Hybrid Nanoparticle-Assisted Chemo-Photothermal Therapy and Photoacoustic Imaging in a Three-Dimensional Breast Cancer Cell Model.

Bioinspired nanoparticles have recently been gaining attention as promising multifunctional nanoplatforms for therapeutic applications in cancer, including breast cancer. Here, the efficiency of the chemo-photothermal and photoacoustic properties of hybrid albumin-modified nanoparticles (HSA-NPs) loaded with doxorubicin was evaluated in a three-dimensional breast cancer cell model. The HSA-NPs showed a higher uptake and deeper penetration into breast cancer spheroids than healthy breast cell 3D cultures. Confocal microscopy revealed that, in tumour spheroids incubated with doxorubicin-loaded NPs for 16 h, doxorubicin was mainly localised in the cytoplasm, while a strong signal was detectable at the nuclear level after 24 h, suggesting a time-dependent uptake. To evaluate the cytotoxicity of doxorubicin-loaded NPs, tumour spheroids were treated for up to 96 h with increasing concentrations of NPs, showing marked toxicity only at the highest concentration of doxorubicin. When doxorubicin administration was combined with laser photothermal irradiation, enhanced cytotoxicity was observed at lower concentrations and incubation times. Finally, the photoacoustic properties of doxorubicin-loaded NPs were evaluated in tumour spheroids, showing a detectable signal increasing with NP concentration. Overall, our data show that the combined effect of chemo-photothermal therapy results in a shorter exposure time to doxorubicin and a lower drug dose. Furthermore, owing to the photoacoustic properties of the NPs, this nanoplatform may represent a good candidate for theranostic applications.

Nanoparticles Design for Theranostic Approach in Cancer Disease.

Presently, there are no conclusive treatments for many types of cancer, mainly due to the advanced phase of the disease at the time of diagnosis and to the side effects of existing therapies. Present diagnostic and therapeutic procedures need to be improved to supply early detection abilities and perform a more specific therapy with reduced systemic toxicity. In this review, improvements in nanotechnology allowing the design of multifunctional nanoparticles for cancer detection, therapy, and monitoring are reported. Nanoparticles, thanks to the nanomaterials they are made of, can be used as contrast agents for various diagnostic techniques such as MRI, optical imaging, and photoacoustic imaging. Furthermore, when used as drug carriers, they can accumulate in tumor tissues through the passive or/and active targeting, protect encapsulated drugs from degradation, raise tumor exposure to chemotherapeutic agents improving treatment effects. In addition, nanocarriers can simultaneously deliver more than one therapeutic agent enhancing the effectiveness of therapy and can co-deliver imaging and therapy agents to provide integration of diagnostics, therapy, and follow-up. Furthermore, the use of nanocarriers allows to use different therapeutic approaches, such as chemotherapy and hyperthermia to exploit synergistic effects. Theranostic approach to diagnose and treat cancer show a great potential to improve human health, however, despite technological advances in this field, the transfer into clinical practice is still a long way off.

Probing Denaturation of Protein A via Surface-Enhanced Infrared Absorption Spectroscopy.

We apply surface-enhanced infrared absorption (SEIRA) spectroscopy to monitor the denaturation process of a surface-bound protein A monolayer. Our proposed platform relies on a plasmonic metasurface comprising different spatial subregions ("pixels") that are engineered to exhibit different resonances covering the infrared region of the electromagnetic spectrum that is matched to the vibrational modes of the Amide groups. Specifically, we are able to determine changes in the Amide I and Amide II vibration coupled modes, by comparing the SEIRA reflectance spectra pertaining to the native state and a denatured state induced by a pH variation. In particular, we observe some evident red-shifts in the principal Amide I mode and the Amide II vibration coupled modes (attributable to the breaking of hydrogen bonds), which result in insurmountable barriers for refolding. Thanks to the strong field localization, and consequent enhancement of the light-matter interactions, our proposed sensing platform can operate with extremely small amounts of an analyte, with an estimated detection limit of about 3 femtomoles of molecules.

Controlled Release of Doxorubicin for Targeted Chemo-Photothermal Therapy in Breast Cancer HS578T Cells Using Albumin Modified Hybrid Nanocarriers.

