Giamaica Conti

PEG-capped, lanthanide doped GdF3 nanoparticles: luminescent and T2 contrast agents for optical and MRI multimodal imaging

A facile method for the synthesis of water dispersible Er(3+)/Yb(3+) and Tm(3+)/Yb(3+) doped upconverting GdF(3) nanoparticles is reported. Strong upconversion emissions are observed in the red (for Er/Yb doped) and near-infrared (for Tm/Yb doped) regions upon laser excitation at 980 nm. The PEG coating ensures a good dispersion of the system in water and reduces the radiationless de-excitation of the excited states of the Er(3+) and Tm(3+) ions by water molecules. The r(2) relaxivity values are quite high with respect to the common T(2)-relaxing agents (22.6 ± 3.4 mM(-1) s(-1) and 15.8 ± 3.4 mM(-1) s(-1) for the Tm/Yb and Er/Yb doped samples, respectively), suggesting that the present NPs can be interesting as T(2) weighted contrast agents for proton MRI purpose. Preliminary experiments conducted in vitro, in stem cell cultures, and in vivo, after subcutaneous injection of the lanthanide-doped GdF(3) NPs, indicate scarce toxic effects. After an intravenous injection in mice, the GdF(3) NPs localize mainly in the liver. The present results indicate that the present Er(3+)/Yb(3+) and Tm(3+)/Yb(3+) doped GdF(3) NPs are suitable candidates to be efficiently used as bimodal probes for both in vitro and in vivo optical and magnetic resonance imaging.

Improved Doxorubicin Encapsulation and Pharmacokinetics of Ferritin–Fusion Protein Nanocarriers Bearing Proline, Serine, and Alanine Elements

A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.

Multifunctional nanoprobes based on upconverting lanthanide doped CaF2: towards biocompatible materials for biomedical imaging

Water dispersible Gd3+,Yb3+,Er3+ and Gd3+,Yb3+,Tm3+ doped CaF2 nanoparticles (NPs) were prepared by one-pot hydrothermal synthesis using citrate ions as capping agents without the need for any post-synthesis reaction. UC emissions are easily observed in the visible and infrared regions upon NIR diode laser excitation at 980 nm. EPR spectroscopy confirms the substitutional nature of the rare-earth doping, while magnetometric studies reveal that the NPs have a useful magnetization. MRI experiments conducted in vivo show that after 40 min from the injection, the NPs localize in the liver and spleen. Electron microscopy images of liver tissue reveal that the NPs are located in the Kupffer cells, although a small amount is also found in the hepatocytes. An excitation with a 980 nm emission on the excised liver and epithelial tissue induces clearly visible UC emission. The local temperature upon 980 nm irradiation was monitored in situ and it was found to increase slowly with the exposure time, maintaining under 1-2 °C for less than 60 second exposure. The NPs show a low toxicity towards cultured HeLa cells and human primary dendritic cells (DCs), and did not induce pro-inflammatory cytokine secretion by cultured human DCs, indicating that the NPs do not cause relevant adverse reactions in immune cells. Therefore, the present NPs are suitable candidates to be efficiently used in surgery applications, where spatial resolution and lack of harmful effects on human health are important issues.

Selective delivery of doxorubicin by novel stimuli-sensitive nano-ferritins overcomes tumor refractoriness

Human ferritin heavy chain (HFt) has been demonstrated to possess considerable potential for targeted delivery of drugs and diagnostic agents to cancer cells. Here, we report the development of a novel HFt-based genetic construct (HFt-MP-PAS) containing a short peptide linker (MP) between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). The peptide linker contains a matrix-metalloproteinases (MMPs) cleavage site that permits the protective PAS shield to be removed by tumor-driven proteolytic cleavage within the tumor microenvironment. For the first time HFt-MP-PAS ability to deliver doxorubicin to cancer cells, subcellular localization, and therapeutic efficacy on a xenogeneic mouse model of a highly refractory to conventional chemotherapeutics type of cancer were evaluated. HFt-MP-PAS-DOXO performance was compared with the novel albumin-based drug delivery system INNO-206, currently in phase III clinical trials. The results of this work provide solid evidence indicating that the stimuli-sensitive, long-circulating HFt-MP-PAS nanocarriers described herein have the potential to be exploited in cancer therapy.

Magnetic nanoparticles from Magnetospirillum gryphiswaldense increase the efficacy of thermotherapy in a model of colon carcinoma.

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.

