1,721,009 research outputs found
Experimental studies aimed to assess the usefulness of nanoparticles as diagnostic and theranostic agents
No full textNell’ultimo decennio, il campo della nanomedicina ha registrato progressi notevoli sia nella sintesi dei nano composti che nello studio delle loro possibili applicazioni biomediche. Questo lavoro di tesi si inquadra nell’ambito delle applicazioni biomediche di nano composti caratterizzati da proprietà diagnostiche e/o terapeutiche. Sono state studiate particelle a base di ossido di Fe caratterizzate dalla capacità di influenzare il segnale di risonanza magnetica di cellule/tessuti e particelle fluorescenti rivelabili tramite tecniche di imaging ottico. Una delle nanoparticelle oggetto della tesi è caratterizzata anche da proprietà terapeutiche dovute alla capacità di sviluppare calore se stimolate da un campo magnetico alternato. Quest’ultimo campo di ricerca è stato recentemente definito teranostica.
Scopo della tesi è stato in generale quello di definire e ottimizzare alcune procedure sperimentali che possano essere di utilità generale nella sperimentazione sull’efficacia diagnostica, sulla tossicità e sull’efficacia terapeutica delle nano particelle. Nello specifico sono stati studiati i seguenti nano composti:
1) Una nano particella a base di ossido di Fe attualmente in sperimentazione clinica per la diagnosi di metastasi linfonodali (P904, Guerbet Francia).
2) Due nano particelle dopate con ioni lantanidi e caratterizzate da proprietà magnetiche e dalla proprietà ottica di upconversion (bimodali).
3) Una nano particella a base di ossido di Fe di origine biologica (magnetosomi) caratterizzata da potenzialità diagnostiche (visibile in Risonanza Magnetica) e terapeutiche (come agente di ipertermia nella terapia dei tumori).
Sebbene molte delle metodiche sperimentali utilizzate negli studi sui nano composti oggetto della tesi siano comuni, si è ritenuto opportuno descrivere in capitoli separati i risultati ottenuti con ciascuna di queste nano particelle. Specificamente nel Cap.3 sono riportati gli studi effettuati in MRI utilizzando P904 su un modello sperimentale di metastasi linfonodale appositamente messo a punto. I risultati ottenuti dimostrano una significativa sensibilità e specificità del P904 nella diagnosi delle metastasi linfonodali. Nel Cap. 4 sono stati descrittistudi effettuati sulla tossicità, sull’uptake cellulare in vitro e sulla biodistribuzione delle nano particelle bimodali. I nostri risultati mostrano scarsa tossicità cellulare e potenziale utilizzo come mezzi di contrasto per imaging cellulare e per MRI. Nel Cap. 5 infine sono stati riportati gli studi effettuati sull’utilizzo dei magnetosomi come mezzi di contrasto per MRI e nella termoterapia dei tumori. I risultati sperimentali ottenuti dimostrano che queste nano particelle innovative e di origine biologica hanno potenziali applicazioni teranostiche come mezzi di contrasto per MRI e come agenti per termoterapia.During the last decade, significant advances have been performed in the field of nanomedicine both in the synthesis of novel nano compounds and in their biomedical applications. This thesis is focused on biomedical applications of nanoparticles characterized by diagnostic and / or therapeutic properties. Classes of nano compounds investigated in this thesis include iron oxide nanoparticles, characterized by the capability to affect the magnetic resonance signal of cells/tissues, and fluorescent particles detectable by optical imaging techniques. One of the nanoparticles investigated is characterized, in addition to diagnostic, by therapeutic properties thanks to its ability to generate heat, when stimulated by an alternating magnetic field. The last research field, joining diagnostic and therapeutic capabilities in a single nanocompound, has been recently defined theranostics.
The aim of the thesis was to define and optimize some experimental procedures, that could be of general utility in testing diagnostic efficacy, toxicity and therapeutic efficacy of nanoparticles. Specifically the following nanocompounds have been studied:
1) iron oxide nanoparticle, presently in clinical trials, for lymph node metastasis detection (P904, Guerbet, France);
2) two lanthanide doped nanoparticles characterized by magnetic and upconversion properties (bimodal nanoparticles);
3) iron oxide nanoparticle, of biological origin (magnetosomes), characterized by diagnostic (detectability in MRI) and therapeutic properties (tumor ablation).