Hybrid nanomaterials have attracted research interest owing to their intriguing properties, which may offer new diagnostic options with triggering features, able to realize a new kind of tunable nanotherapeutics. Hybrid silica/melanin nanoparticles (NPs) containing silver seeds (Me-laSil_Ag-HSA NPs) disclosed relevant photoacoustic contrast for molecular imaging. In this study we explored therapeutic function in the same nanoplatform. For this purpose, MelaSil_Ag-HSA were loaded with doxorubicin (DOX) (MelaSil_Ag-HSA@DOX) and tested to assess the efficiency of drug delivery combined with concurrent photothermal treatment. The excellent photothermal properties allowed enhanced cytotoxic activity at significantly lower doses than neat chemotherapeutic treatment. The results revealed that MelaSil_Ag-HSA@DOX is a promising platform for an integrated photothermal (PT) chemotherapy approach, reducing the efficacy concentration of the DOX and, thus, potentially limiting the several adverse side effects of the drug in in vivo treatments.

Advanced DNA Detection via Multispectral Plasmonic Metasurfaces.

We propose and demonstrate a sensing platform based on plasmonic metasurfaces for the detection of very low concentrations of deoxyribo-nucleic acid (DNA) fragments. The platform relies on surface-enhanced infrared absorption spectroscopy, implemented via a multispectral metasurface. Specifically, different regions ("pixels") are engineered so as to separately cover the medium-infrared range of the electromagnetic spectrum extending from the functional-groups to the fingerprint region of a single analyte. In conjunction with a suitable bio-functionalization, this enables univocal and label-free recognition of specific molecules. For experimental validation, we fabricate a large-area gold metasurface on a silicon chip, and functionalize it with a recognition layer of peptide nucleic acid (PNA). Our experimental results indicate the possibility to detect complementary DNA fragments in concentrations as low as 50 fM, i.e., well below the value attained by standard methods, with additional advantages in terms of processing time, versatility and ease of implementation/operation.

Nanoparticle Surface Functionalization: How to Improve Biocompatibility and Cellular Internalization.

The use of nanoparticles (NP) in diagnosis and treatment of many human diseases, including cancer, is of increasing interest. However, cytotoxic effects of NPs on cells and the uptake efficiency significantly limit their use in clinical practice. The physico-chemical properties of NPs including surface composition, superficial charge, size and shape are considered the key factors that affect the biocompatibility and uptake efficiency of these nanoplatforms. Thanks to the possibility of modifying physico-chemical properties of NPs, it is possible to improve their biocompatibility and uptake efficiency through the functionalization of the NP surface. In this review, we summarize some of the most recent studies in which NP surface modification enhances biocompatibility and uptake. Furthermore, the most used techniques used to assess biocompatibility and uptake are also reported.

Albumin-Modified Melanin-Silica Hybrid Nanoparticles Target Breast Cancer Cells via a SPARC-Dependent Mechanism.

Bioconjugation of a recently developed photoacoustic nanoprobe, based on silica-templated eumelanin-silver hybrid nanoparticles (MelaSil_Ag-NPs), with human serum albumin (HSA) is disclosed herein as an efficient and practical strategy to improve photostability and to perform SPARC mediated internalization in breast cancer cells. Modification of NPs with HSA induced a slight viability decrease in breast cancer cells (HS578T) and normal breast cells (MCF10a) when incubated with HSA-NPs up to 100 µg/mL concentration for 72 h and a complete suppression of hemotoxicity for long incubation times. Uptake experiments with MelaSil_Ag-HSA NPs indicated very high and selective internalization via SPARC in HS578T (SPARC positive cells) but not in MCF10a (SPARC negative cells), as evaluated by using endocytosis inhibitors. The binding of SPARC to HSA was confirmed by Co-IP and Dot-blot assays. Additional studies were performed to analyze the interaction of MelaSil_Ag-HSA NPs with protein corona. Data showed a dramatic diminution of interacting proteins in HSA conjugated NPs compared to bare NPs. HSA-coated MelaSil_Ag-NPs are thus disclosed as a novel functional nanohybrid for potential photoacoustic imaging applications.

Silver-nanoparticles as plasmon-resonant enhancers for eumelanin's photoacoustic signal in a self-structured hybrid nanoprobe.

