In vivo imaging techniques for bone tissue engineering.

Bone is a dynamic tissue that constantly undergoes modeling and remodeling. Bone tissue engineering relying on the development of novel implant scaffolds for the treatment of pre-clinical bone defects has been extensively evaluated by histological techniques. The study of bone remodeling, that takes place over several weeks, is limited by the requirement of a large number of animals and time-consuming and labor-intensive procedures. X-ray-based imaging methods that can non-invasively detect the newly formed bone tissue have therefore been extensively applied in pre-clinical research and in clinical practice. The use of other imaging techniques at a pre-clinical level that act as supportive tools is convenient. This review mainly focuses on nuclear imaging methods (single photon emission computed tomography and positron emission tomography), either alone or used in combination with computed tomography. It addresses their application to small animal models with bone defects, both untreated and filled with substitute materials, to boost the knowledge on bone regenerative processes.

Evaluation of ανß3-mediated tumor expression with a 99mTc-labeled ornithine-modified RGD derivative during glioblastoma growth in vivo.

In this study, a novel way of distinguishing the intrinsic relationship between ανß3 integrin targeting and detection of tumor growth by using a radiolabeled tracer based on a cyclic Arg-Gly-Asp (RGD) peptide was provided. The potential of the in vivo scintigraphic imaging of the developing vasculature from the early stage of tumor growth was evaluated. Alongside with the scintigraphic images, biodistribution studies were performed at distinct time points to validate this noninvasive imaging approach. The ability to noninvasively assess the tumor growth of ανß3 integrin-positive glioblastoma tumors provides a method to better understand tumor angiogenesis in vivo and allows for a direct assessment of anti-integrin treatment efficacy.

Hollow microspheres based on - Folic acid modified - Hydroxypropyl Cellulose and synthetic multi-responsive bio-copolymer for targeted cancer therapy: controlled release of daunorubicin, in vitro and in vivo studies.

HYPOTHESIS: Conventional chemotherapy drugs such as anthracyclines show no specific activity. They destroy cancer cells but also and the healthy ones, and for that reason exhibit high toxicity. In order to alleviate the toxic effects of chemotherapeutic drugs, the administration dose is being minimized, while their reactivity against tumor cells is lessened. This problem can be overcome or at least reduced by using nanoscale drug delivery systems to target the pathogenic area. The present work deals with the synthesis, characterization and biological evaluation of multi-responsive hollow microspheres coated with Hydroxypropyl Cellulose (HPC)-a biocompatible and thermosensitive polysaccharide-conjugated with folic acid as well promising drug vehicles for targeted cancer therapy. EXPERIMENTS: The synthetic route consists of two steps. In the first step, a single layer of sensitive copolymers is ((Methacrylic acid (MAA), N-(2-Hydroxypropyl) methacrylamide (HPMA) and N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(2-methylacrylamide) (DSBMA)) fabricated on a sacrificial template of SiO2 and in the second step, an additional layer of the folic acid modified HPC coat the microspheres' surface. The layers fabrication is performed through a combination of distillation precipitation co-polymerization and chemical deposition method. The loading capacity (% LC) and encapsulation efficiency (% EE) percentages of the chemotherapeutic agent daunorubicin (DNR) in the fabricated microspheres were calculated through the standard curve methodology. In addition, the releasing properties of the resulting spheres are investigated, using the above mentioned methodology. It is worth mentioning that, spheres release the entrapped drug under combined conditions such acidic and reductive environment along with conventional hyperthermia. Cytotoxic activity of the synthesized spheres was investigated by using the well-established method of MTT assay in MCF-7 (breast cancer), HeLa (cervical cancer) and HEK 293 (Human Embryonic Kidney healthy cells) cell lines. Confocal and fluorescence microscopy were used to confirm the in vitro targeted ability of folic acid modified drug loaded microspheres in HeLa, to that overexpress folate receptors, MCF-7 and 3T3 cells, as negative folate cell substrate. Finally, radiolabelling of the spheres is performed, with a gamma emitting radionuclide ((99m)Tc), to assess their in vivo profile by means of scintigraphic imaging and biodistribution studies. FINDINGS: Hollow spheres release the encapsulated drug under acidic environment, conventional hyperthermia or in the presence of glutathione (reductive environment). The ability of modified drug carriers to target the HeLa cells, was confirmed by confocal and fluorescence microscopy. The resulting spheres are observed to be promising drug-carriers for cancer treatment due to their releasing properties under tumor's environment and high concentration in HeLa cells via endocytosis. In addition, the empty vehicles have no toxicity in healthy cells and present antimicrobial activity.

In vivo anticancer evaluation of the hyperthermic efficacy of anti-human epidermal growth factor receptor-targeted PEG-based nanocarrier containing magnetic nanoparticles.

Polymeric nanoparticles with targeting moieties containing magnetic nanoparticles as theranostic agents have considerable potential for the treatment of cancer. Here we report the chemical synthesis and characterization of a poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-based nanocarrier containing iron oxide nanoparticles and human epithelial growth factor receptor on the outer shell. The nanocarrier was also radiolabeled with (99m)Tc and tested as a theranostic nanomedicine, ie, it was investigated for both its diagnostic ability in vivo and its therapeutic hyperthermic effects in a standard A431 human tumor cell line. Following radiolabeling with (99m)Tc, the biodistribution and therapeutic hyperthermic effects of the nanosystem were studied noninvasively in vivo in tumor-bearing mice. A substantial decrease in tumor size correlated with an increase in both nanoparticle concentration and local temperature was achieved, confirming the possibility of using this multifunctional nanosystem as a therapeutic tool for epidermoid carcinoma.