The results obtained with the above mentioned nanoparticles have been reported in different chapters of the thesis. In particular, in Capther 3, experimental studies, performed with P904 used as MRI diagnostic agent, in a murine model of lymph node metastasis, have been reported. Results indicate high sensitivity of P904 in the detection of lymph node metastasis. In Chapter 4, experiments performed with upconverting lanthanide doped nanoparticles are described. Studies about cellular toxicity and cellular uptake in vitro as well as biodistribution studies in vivo have been performed. Our results demonstrate that the upconverting lanthanide doped nanoparticles are characterized by low toxicity and by the capacity to label cells in vitro. Moreover, our studies suggest that lanthanide doped nanoparticles have potential usefulness, in vivo, as liver MRI contrast agents. In Chapter 5, in vitro studies on magnetosomes interaction with cancer cell and the in vivo experiments with magnetosomes used as MRI contrast agent and theranostic agent, have been described. The experimental results demonstrate that magnetosomes, innovative and biologically synthesized nanoparticles, may have interesting theranostic properties as they are easily detectable in MRI and, at the same time, have the capability of release heat in the tumor tissue when stimulated by proper, oscillating magnetic fields
Hyaluronic Acid (HA) Scaffolds and Multipotent Stromal Cells (MSCs) in Regenerative Medicine
No full textTraditional 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
Laser generated gold nanocorals with broadband plasmon absorption for photothermal applications
No full textGold nanoparticles with efficient plasmon absorption in the visible and near infrared (NIR) regions, biocompatibility
and easy surface functionalization are of interest for photothermal applications. Herein we
describe the synthesis and photothermal properties of gold “nanocorals” (AuNC) obtained by laser
irradiation of Au nanospheres (AuNS) dispersed in liquid solution. AuNC are formed in two stages: by
photofragmentation of AuNS, followed by spontaneous unidirectional assembly of gold nanocrystals. The
whole procedure is performed without chemicals or templating compounds, hence the AuNC can be
coated with thiolated molecules in one step. We show that AuNC coated with thiolated polymers are
easily dispersed in an aqueous environment or in organic solvents and can be included in polymeric
matrixes to yield a plasmonic nanocomposite. AuNC dispersions exhibit flat broadband plasmon absorption
ranging from the visible to the NIR and unitary light-to-heat conversion. Besides, in vitro biocompatibility
experiments assessed the absence of cytotoxic effects even at a dose as high as 100 μg mL−1.
These safe-by-designed AuNC are promising for use in various applications such as photothermal cancer
therapy, light-triggered drug release, antimicrobial substrates, optical tomography, obscurant materials
and optical coatings
Comparative technical analysis of lipoaspirate mechanical processing devices
No full textFat grafting is a well-established procedure in reconstructive, aesthetic, and regenerative medicine, in particular due to the presence in the adipose tissue of a high concentration of mesenchymal stem cells. The need to reduce fat processing times, for an immediate clinical use and regulatory restrictions on the degree of manipulation of human tissues have led to the development of numerous devices for the mechanical, non-enzymatic processing of adipose tissue. The aim of this study is to describe the state of the art of mechanical devices used for fat processing, performing a technical analysis of the currently commercially available devices. This should facilitate the development of new devices that improve therapeutic results
The post-adipocytic phase of the adipose cell cycle.
No full textSubcutaneous white adipose tissue harvested by liposuction has been studied with the aim to understand how the adipocytes modify their morphology when subjected to the passage in a needle for liposuction and to cryopreservation. The work try to clarify the ultrastructural aspects of adipose tissue, in the conditions described before, examining samples of body fat employed in fat graft procedures, and samples after cryopreservation. Scanning and transmission electron microscopy show that the first event that occur in the adipocytes is a lesion of mild degree detectable early in the samples fixed immediately after liposuction. The sequence of events following the adipocyte stress appeared composed by different phases: plasmatic membrane interruption, loss of lipid charge, formation of cup-like adipocytes and formation of post-adipocytes (i.e. cells that survive to traumatic events and restart to internalize lipid droplets). In conclusion, the study suggests that the loss of lipid charge in adipose cell is an active process that can be due to a small hole in the cytoplasmic membrane with the preservation of a large part of the cytoplasmatic content and that at the end of the process of lipid extrusion the cell can maintain viability
Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells
Full text linkMixed 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
Heterogeneous enhancement pattern in DCE-MRI reveals the morphology of normal lymph nodes: an experimental study
Full text linkPurpose: To investigate the heterogeneous enhancement pattern in normal lymph nodes of healthy mice by different albumin-binding contrast agents. Methods: The enhancement of normal lymph nodes was assessed in mice by dynamic contrast-enhanced MRI (DCE-MRI) after the administration of two contrast agents characterized by different albumin-binding properties: gadopentetate dimeglumine (Gd-DTPA) and gadobenate dimeglumine (Gd-BOPTA). To take into account potential heterogeneities of the contrast uptake in the lymph nodes, k-means cluster analysis was performed on DCE-MRI data. Cluster spatial distribution was visually assessed. Statistical comparison among clusters and contrast agents was performed on semiquantitative parameters (AUC, wash-in rate, and wash-out rate) and on the relative size of the segmented clusters. Results: Cluster analysis of DCE-MRI data revealed at least two main clusters, localized in the outer portion and in the inner portion of each lymph node. With both contrast agents, AUC (p < 0.01) and wash-in (p < 0.05) rates were greater in the inner cluster, which also showed a steeper wash-out rate than the outer cluster (Gd-BOPTA, p < 0.01; Gd-DTPA, p=0.056). The size of the outer cluster was greater than that of the inner cluster by Gd-DTPA (p < 0.05) and Gd-BOPTA (p < 0.01). The enhancement pattern of Gd-DTPA was not significantly different from the enhancement pattern of Gd-BOPTA. Conclusion: DCE-MRI in normal lymph nodes shows a characteristic heterogeneous pattern, discriminating the periphery and the central portion of the lymph nodes. Such a pattern deserves to be investigated as a diagnostic marker for lymph node staging
Morphogenetic events in the perinodal connective tissue in a metastatic cancer model.
