Silver enhancement for transmission electron microscopy imaging of antibody fragment-gold nanoparticles conjugates immobilized on ordered mesoporous silica

Ordered mesoporous silica (OMS) materials are receiving great attention as possible carriers for valuable but unstable drugs as, for example, therapeutic proteins. A key issue is to prove that the therapeutic protein is effectively able to penetrate the pores of OMS during the adsorption step. Here, we immobilized an antibody fragment [F(ab')GAMIgG] conjugated with ultrasmall gold nanoparticles (GNPs) onto amino-functionalized SBA-15 (SBA-NH2) mesoporous silica. The aim of this work is the visualization of the location of the conjugates adsorbed onto SBA-NH2 with transmission electron microscopy (TEM). Because of the ultrasmall size of GNPs (<1 nm), we use the silver enhancement procedure to amplify their size. In this procedure, ultrathin sections of conjugate-loaded SBA-NH2 particles are prepared by a ultramicrotome sectioning technique. The ultrasmall GNPs located on the top side of the 70-90 nm thick slices act as microcrystallization nucleation sites for the deposition of reduced metallic silver. Consequently, the ultrasmall GNPs increase their size. This allows for the direct imaging of the conjugates adsorbed. We clearly localize the F(ab')GAMIgG-GNPs conjugates either on the external surface of the particles or inside the mesopores of SBA-NH2 through TEM

Release of small hydrophilic molecules from polyelectrolyte capsules : Effect of the wall thickness

Polymer nanocapsules assembled on cationic liposomes have been built through the layer-by-layer (LbL) technique. Chitosan and alginate, two biocompatible polyelectrolytes, were used to cover the template, where the Rhodamine B was previously loaded. The multishell formed with the alternate deposition of the polyelectrolytes, according to the principles of the LbL assembly, was supposed to change the permeability of the capsule wall. The thickness of the multishell was seen increasing with the number of layers deposited through the observations with the Transmission Electron Microscope. The permeability of the capsules was studied through Rhodamine B release assays. Nanocapsules with seven layers of polyelectrolytes released the dye slowly compared to the capsules with three or five layers. The Ritger-Peppas model was applied to investigate the release mechanisms and a non-Fickian transport behavior was detected regardless of the number of layers. Values of diffusion coefficients of Rhodamine B through the capsule wall were also calculated. (C) 2014 Elsevier Inc. All rights reserved

Loading and Protection of Hydrophilic Molecules into Liposome-Templated Polyelectrolyte Nanocapsules

Compartmentalized systems produced via the layer-by-layer (LbL) self-assembly method have been produced by alternatively depositing alginate and chitosan layers onto cores of liposomes. The combination of dynamic light scattering (DLS), zeta potential, and transmission electron microscopy (TEM) techniques provides detailed information on the stability, dimensions, charge, and wall thickness of these polyelectrolyte globules. TEM microphotographs demonstrate the presence of nanocapsules with an average diameter of below 300 nm and with a polyelectrolyte wall thickness of about 20 nm. The possibility of encapsulating and releasing molecules from this type of nanocapsule was demonstrated by loading FITC-dextrans of different molecular weights in the liposome system. The release of the loaded molecules from the nanocapsule was demonstrated after liposome core dissolution. Even at low molecular weight (20 kDa), the nanocapsules appear to be appropriate for prolonged molecule compartmentalization and protection. By means of the Ritger-Peppas model, non-Fickian transport behavior was detected for the diffusion of dextran through the polyelectrolyte wall. Values of the diffusion coefficient were calculated and yield useful information regarding chitosan/alginate hollow nanocapsules as drug-delivery systems. The influence of the pH on the release properties was also considered. The results indicate that vesicle-templated hollow polyelectrolyte nanocapsules show great potential as novel controllable drug-delivery devices for biomedical and biotechnological applications

Predictors of Outcome after (Chemo)Radiotherapy for Node-Positive Oropharyngeal Cancer: The Role of Functional MRI

The prognosis of a subset of patients with locally advanced oropharyngeal cancer (LA-OPC) is still poor despite improvements in patient selection and treatment. Identifying specific patient- and tumor-related factors can help to select those patients who need intensified treatment. We aimed to assess the role of historical risk factors and novel magnetic resonance imaging (MRI) biomarkers in predicting outcomes in these patients. Patients diagnosed with LA-OPC were studied with diffusion-weighted imaging (DWI) and dynamic-contrast enhanced MRI at baseline and at the 10th radiotherapy (RT) fraction. Clinical information was collected as well. The endpoint of the study was the development of disease progression, locally or distantly. Of the 97 patients enrolled, 68 were eligible for analysis. Disease progression was recorded in 21 patients (11 had loco-regional progression, 10 developed distant metastases). We found a correlation between N diameter and disease control (p = 0.02); features such as p16 status and extranodal extension only showed a trend towards statistical significance. Among perfusion MRI features, higher median values of Kep both in primary tumor (T, p = 0.016) and lymph node (N, p = 0.003) and lower median values of ve (p = 0.018 in T, p = 0.004 in N) correlated with better disease control. Kep P90 and N diameter were identified by MRMR algorithm as the best predictors of outcome. In conclusion, the association of non-invasive MRI biomarkers and patients and tumor characteristics may help in predicting disease behavior and patient outcomes in order to ensure a more customized treatment

Mesoporous silica nanoparticles functionalized with hyaluronic acid and chitosan biopolymers. Effect of functionalization on cell internalization