Developing safe and high efficiency contrast tools is an urgent need to allow in vivo applications of photoacoustics (PA), an emerging biomolecular imaging methodology, with poor invasiveness, deep penetration, high spatial resolution and excellent endogenous contrast. Eumelanins hold huge promise as biocompatible, endogenous photoacoustic contrast agents. However, their huge potential is still unexplored due to the difficulty to achieve at the same time poor aggregation in physiologic environment and high PA contrast. This study addresses both issues through the design of a biocompatible photoacoustic nanoprobe, named MelaSil_Ag-NPs, relying on silica-templated eumelanin formation as well as eumelanins redox and metal chelating properties to reduce Ag+ ions and control the growth of generated metal nanoparticles. This strategy allowed self-structuring of the system into a core-shell architecture, where the Ag core was found to boost PA signal, despite the poor eumelanin content. Obtained hybrid nanoplatforms, showed stable photoacoustic properties even under long irradiation. Furthermore, conjugation with rhodamine isothiocyanate allowed particles detection through fluorescent imaging proving their multifunctional potentialities. In addition, they were stable towards aggregation and efficiently endocytosed by human pancreatic cancer cells (BxPC3 and Panc-1) displaying no significant cytotoxicity. Such numerous features prove huge potential of those nanoparticles as a multifunctional platform for biomedical applications.

Optical Biosensors Based on Photonic Crystals Supporting Bound States in the Continuum.

A novel optical label-free bio-sensing platform based on a new class of resonances supported in a photonic crystal metasurface is reported herein. Molecular binding is detected as a shift in the resonant wavelength of the bound states in the continuum of radiation modes. The new configuration is applied to the recognition of the interaction between protein p53 and its protein regulatory partner murine double minute 2 (MDM2). A detection limit of 66 nM for the protein p53 is found. The device provides an excellent interrogation stability and loss-free operation, requires minimal optical interrogation equipment and can be easily optimized to work in a wide wavelength range.

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging.

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non-toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label-free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co-localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses.

Nanoparticle-based strategy for personalized B-cell lymphoma therapy.

B-cell lymphoma is associated with incomplete response to treatment, and the development of effective strategies targeting this disease remains challenging. A new personalized B-cell lymphoma therapy, based on a site-specific receptor-mediated drug delivery system, was developed in this study. Specifically, natural silica-based nanoparticles (diatomite) were modified to actively target the antiapoptotic factor B-cell lymphoma/leukemia 2 (Bcl2) with small interfering RNA (siRNA). An idiotype-specific peptide (Id-peptide) specifically recognized by the hypervariable region of surface immunoglobulin B-cell receptor was exploited as a homing device to ensure specific targeting of lymphoma cells. Specific nanoparticle uptake, driven by the Id-peptide, was evaluated by flow cytometry and confocal microscopy and was increased by approximately threefold in target cells compared with nonspecific myeloma cells and when a random control peptide was used instead of Id-peptide. The specific internalization efficiency was increased by fourfold when siRNA was also added to the modified nanoparticles. The modified diatomite particles were not cytotoxic and their effectiveness in downregulation of gene expression was explored using siRNA targeting Bcl2 and evaluated by quantitative real-time polymerase chain reaction and Western blot analyses. The resulting gene silencing observed is of significant biological importance and opens new possibilities for the personalized treatment of lymphomas.

Bioengineered Silicon Diatoms: Adding Photonic Features to a Nanostructured Semiconductive Material for Biomolecular Sensing.

Native diatoms made of amorphous silica are first converted into silicon structures via magnesiothermic process, preserving the original shape: electron force microscopy analysis performed on silicon-converted diatoms demonstrates their semiconductor behavior. Wet surface chemical treatments are then performed in order to enhance the photoluminescence emission from the resulting silicon diatoms and, at the same time, to allow the immobilization of biological probes, namely proteins and antibodies, via silanization. We demonstrate that light emission from semiconductive silicon diatoms can be used for antibody-antigen recognition, endorsing this material as optoelectronic transducer.

Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A.

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.

Surface bioengineering of diatomite based nanovectors for efficient intracellular uptake and drug delivery.