No full textBackground: The modifications of connective tissue surrounding metastatic lymph nodes in a murine model of rectal cancer are described. Methods: Athymic nude mice (n = 36) were inoculated with 10 × 10 5 ht-29 cancer cells into the submucosal layer of the rectum. Control mice (n = 5) were treated with a sterile buffer. Tumor and the involved lymph nodes were visualized in vivo by magnetic resonance imaging at 1 to 4 weeks after cell injection. After the sacrifice, the excised samples were processed for histology. Results: After one week from cell injection all treated animals developed rectal cancer. Since the first week, neoplastic cells were visible in the nodes. In the surrounding connective tissue, the diameter of the adipocytes was reduced and a mesenchymal-like pattern with stellate cells embedded in an oedematous environment was visible. Since the second week, in the perinodal connective an enlargement of the stroma was present. The tissue was organized in cords and areas with extracellular accumulation of lipids were found. At the fourth week, we observed an enlargement of multilocular areas and lobules of elongated elements almost devoid of lipid droplets. In control animals, in absence of neoplastic masses, pelvic nodes were surrounded by a typical connective tissue characterized by unilocular adipocytes with groups of multilocular adipocytes. Conclusions: We have developed a model of rectal cancer with nodal metastases. Using this model, the work demonstrates that around secondary lesions, the morphogenetic events follow a standard evolution characterized by an early phase with lipolysis and mesenchymalization and later phases with a brown-like phenotype acquisition
Evaluation of lung inflammation induced by intratracheal administration of LPS in mice: comparison between MRI and histology.
No full textPURPOSE: To study the in vivo effect of intratracheal administration of lipopolysaccharide (LPS) in mice by magnetic resonance imaging (MRI) and to investigate the correlation with ex vivo histological evaluation of lung inflammation and oedema. MATERIALS AND METHODS: LPS (or phosphate buffered saline) was administered intratracheally to thirty male Balb/C mice at a concentration of 0.3 mg/ml in a total volume of 100 microl. Animals were divided into fifteen LPS-treated and fifteen control mice. MR images were acquired 24 h after challenge in freely breathing animals with standard ECG-gated Gradient-Echo (GRE) sequences and, in a limited number of animals, with ECG-gated Ultrashort-echo time (UTE) sequences. After MRI, animals were sacrificed, and lungs were fixed and processed for histological analysis of the total volume of healthy lung tissue. RESULTS: GRE images revealed the presence of high intensity signal in lungs of LPS-treated mice that was attributable to oedema caused by alveolar inflammation. In histological slices, regions of alterations in the normal alveolar microstructure were observed that could account for MRI findings. A good correlation was observed between the volumes of lesioned tissue measured by MRI and by histology. The volume of the lesion detected by GRE sequences was lower than the volume detected by UTE sequences. CONCLUSIONS: The effect of intratracheal administration of LPS in mice was investigated by MRI and histology. A good correlation was observed between GRE-MRI and histological findings. MR images obtained with UTE sequences appear to be more sensitive to the presence of lesions than those obtained by standard GRE acquisitions
Negative pressure from an internal spiral tissue expander generates new subcutaneous adipose tissue in an in vivo animal model
No full textBackground: Tissue expanders are widely utilized in plastic surgery. Traditional expanders usually are “inflatable balloons,” which are planned to grow additional skin and/or to create space to be filled, for example, with an implant. In very recent years, reports suggest that negative pressure created by an external device (ie, Brava) induces both skin expansion and adipogenesis. Objectives: The authors evaluated and assessed the adipogenetic potential of a novel internal tissue expander in an in vivo animal model. Methods: New Zealand female rabbits were enrolled in the study. A prototype spiral inner tissue expander was employed. It consisted of a-dynamic conic expander (DCE) with a valve at the end: when empty, it is flat (Archimedean spiral), whereas when filled with a fluid, it takes a conic shape. Inside the conic spiral, a negative pressure is therefore created. DCE is implanted flat under the latissimus dorsi muscle in experimental animals (rabbit) and then filled to reach the conical shape. Animals were investigated with magnetic resonance imaging, histology, and transmission electronic microscopy at 3, 6, and 12 months. Results: Magnetic resonance imaging revealed a marked increase in newly formed adipose tissue, reaching its highest amount at 12 months after the DCE implantation. Histology confirmed the existence of new adipocytes, whereas transmission electronic microscopy ultrastructure confirmed that most of these new cells were mature adipocytes. Conclusions: Tensile stress, associated with negative-pressure expanders, generated newly white subcutaneous adipose tissue
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