Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2.

The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.

[18F]FDG-PET/CT and long-term responses to everolimus in advanced neuroendocrine neoplasia.

PURPOSE: This study aims to identify in patients with neuroendocrine neoplasia (NEN) the potential correlation between FDG-PET findings and responses to everolimus therapy to identify predictors of long-term efficacy. METHODS: Retrospective analysis of patients with sporadic, advanced, progressive NEN treated with everolimus was performed based on the available data on FDG-PET patients obtained before commencing therapy. Data are expressed as the median (25-75th IQR). Risk factor analysis and survival analysis were performed by logistic regression and Cox proportional hazard regression and the determination of Kaplan-Meier curves, as appropriate. RESULTS: Sixty-six patients were evaluated (NET G1 19.7%, NET G2 75.7%, and NET G3 4.6%), including 45.4% with positive FDG-PET findings. Overall, disease stabilization and a partial response were achieved for 71.2% and 6% of patients, respectively. A long-term response (> 24 months) was observed in 33% of patients. Ki67 was the only predictor of tumor progression (p = 0.03). No significant difference in clinical outcomes was observed between patients with positive or negative FDG-PET findings (median PFS was 24 months and 18 months, respectively, p = 0.337; the disease control rate was 83.3% and 70%, respectively, p = 0.245). CONCLUSIONS: Everolimus is a valid therapeutic option for advanced, progressive, well-differentiated NEN, even in patients with positive FDG-PET findings.

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model.

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

Does conjugation strategy matter? Cetuximab-conjugated gold nanocages for targeting triple-negative breast cancer cells.

The efficient targeting of cancer cells depends on the success of obtaining the active targeting of overexpressed receptors. A very accurate design of nanoconjugates should be done via the selection of the conjugation strategy to achieve effective targeted nanoconjugates. Here, we present a detailed study of cetuximab-conjugated nonspherical gold nanocages for the active targeting of triple-negative breast cancer cells, including MDA-MB-231 and MDA-MB-468. A few different general strategies were selected for monoclonal antibody conjugation to the nanoparticle surface. By varying the bioconjugation conditions, including antibody orientation or the presence of a polymeric spacer or recombinant protein biolinker, we demonstrate the importance of a rational design of nanoconjugates. A quantitative study of gold content via ICP-AES allowed us to compare the effectiveness of cellular uptake as a function of the conjugation strategy and confirmed the active nature of nanoparticle internalization in cancer cells via epidermal growth factor receptor recognition, corroborating the importance of the rational design of nanomaterials for nanomedicine.

H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

Light scattered by model phantom bacteria reveals molecular interactions at their surface.

Testing molecular interactions is an ubiquitous need in modern biology and molecular medicine. Here, we present a qualitative and quantitative method rooted in the basic properties of the scattering of light, enabling detailed measurement of ligand-receptor interactions occurring on the surface of colloids. The key factor is the use of receptor-coated nanospheres matched in refractive index with water and therefore optically undetectable ("phantom") when not involved in adhesion processes. At the occurrence of ligand binding at the receptor sites, optically unmatched material adsorbs on the nanoparticle surface, giving rise to an increment in their scattering cross section up to a maximum corresponding to saturated binding sites. The analysis of the scattering growth pattern enables extracting the binding affinity. This label-free method has been assessed through the determination of the binding constant of the antibiotic vancomycin with the tripeptide l-Lys-d-Ala-d-Ala and of the vancomycin dimerization constant. We shed light on the role of chelate effect and molecular hindrance in the activity of this glycopeptide.

Current role of immunoscintigraphy in malignant melanoma follow-up. A study of 114 patients.

Although its role and importance is debated, Immunoscintigraphy (IS) remains a popular technique in Malignant Melanoma (MM) follow-up for postoperative assessment. Between April 1990 and December 1996, 287 consecutive patients underwent 650 IS examinations at our Department. Only data on 114 patients, operated up to December 1993, with a follow-up of at least 7 years are given here. IS results were compared to physical examination, to other imaging modalities and, where available, to histology. IS specificity and sensivity, concerning detection of melanoma metastases in lymph nodes, skin, brain, lung, visceral sites and bone, ranged from 37.5% and 22% to 100%. Our results indicate a high diagnostic accuracy of IS only for lymph node examination, but not for other possible locations of melanoma metastases. In our experience IS cannot be recommended for staging of melanoma patients, and should only be used in association with other imaging modalities.

Sternal wound infection revisited.