The trochanteric fat pad

Technological developments based on the use of autologous white adipose tissue (WAT) attracted attention to minor fat depots as possible sources of adipose tissue. In plastic surgery, the trochanteric fatty pad is one of the most used WAT depots for its location and organoleptic characteristics that make it particularly suitable for reconstructive procedures. Despite its wide use in clinic, the structure of this depot has never been studied in detail and it is not known if structural differences exist among trochanteric fat and other subcutaneous WAT depots. The present study was performed on trochanteric fat pad with the aim to clarify the morphology of its adipocytes, stroma and microcirculation, with particular reference to the stem niches. Histological and ultrastructural studies showed that the main peculiar feature of the trochanteric fat concerns its stromal component, which appears less dense than in the other subcutaneous WATs studied. The intra-parenchymal collagen stroma is poor and the extracellular compartment shows large spaces, filled with electron-light material, in which isolated collagen bundles are present. The adipocytes are wrapped in weak and easily detachable collagen baskets. These connective sheaths are very thin compared to the sheaths in other subcutaneous WAT depots. The capillaries are covered by large, long and thin elements surrounded by an external lamina; these perivascular cells are poor in organelles and mainly contain poly-ribosomes. In conclusion, when compared to other WAT deposits, the trochanteric fatty pad shows structural peculiarities in its stroma and microcirculation suggesting a high regenerative potential. Resistance, dissociability, microvascular weft and high regenerative potential make the trochanteric fatty pad a privileged source for harvesting in autologous WAT-based regenerative procedures.

Highly sensitive electrochemiluminescence detection of a prostate cancer biomarker

Prostate-specific membrane antigen (PSMA), a glycoprotein expressed in the prostatic epithelium endowed with enzymatic activity, is a very promising diagnostic marker for the early detection of prostate cancer. In this study, we report a novel electrochemiluminescence ELISA-like immunosensor based on carbon nanotubes and a highly specific sandwich immunoassay for the PSMA detection. To fabricate the device, an optically transparent electrode was modified with doubly functionalized multi-walled carbon nanotubes carrying amine groups and a monoclonal anti-PSMA antibody. Subsequently, to complete the sandwich immunosensing device, a second specific monoclonal anti-PSMA antibody was labelled with a electrochemiluminescent probe. Under optimized experimental conditions, the proposed sensing device exhibits a performance exceeding that of the state of-the-art in terms of the limit of detection (LOD) and limit of quantification (LOQ) as good as 0.88 ng mL-1 and 2.60 ng mL-1, respectively, in real complex samples such as cell lysates. In addition, the unique role of carbon nanotubes is also discussed by comparison with an analogue sensor assembled without the nanocarbon-based material.

Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells

Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2) receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents.

Hyaluronic Acid (HA) Scaffolds and Multipotent Stromal Cells (MSCs) in Regenerative Medicine

Traditional methods for tissue regeneration commonly used synthetic scaffolds to regenerate human tissues. However, they had several limitations, such as foreign body reactions and short time duration. In order to overcome these problems, scaffolds made of natural polymers are preferred. One of the most suitable and widely used materials to fabricate these scaffolds is hyaluronic acid. Hyaluronic acid is the primary component of the extracellular matrix of the human connective tissue. It is an ideal material for scaffolds used in tissue regeneration, thanks to its properties of biocompatibility, ease of chemical functionalization and degradability. In the last few years, especially from 2010, scientists have seen that the cell-based engineering of these natural scaffolds allows obtaining even better results in terms of tissue regeneration and the research started to grow in this direction. Multipotent stromal cells, also known as mesenchymal stem cells, plastic-adherent cells isolated from bone marrow and other mesenchymal tissues, with self-renew and multi-potency properties are ideal candidates for this aim. Normally, they are pre-seeded onto these scaffolds before their implantation in vivo. This review discusses the use of hyaluronic acid-based scaffolds together with multipotent stromal cells, as a very promising tool in regenerative medicine.

Characterization of magnetic nanoparticles from Magnetospirillum Gryphiswaldense as potential theranostics tools.

We investigated the theranostic properties of magnetosomes (MNs) extracted from magnetotactic bacteria, promising for nanomedicine applications. Besides a physico-chemical characterization, their potentiality as mediators for magnetic fluid hyperthermia and contrast agents for magnetic resonance imaging, both in vitro and in vivo, are here singled out. The MNs, constituted by magnetite nanocrystals arranged in chains, show a superparamagnetic behaviour and a clear evidence of Verwey transition, as signature of magnetite presence. The phospholipid membrane provides a good protection against oxidation and the MNs oxidation state is stable over months. Using an alternate magnetic field, the specific absorption rate was measured, resulting among the highest reported in literature. The MRI contrast efficiency was evaluated by means of the acquisition of complete NMRD profiles. The transverse relaxivity resulted as high as the one of a former commercial contrast agent. The MNs were inoculated into an animal model of tumour and their presence was detected by magnetic resonance images two weeks after the injection in the tumour mass.