Comparative in vitro stability and scintigraphic imaging for trafficking and tumor targeting of a directly and a novel 99mTc(I)(CO)3 labeled liposome.

Liposomes radiolabelling with diagnostic radionuclides is an excellent tool for studying pharmacokinetics with the view of developing liposome-based drug delivery agents. The aim of the present study is to evaluate the in vitro and in vivo behavior of a (99m)Tc-labeled liposome by applying either a direct labeling strategy via a carboxyl group, LP-COOH, or a surface chelating method via pyridyl ethyl cysteine compound (with the intermediate [99mTc(I)(CO)3(H2O)(3)](+)), LP-PEC. 99mTc-LP-COOH was obtained in high radiolabelling yield and radiochemical purity, while 99mTc(I)(CO)3-LP-PEC was initially purified before being in vitro and in vivo evaluated. 99mTc-LP-COOH was less stable in the presence of competitive for 99mTc ligands than 99mTc(I)(CO)3-LP-PEC. According to DLS measurements, the presence of serum as well as the applied radiolabelling conditions did not affect the liposomes' size. The different radiolabelling methods seemed to exert an influence on the biodistribution pattern of the liposomes with the 99mTc(I)(CO)3-LP-PEC showing slow blood clearance, which was also confirmed by in vivo scintigraphic imaging. Nevertheless, passive tumor targeting was attained at a similar extent no matter which radiolabelling technique was followed.

Structural modifications of 99mTc-labelled bombesin-like peptides for optimizing pharmacokinetics in prostate tumor targeting.

PURPOSE: The main goal of the present study was to investigate the importance of the addition of a positively charged aa in the naturally occurring bombesin (BN) peptide for its utilization as radiodiagnostic agent, taking into consideration the biodistribution profile, the pharmacokinetic characteristics and the tumor targeting ability. METHODS: Two BN-derivatives of the general structure [M-chelator]-(spacer)-BN(2-14)-NH(2), where M: (99m)Tc or (185/187)Re, chelator: Gly-Gly-Cys-, spacer: -(arginine)(3)-, M-BN-A; spacer: -(ornithine)(3)-, M-BN-O; have been prepared and evaluated as tumor imaging agents. RESULTS: The peptides under study presented high radiolabelling efficiency (>98%), significant stability in human plasma (>60% intact radiolabelled peptide after 1h incubation) and comparable receptor binding affinity with the standard [(125)I-Tyr(4)]-BN. Their internalization rates in the prostate cancer PC-3 cells differed, although the amount of internalized peptide was the same. The biodistribution and the dynamic γ-camera imaging studies in normal and PC-3 tumor-bearing SCID mice have shown significant tumor uptake, combined with fast blood clearance, through the urinary pathway. CONCLUSION: The addition of the charged aa spacer in the BN structure was advantageous for biodistribution, pharmacokinetics and tumor targeting ability, because it reduced the upper abdominal radioactivity levels and increased tumor/normal tissue contrast ratios.

Spacer site modifications for the improvement of the in vitro and in vivo binding properties of (99m)Tc-N(3)S-X-bombesin[2-14] derivatives.

It has been shown that gastrin releasing peptide receptors (GRPRs) are overexpressed in various types of cancer cells. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPRs. Significant research efforts have been lately devoted to the design of radiolabeled 8 or 14 aminoacid bombesin (BN) peptides for the detection (either with gamma or positron emitting radionuclides) and therapy (with beta(-) emitting radionuclides) of cancer. The specific aim of the present study was to further investigate the radiolabeled peptide structure and to determine whether the total absence of a linker or the use of a basic diverse amino acid linker could influence the biodistribution profile of the new compounds for specific targeting of human prostate cancer. Thus, two new derivatives with the structure Gly-Gly-Cys-X-BN[2-14], where linker X is either zero (I) or Orn-Orn-Orn (Orn: ornithine) (II) were designed and synthesized. The corresponding (99m)Tc-BN derivatives were obtained with high radiochemical yield (>98%) and had almost identical retention times in RP-HPLC with the (185/187)Re complexes, which were also characterized by ESI-MS. Metabolic stability was found to be high in human plasma, moderate in PC-3 cells, and rather low in mouse liver and kidney homogenates for both BN derivatives studied. The BN derivative without the spacer was less stable in cell culture and liver homogenates. A satisfactory binding affinity to GRPRs, in the nanomolar range, was obtained for both BN derivatives as well as for their Re complexes, with BN (II) demonstrating the highest one. In vitro internalization/externalization assays indicated that approximately 6% of BN (I) and approximately 25% of BN (II) were internalized into PC-3 cells. In vivo evaluation in normal Swiss mice and in tumor bearing SCID mice showed that BN (II) presented higher tumor and pancreas uptake than BN (I). Small animal SPECT dynamic imaging, carried out after an injection of BN (II) in mice bearing PC-3 tumors, resulted in PC-3 tumor delineation with low background activity. Overall, this study performed for two new N(3)S-X-BN[2-14] derivatives indicated that hydrophilicity and charge strongly affected the in vitro and in vivo binding properties and the biodistribution pattern. This finding is confirmed by SPECT imaging of BN (II), which is under further in vivo evaluation for detecting cancer-positive GRPRs.