Sternal wound infections (SWIs) can be subdivided into two types, superficial or deep, that require different treatments. The clinical diagnosis of superficial SWI is normally easy to perform, whereas the involvement of deep tissues is frequently difficult to detect. Therefore, there is a need for an imaging study that permits the assessment of SWIs and is able to distinguish between superficial and deep SWI. The present work was a prospective study aiming to evaluate the role of technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO) labelled leucocyte scan in SWI management. Twenty-eight patients with suspected SWIs were included in the study. On the basis of clinical examination they were subdivided into three groups: patients with signs of superficial SWI (group 1), patients with signs of superficial SWI and suspected deep infection (group 2) and patients with suspected deep SWI without superficial involvement (group 3). Ten patients previously submitted to median sternotomy, but without suspected SWI, were also included in the study as a control group (group 4). All patients with suspected SWI had bacteriological examinations of wound secretion, if present. In addition 99mTc-HMPAO labelled leucocyte scan was performed in all patients. The patients of groups 1, 2 and 3 were treated on the basis of the clinical signs and microbiological findings, independently of the scintigraphic results. The patients of group 4 did not receive treatment. The final assessment of infection was based on histological and microbiological findings or on long-term clinical follow-up. Sensitivity, specificity, accuracy and positive and negative predictive values for scintigraphic and non-scintigraphic results were calculated. In the diagnosis of superficial and deep SWI, clinical and microbiological examination (combined) yielded, respectively, a sensitivity of 68.7% and 100%, a specificity of 77.3% and 80.8%, an accuracy of 73.7% and 86.8%, a positive predictive value of 68.7% and 70.6% and a negative predictive value of 77.3% and 100%. The scintigraphic results obtained in superficial SWI yielded a sensitivity of 56.2%, a specificity of 90.9%, an accuracy of 76.3%, a positive predictive value of 81.8% and a negative predictive value of 74.1%, while, by contrast, in deep SWI all of these values were 100%. Therefore, one can conclude that 99mTc-HMPAO labelled leucocyte scan permits accurate diagnosis of deep SWI, solving the main clinical problem in this field. In the present study the categorisation of patients without taking into account 99mTc-HMPAO labelled leucocyte planar scan findings caused a non-negligible number of cases of superficial SWI to be treated as though they were deep SWI. This "overestimation" led to unnecessary surgery, increased and prolonged use of antibiotics with more (higher) toxicity and additional expense.

[Computerized tomography, magnetic resonance, and nuclear medicine in the non-invasive diagnosis of focal nodular hyperplasia of the liver]. / Tomografia Computerizzata, Risonanza Magnetica e medicina nucleare nella diagnosi non invasiva dell'iperplasia nodulare focale del fegato.

INTRODUCTION: Focal nodular hyperplasia (FNH) is a benign liver lesion requiring a prompt diagnosis and a conservative management. Aim of our study was to prospectively integrate enhanced CT, MRI, nuclear medicine in the noninvasive diagnosis of FNH. MATERIAL AND METHODS: 20 FNH lesions (diameter ranging 1.5-13 cm) in 18 asymptomatic patients were investigated with MRI and nuclear medicine. MRI examinations were performed with a 1.5 T superconducting system (Philiphs NT) by acquiring T1-weighted, T2-weighted, T2-weighted fat-suppressed Turbo Spin-Echo and dynamic MRI sequences, with breath-hold T1-weighted Turbo Field Echo and Gd-DTPA. Nuclear medicine included in all cases 99mTc sulfur colloid or 99mTc iminodiacetic acid studies. Diagnostic sensitivity of MRI was compared with that of nuclear medicine, and the sensitivity of the two modalities combined. The definitive diagnosis was made by percutaneous core-needle biopsy (12 lesions), surgery (2 lesions) and longterm follow-up (6 lesions). RESULTS: In 13 lesions larger than 3.5 cm typical findings, such as the central scar and homogeneous pattern, were observed in 9 cases (75%) with enhanced CT, in 10 cases (83%) with unenhanced MRI and in 11 cases (91%) after gadolinium injection. Hepatobiliary scintigraphy showed increased tracer uptake in delayed scans in 10/12 cases (83%) while sulfur colloid studies were diagnostic in 2/12 cases only (16%), showing the same tracer uptake than the surrounding liver parenchyma. In 7 lesions smaller than 3.5 cm, only 1 case showed typical findings; dynamic MRI showed typical early hypervascularity in 5 of the 6 remaining lesions (71%) which persisted on late images. In lesions smaller than 3.5 cm, sulfur colloid studies were diagnostic in 1/7 lesions (14%) and hepatobiliary scintigraphy in 3/7 lesions (42%). Considering all 20 lesions, MRI-nuclear medicine integration performed better than MRI alone. CONCLUSIONS: Enhanced MRI and biliary scintigraphy are important tools to make the final diagnosis of FNH. In small lesions both examinations should performed, but in large lesions MRI may be sufficient to make an unquestionable diagnosis, limiting hepatobiliary scintigraphy to questionable cases.