Diatomite is a natural porous silica material of sedimentary origin. Due to its peculiar properties, it can be considered as a valid surrogate of synthetic porous silica for nano-based drug delivery. In this work, we exploit the potential of diatomite nanoparticles (DNPs) for drug delivery with the aim of developing a successful dual-biofunctionalization method by polyethylene glycol (PEG) coverage and cell-penetrating peptide (CPP) bioconjugation, to improve the physicochemical and biological properties of the particles, to enhance the intracellular uptake in cancer cells, and to increase the biocompatibility of 3-aminopropyltriethoxysilane (APT) modified-DNPs. DNPs-APT-PEG-CPP showed hemocompatibility for up to 200 µg mL(-1) after 48 h of incubation with erythrocytes, with a hemolysis value of only 1.3%. The cytotoxicity of the modified-DNPs with a concentration up to 200 µg mL(-1) and incubation with MCF-7 and MDA-MB-231 breast cancer cells for 24 h, demonstrated that PEGylation and CPP-bioconjugation can strongly reduce the cytotoxicity of DNPs-APT. The cellular uptake of the modified-DNPs was also evaluated using the above mentioned cancer cell lines, showing that the CPP-bioconjugation can considerably increase the DNP cellular uptake. Moreover, the dual surface modification of DNPs improved both the loading of a poorly water-soluble anticancer drug, sorafenib, with a loading degree up to 22 wt%, and also enhanced the drug release profiles in aqueous solutions. Overall, this work demonstrates that the biofunctionalization of DNPs is a promising platform for drug delivery applications in cancer therapy as a result of its enhanced stability, biocompatibility, cellular uptake, and drug release profiles.

New insights on the interaction between the isoforms 1 and 2 of human translation elongation factor 1A.

The eukaryotic translation elongation factor 1A (eEF1A) is a moonlighting protein that besides to its canonical role in protein synthesis is also involved in many other cellular processes such as cell survival and apoptosis. In a previous work, we identified eEF1A Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and apoptosis of human cancer cells. We proposed that the phosphorylation of eEF1A by C-Raf required the presence of both eEF1A isoforms thus suggesting the formation of a potential eEF1A heterodimer owning regulatory properties. This study aimed at investigating the cellular localization and interaction between two eEF1A isoforms. To this end, we developed chimera proteins by adding at the N-terminal end of both eEF1A1 and eEF1A2 cyan fluorescence protein (mCerulean) and yellow fluorescence protein (mVenus), respectively. The fluorescent eEF1A1 and eEF1A2 chimeras were both addressed to COS-7 cells and found co-localized in the cytoplasm at the level of cellular membranes. We highlighted FRET between the labeled N-termini of eEF1A isoforms. The intra-molecular FRET of this chimera was about 17%. Our results provide novel information on the intracellular distribution and interaction of eEF1A isoforms.

Ectopic expression of gastrokine 1 in gastric cancer cells up-regulates tight and adherens junction proteins network.

Gastrokine 1 (GKN1) is a stomach-specific protein important in the replenishment of the surface lumen epithelial cell layer and in maintaining mucosal integrity. A role in cell proliferation and differentiation has also been hypothesized. Despite these findings, the function(s) as well as the cellular localization of GKN1 in the cellular machinery are currently not clarified. The investigation of subcellular localization of GKN1 in gastric cancer cells can provide insights into its potential cellular roles. Subcellular fractions of gastric cancer cells (AGS) transfected with full-length GKN1 (flGKN1) or incubated with recombinant GKN1 (rGKN1) lacking the first 20 amino acids at N-terminal were analyzed by Western blot and confocal microscopy and compared with those from normal gastric tissue. Wild type GKN1 (wtGKN1) and flGKN1 were revealed in the cytoplasm and in the membrane fractions of gastric cells, whereas rGKN1 was revealed in the cytoplasmic fractions, but a high amount was detected in the membrane pellet of the AGS lysate. The cellular distribution of GKN1 was also confirmed by confocal microscopy. The purified protein was also used to highlight its possible association with actin through confocal microscopy, pelleting assay, and size-exclusion chromatography. GKN1 co-localizes with actin in normal gastric tissue, but no direct interaction was observed between the two proteins in vitro. Most likely, GKN1 indirectly participates in actin stabilization since its overexpression in gastric cancer cells strongly increases the expression of tight and adherens junction proteins.

B-cell receptor-guided delivery of peptide-siRNA complex for B-cell lymphoma therapy.