Mesoporous silica nanoparticles (MSNs), based on the MCM-41 matrix, were functionalized with amino groups, and then with hyaluronic acid (HA) or chitosan (CHIT) to fabricate bioactive conjugates. The role of the functional groups toward cytotoxicity and cellular uptake was investigated using 3T3 mouse fibroblast cells. A very high biocompatibility of MSN-NH2, MSN-HA and MSN-CHIT matrices was assessed through the MTS biological assay and Coulter counter evaluation. No significant differences in cytotoxicity data arise from the presence of different functional groups in the investigated MSNs. Fluorescence microscopy experiments performed using fluorescein isothiocyanate-conjugated MSN-NH2, MSN-HA, and MSN-CHIT, and transmission electron microscopy experiments performed on slices of the investigated systems embedded in epoxy resins give evidence of significant differences due to type of functionalization in terms of cellular uptake and stability of the particles in the biological medium. MSN-NH2 and MSN-HA conjugates are easily internalized, the uptake of the HA-functionalized MSNs being much higher than that of the -NH2-functionalized MSNs. Differently, MSN-CHIT conjugates tend to give large aggregates dispersed in the medium or localized at the external surface of the cell membranes. Both fluorescence microscopy and TEM images show that the MSNs are distributed in the cytoplasm of the cells in the case of MSN-NH2 and MSN-HA, whereas only a few particles are internalized in the case of MSN-CHIT. Flow cytometry experiments confirmed quantitatively the selectively high cellular uptake of MSN-HA particle

Interactions between bovine serum albumin and mesoporous silica nanoparticles functionalized with biopolymers

Biomedical application of nanoparticles is largely associated to their fate in biological media which, in turn, is related to their surface properties. Surface functionalization plays a key role in determining biodegradation, cytotoxicity and biodistribution through interactions which may be mediated by the macromolecules occurring in biological media. A typical example is given by several proteins which lead to the formation of coated nanoparticles referred as protein corona. In this work we focus on mesoporous silica nanoparticles which, due to their intrinsic textural features, show potential as carriers for sustained drug release. Mesoporous silica nanoparticles functionalized by different biopolymers such as hyaluronic acid and chitosan were synthesized and characterized through small angle X-rays scattering, thermal analysis, and infrared spectroscopy. Biopolymer-coated mesoporous silica nanoparticles were used to investigate the interaction with bovine serum albumin, and to point out the role of different biopolymer coating. Gold-conjugated-bovine serum albumin was used to gain evidence on the occurrence of surface bound proteins enabling direct observation by transmission electron microscopy. Our findings provide insights on how different biopolymers affect the formation of a protein corona around functionalized mesoporous silica nanoparticles

Brain atrophy pattern in patients with mild cognitive impairment: MRI study.

We evaluated the accuracy of the quantitative and semiquantitative analysis in detecting regional atrophy patterns and differentiating mild cognitive impairment patients who remain stable (aMCI-S) from patients who develop Alzheimer's disease (aMCI-AD) at clinical follow-up. Baseline magnetic resonance imaging was used for quantitative and semiquantitative analysis using visual rating scales. Visual rating scores were related to gray matter thicknesses or volume measures of some structures belonging to the same brain regions. Receiver operating characteristic (ROC) analysis was performed to assess measures' accuracy in differentiating aMCI-S from aMCI-AD. Comparing aMCI-S and aMCI-AD patients, significant differences were found for specific rating scales, for cortical thickness belonging to the middle temporal lobe (MTL), anterior temporal (AT), and fronto-insular (FI) regions, for gray matter volumes belonging to MTL and AT regions. ROC curve analysis showed that middle temporal atrophy, AT, and FI visual scales showed better diagnostic accuracy than quantitative measures also when thickness measures were combined with hippocampal volumes. Semiquantitative evaluation, performed by trained observers, is a fast and reliable tool in differentiating, at the early stage of disease, aMCI patients that remain stable from those patients that may progress to AD since visual rating scales may be informative both about early hippocampal volume loss and cortical thickness reduction

Surgical treatment of cerebellar metastases in elderly patients: A threshold that moves forward?

The impact of surgery for cerebellar brain metastases in elderly population has been the object of limited studies in literature. Given the increasing burden of their chronic illnesses, the decision to recommend surgery remains difficult.All patients aged ≥65 years, who underwent surgical resection of a cerebellar brain metastasis from May 2000 and May 2021 at IRCCS National Cancer Institute “Regina Elena”, were analyzed. The study cohort includes 48 patients with a mean age of 70.8 years. 7 patients belonged to the II Class according to the RPA classification, 41 to the III Class; the median GPA classification was 1.5. Median pre-operative and post-operative KPS was 60. Median Charlson Comorbidity Index (CCI) was 11; median 5-variable modified Frailty Index was 2. Overall, 14 patients (29%) presented perioperative neurologic and systemic complications. 34 patients (71%) were able to perform adjuvant therapies as RT and/or CHT after surgery. A higher CCI predicted complications occurrence (p = 0.044), while significant factors for a post-operative KPS ≥70, were i) hemispheric location of the metastasis, ii) higher pre-operative KPS, iii) RPA II classification. Median Overall Survival was 7 months. A post-operative KPS <70 (p = 0.004) and a short time interval between diagnosis of the primary tumor and cerebellar metastasis appearance, were predictive for a worse outcome (p = 0.012). Our study suggests that selected elderly patients with cerebellar metastases may benefit from microsurgery to continue their adjuvant therapies, although a high complications rate should be taken in account