[Assessment with magnetic resonance with Turbo-FLAIR sequences and volumetric analysis of the hippocampal region in drug-resistant temporal epilepsy]. / Valutazione con Risonanza Magnetica con sequenze Turbo-FLAIR e analisi volumetrica della regione ippocampale nell'epilessia temporale farmaco-resistente.

INTRODUCTION: We studied the hippocampal region using Fluid Attenuation Inversion Recovery (Turbo-FLAIR) sequences to detect signal intensity abnormalities, and volumetric sequences to detect cortical thickness changes, in patients with drug-resistant temporal epilepsy. MATERIALS AND METHODS: We examined 30 patients with drug-resistant temporal epilepsy with a 1.5 Tesla unit (NT 15 Philips Gyroscan). Conventional SE, Turbo SE, IR, Turbo-FLAIR, volumetric 3D sequences on coronal plane, PD, T2-weighted SE sequences on axial plane, T1-weighted SE on sagittal plane were performed. Signal intensity and volumetric computerized measurements were obtained using the SUN system. RESULTS: Differences in signal intensity values between the two hippocampal regions were found in 18 patients with Turbo-FLAIR sequences. In 6 of these patients no significant differences in computerized evaluation of signal intensity were detected with either conventional or Turbo-SE sequences. Volumetric analysis showed hippocampal cortex thinning in 9 of 18 patients with hippocampal signal intensity abnormalities. CONCLUSIONS: Turbo-FLAIR were the best sequences for the detection of signal intensity changes in the hippocampal region. Such changes are strongly suggestive of hippocampal sclerosis, especially when associated with cortical atrophy.

Clinical usefulness of technetium-99m-HMPAO-labeled leukocyte scan in prosthetic vascular graft infection.

UNLABELLED: The infection of a prosthetic vascular graft (PVGI), although rare, is the most severe complication in reconstructive vascular surgery. The early diagnosis of this complication reduces the death rate from surgery. Aortofemoral graft infections differ clinically from peripheral graft infections in significant ways. The aim of this article is to evaluate separately the reliability of the 99mTc-HMPAO-labeled leukocyte scan or white blood cell count (WBC) in the early detection of both aortofemoral and peripheral graft infections. METHODS: One hundred sixty-two WBCs were performed on 129 consecutive patients with suspected aortofemoral (122 scans) and peripheral (40 scans) graft infection and in a 12-patient control group. Patients with suspected PVGI were categorized into three groups on the basis of their signs and symptoms on readmission: (a) patients with specific signs of graft infection (Group A); (b) patients with nonspecific signs of graft infection (Group B); and (c) patients with anastomotic aneurysms (Group C). Gram's stains of the perigraft exudate and graft cultures were performed and used as the gold standard in patients who underwent surgery. An 18-mo clinical follow-up was done to assess the presence or absence of graft infection in patients who did not have surgery. RESULTS: In patients with suspected aortofemoral graft infections, the overall sensitivity, specificity and accuracy of WBCs (Groups A, B, C) were 100%, 92.5% and 97.5%, respectively, whereas sensitivity, specificity and accuracy calculated in the patients with nonspecific signs of graft infection (Groups B, C) were 100%, 92.3% and 96.9%, respectively. In patients with suspected peripheral graft infections, sensitivity, specificity and accuracy were 100%. CONCLUSION: The white blood cell scan seems a reliable diagnostic method for early diagnosis of PVGI, and it is more useful in aortofemoral graft infections.

Barriers to mental health service utilization.

This study examines the system dimensions of need, barriers to receiving services, and utilization within a single mental health service area. The gap between estimates of service need and service utilization is conceptualized as due to a set of specific barriers covering the access attributes of availability, accessibility, acceptability and affordability. Data from community telephone surveys (N = 2183) of mental health need are analyzed to determine the relationship between the system dimensions of need, barriers and utilization. Respondents were able to distinguish among different types of service barriers. Those in the service gap were potentially more influenced by barriers than the rest of the sample, as were, paradoxically, those who had utilized services within the past year. The implications of these findings for service provision and system design are discussed.