BACKGROUND: Despite the clinical response of conventional anticancer therapy, including chemotherapeutic treatments, radiation therapy and corticosteroids, tumorigenic B-cell lymphomas show an incomplete response to clinical practices that result in a minimal residual disease (MRD) where few residual neoplastic cells undetected in vivo, replenish the cancer cell reservoir. This scenario, which is also shared with other cancer diseases, requires the development of strategies to advance in novel, selective targeting toward the tumorigenic cells that survive to the anticancer agents. METHODS: Here, we have taken advantage of the therapeutic properties of an idiotype specific peptide (pA20-36) that bind specifically to murine B-lymphoma cells in the setting of an anti cancer strategy, based on the selected delivery of electrostatic-based complex, peptide-siRNA. To this end, two engineered, arginine rich, peptides that included the pA20-36 targeting sequence were designed to bind fluorescent-labelled siRNA. One peptide presented 9 Arg at the C-terminal of pA20-36 whereas the other included 5 Arg at the N- and C-terminus, respectively. RESULTS: Compared to the control and random peptide-siRNA complexes, both pA20-36-siRNA complexes were endowed with the selective delivering of fluorescent-labelled siRNA toward the A20 murine B-cell lymphoma, as evaluated by cytofluorimetry and confocal microscopy, whereas fluorescent-labelled siRNA alone was not internalized in the selected cells. Compared to peptide controls, the use of the modified pA20-36 peptides complexed with siRNA anti-GAPDH and anti-Bcl2 showed a down-regulation in the expression levels of the corresponding genes. CONCLUSIONS: Peptide-siRNA complex can be suitable tool for both selective peptide-driven cell targeting and gene silencing. In this setting, the improvement of this strategy is expected to provide a safe and non-invasive approach for the delivery of therapeutic molecules.

A new strategy for label-free detection of lymphoma cancer cells.

In this paper, a new strategy for highly selective and sensitive direct detection of lymphoma cells by exploiting the interaction between a peptide and its B-cell receptor, has been evaluated. In particular, an idiotype peptide, able to specifically bind the B-cell receptor of A20 cells in mice engrafted with A20 lymphoma, has been used as molecular probe. The new detection technique has been demonstrated on a planar crystalline silicon chip. Coverage of 85% of silicon surface and detection efficiency of 8.5 × 10(-3) cells/µm(2) were obtained. The recognition strategy promises to extend its application in studying the interaction between ligands and their cell-surface receptors.

Diatomite biosilica nanocarriers for siRNA transport inside cancer cells.

BACKGROUND: Diatomite is a natural porous biomaterial of sedimentary origin, formed by fragments of diatom siliceous skeletons, called "frustules". Due to large availability in many areas of the world, chemical stability, and non-toxicity, these fossil structures have been widespread used in lot of industrial applications, such as food production, water extracting agent, production of cosmetics and pharmaceutics. However, diatomite is surprisingly still rarely used in biomedical applications. In this work, we exploit diatomite nanoparticles for small interfering ribonucleic acid (siRNA) transport inside human epidermoid cancer cells (H1355). METHODS: Morphology and composition of diatomite microfrustules (average size lower than 40µm) are investigated by scanning electron microscopy equipped by energy dispersive X-ray spectroscopy, Fourier transform infrared analysis, and photoluminescence measurements. Nanometric porous particles (average size lower than 450nm) are obtained by mechanical crushing, sonication, and filtering of micrometric frustules. siRNA bioconjugation is performed on both micrometric and nanometric fragments by silanization. RESULTS: In-vitro experiments show very low toxicity on exposure of the cells to diatomite nanoparticle concentration up to 300µg/ml for 72h. Confocal microscopy imaging performed on cancer cells incubated with siRNA conjugated nanoparticles demonstrates a cytoplasmatic localization of vectors. Gene silencing by delivered siRNA is also demonstrated. CONCLUSION: Our studies endorse diatomite nanoparticles as non-toxic nanocarriers for siRNA transport in cancer cells. GENERAL SIGNIFICANCE: siRNA is a powerful molecular tool for cancer treatment but its delivery is inefficient due to the difficulty to penetrate the cell membrane. siRNA-diatomite nanoconjugate may be well suited for delivery of therapeutic to cancer cells.