACKGROUND: The Zolfino bean is a variety of Phaseolus vulgaris, which is cultivated in a limited area of Tuscany, Italy, and is widely appreciated for its flavor and culinary uses. OBJECTIVES: A yellow Zolfino landrace cultivated in the Leccio-Reggello area was characterized and compared with three other varieties of Phaseolus vulgaris (i.e. the Borlotto, Cannellino, and Corona beans) in terms of its general features and potential as an antioxidant/anti-inflammatory agent. DESIGN: The length, width, thickness, equatorial section surface, weight, volume, and seed coat section were measured in all the beans. The seed surface area was also estimated by an original empirical method. The ability of the different beans to interfere with the enzymes of the polyol pathway (that is, aldose reductase (AR) and sorbitol dehydrogenase) was tested using the supernatant after soaking the beans at room temperature and after thermal treatment, which simulated the bean-cooking process in a controlled fashion. RESULTS: Concerning the general features, Zolfino was comparable with other beans, except Corona, in terms of surface-volume ratio, which possesses the lowest tegument thickness. Moreover, Zolfino appears the most effective in inhibiting AR activity. The inhibitory ability is unaffected by thermal treatment and appears to be associated with compound(s) present in the coat of the bean. CONCLUSIONS: The ability of Zolfino to inhibit AR, thus reducing the flux of glucose through the polyol pathway, highlights the features of Zolfino as a functional food, potentially useful in treating the dysfunctions linked to the hyperactivity of AR, such as diabetic complications or inflammatory responses.

Calmodulin antagonists do not inhibit IK(Ca) channels of human erythrocytes.

Patch-clamp recordings were performed to study the effects of three calmodulin (CaM) antagonists on the gating of intermediate calcium-activated K(+) channels (IK(Ca)) of human erythrocytes. In the cell-attached configuration, both opening frequency and open probability of IK(Ca) channels were not significantly different in control cells and in those incubated with calmidazolium, trifluoperazine or W7. IK(Ca) channels in excised membrane patches, were normally activated by the calcium bathing the cytoplasmic side in the presence of CaM antagonists, at calcium concentrations ranging from 10(-7) to 10(-3) M. The activity of IK(Ca) channels, which had been previously up-modulated by an endogenous cAMP-dependent protein kinase, was not inhibited when perfused with CaM antagonists. The results presented in this study demonstrate that calmodulin antagonists do not inhibit the activity of native IK(Ca) channels of human erythrocytes. These data are in accordance with findings on the cloned IK(Ca) indicating that calmodulin is constitutively associated with these channels.

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C.

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.

Human dermal fibroblasts HDFa can be used as an appropriate healthy control for PMMA nanoparticles-survivin molecular beacon cellular uptake studies.

Survivin is a member of the inhibitor of apoptosis protein family (IAPs); besides its inhibitory action on apoptosis, it is also involved in the regulation of cell division. The protein expression is up-regulated in several cancers, being involved in the tumor progression and evasion of apoptosis. In line with its physiological roles, it is expressed also in several healthy tissues. The high expression of survivin in cancer cells correlates to poor prognosis and resistance to chemotherapeutic treatment, thus making this protein an attractive target in anticancer therapy. The dual role of survivin in cells, regulation of cell division and inhibition of apoptosis, combined with controversial data concerning the expression in normal tissues, emphasize the need to have an appropriate healthy control for in vitro studies. Aim of this study is to highlight this problem and to clarify the experimental conditions in which HDFa fibroblasts represent a negative control for survivin mRNA expression while ensuring the NPs-MB uptake. In this paper, by using confocal microscopy analysis supported by RT- and real-time-PCR experiments studies, we showed that HDFa fibroblasts represent a healthy negative control for survivin mRNA expression, only at very low cell density or at confluence. At the same time, we demonstrated that HDFa at any cell density are able to internalize NPs-MB after 6h of treatment.